Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

A Large Number of Reviews on Physician Rating Websites May Reflect Reputation Management

BackgroundPhysicians with a large number of reviews and a high rating may be employing reputation management strategies. Specialists may be more likely than non-specialists to employ such strategies. This should be apparent in a study of online physician reviews on physician rating websites (PRW).MethodsUsing one physician rating website, we gathered orthopedic surgeon and family physician reviews. We measured Spearman correlations between the number of reviews and average numerical rating and used chi-squared to test threshold relationships.ResultsThere were very small negative Spear-man correlations between the number of online reviews and the average numerical rating for orthopedic surgeons (p= -0.097, p-value=<0.001) family medicine physicians (p= -0.170, p-value=<0.001; Figure 2). Physicians with more than 100 reviews had a greater average numerical rating than physicians with fewer than 50 reviews. Orthopedic surgeons are more likely than family medicine physicians to have a large number of reviews and average numerical rating greater than 3.Open in a separate windowFigure 2.Family medicine physicians average rating plotted against number of reviews.ConclusionThe small fraction of physician with a high number of reviews may be utilizing reputation management strategies, and this seems relatively specific to specialists rather than non-specialists. Level of Evidence: III     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Diversity in spindle morphology in Arabidopsis root tip

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.Key words: spindle pole, microtubule, morphogenesis, cell type, metaphaseThe cellular apparatus for chromosome segregation during mitosis is typically described as a spindle composed of microtubules and microtubule-associated proteins. Research on the structure and function of the spindle is usually conducted under the assumption that spindles are structurally the same or alike in different cell types in an organism. If the assumption is true, it would indicate that either the intracellular conditions in different dividing cells are very similar or the assembly and maintenance of the spindle are insensitive to otherwise variable intracellular conditions. But experimental evidence related to this assumption is relatively sparse.The root tip in Arabidopsis, as in other higher plants, contains dividing cells of different shapes and sizes. These cells include both meristem initial and derivative cells, with the former and latter being proximal and distal to the quiescent center, respectively.¹ The diversity in dividing cells in the root tip provides an opportunity for testing whether the spindles also exhibit diversity in morphology. To visualize the spindles at the metaphase stage in the root tip cells, we conducted indirect immunofluorescence labeling of the β-tubulin in single cells prepared from wild-type Arabidopsis (in Col-0 background) root tips as previously described in references ² and ³. The spindles in cells of different morphologies were then observed under a confocal laser scanning microscope.³ Three types of spindle were detected. The first type (Fig. 1A) was the smallest in width and length and had the most-pointed poles among the three types. The second type (Fig. 1B) was wider and longer than the first type but with less-pointed poles than the first type. The third type (Fig. 1C) was similar in height to the second type but lacked the pointed poles. In fact, the third type is shaped more like a barrel than a spindle. The first type was found in cells narrow in the direction parallel to the equatorial plane of the spindle, a situation opposite to that of the third type whose cells were wide in the equatorial direction. The wide cells containing the barrel-shaped spindles likely belonged to the epidermal layer in the root tip.¹ The second type was found in cells intermediate in width. Examples of metaphase spindles morphologically resembling the three types of spindles in Arabidopsis root can also be found in a previous report by Xu et al. even although spindle diversity was not the subject of the report.⁴ In Xu et al.''s report, type 1- or 2-like metaphase spindles can be identified in Figures 2B and 3A, and type 3-like metaphase spindles can be identified in Figures 1A and 3B. These observations indicate that at least three types of spindles exist in the root cells.Open in a separate windowFigure 1Spindles in wild-type root cells. (A) Type-1 spindle. (B) Type-2 spindle. (C) Type-3 spindle. The spots without fluorescence signals in the middle of the spindles are where the chromosomes were located. Scale bar for all the figures = 20 µm.Open in a separate windowFigure 2Spindles in cyd1 root cells. (A) Type-1 spindle. Arrows indicate the upper and lower boundaries of the cell. (B and C) Two type-2 spindles. (D and E) Two type-3 spindles. (F) DAPI-staining image corresponding to (E), showing chromosomes at the equatorial plane. Scale bar for the images = 20 µm.The above observations suggest that either the cell size or the cell type (shape) might be a factor in the type of spindle found in a specific cell. To further investigate the relationship between cell morphology and spindle morphology, we studied metaphase spindles in root cells of the cytokinesis defective1 (cyd1) mutant.⁵ Because the root cells in cyd1 were larger than corresponding cells in the wild type, presumably due to abnormal polyploidization prior to the collection of the root cells,^5,6 this investigation might reveal a relationship between increasing cell size and altered spindle morphology. A pattern of different spindle types in different cell types similar to that in the wild type was observed in cyd1 (Fig. 2). Figures 2A–C show narrow cells that contained spindles with pointed poles even though the spindles differed in size and focus. Figure 2D shows a barrel-shaped spindle in a wide cell, resembling Figure 1C in overall appearance. The large number of chromosomes at metaphase (more than the diploid number of 10) in Figure 2F indicates that the cells in Figure 2 were polyploid. These figures thus demonstrate that the enlargement in cell size did not alter the pattern of types 1 and 2 spindles in narrow cells, as well as type 3 spindles in wide cells. Moreover, the edges of the spindles in Figure 2B and E were similarly distanced to the cell walls in the equatorial plane, and yet they differ greatly in shape with the former being type 2 and the latter being type 3. This finding argues against that the cell width in the equatorial direction dictates the spindle shape. On the other hand, the cells in Figure 2B and E are obviously of different types. Taken together, these observations suggest that the spindle diversity in both wild type and cyd1 is associated with cell-type diversity.It is unclear whether the different spindle types have different functions in their respective cell types, in addition to the usual role for chromosome segregation. One possibility is that, at the ensuing telophase, the pointed spindles result in compact chromosomal congregation at the poles whereas the barrel-shaped spindles result in loose chromosomal congregation at the poles, which in turn may differentially affect the shape of the subsequently formed daughter nuclei and their organization. Different nuclear shape and organization are likely to be integrated into the processes that confer cell differentiation.     

A photoreceptor going nowhere but the nucleus

Cryptochrome 2 (CRY2) is a blue/UV-A light receptor that regulates light inhibition of cell elongation and photoperiodic promotion of floral initiation in Arabidopsis. We and others have previously shown that CRY2 is a nuclear protein that regulates gene expression to affect plant development. We also showed that CRY2 is phosphorylated in response to blue light and the phosphorylated CRY2 is most likely active and degraded in blue light. Given that protein translation (and probably chromophore attachment) takes place in the cytosol and that a photoreceptor would absorb photon instantaneously, it would be interesting to know where those inter-connected events occur in the cell. Our results showed that freshly synthesized CRY2 photoreceptor is inactive in the cytosol although it may be photon-excited, it is imported into the nucleus where the photoreceptor is phosphorylated, performs its function, becomes ubiquitinated, and eventually gets degraded (Fig. 1).¹ To our knowledge, this is the first example in any organism that a photoreceptor is shown to complete its post-translational life cycle in a single subcellular compartment.Open in a separate windowFigure 1A model depicting the post-translational life cycle of CRY2. Pi, phosphate group; Ubq, ubiquitin.Key words: blue light, cryptochrome, ubiquitination, phosphorylation, Arabidopsis     

Conserved residues in the ankyrin domain of VAPYRIN indicate potential protein-protein interaction surfaces

Plant VAPYRINs are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINs of dicots, monocots and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.Key words: VAPYRIN, arbuscular mycorrhiza, petunia, symbiosis, glomus, ankyrin, major sperm protein, VAPPlants engage in mutualistic interactions such as root nodule symbiosis (RNS) with rhizobia and arbuscular mycorrhiza (AM) with Glomeromycotan fungi. These associations are referred to as endosymbioses because they involve transcellular passage through the epidermis and intracellular accommodation of the microbial partner within root cortical cells of the host.^1,2 Infection by AM fungi and rhizobia is actively promoted by the plant and requires the establishment of infection structures namely the prepenetration apparatus (PPA) in AM and a preinfection thread in RNS, respectively.^3–5 In both symbioses the intracellular microbial accommodation in epidermal and root cortical cells involves rebuilding of the cytoskeleton and of the entire membrane system.^6–8 Recently, intracellular accommodation of rhizobia and AM fungi, and in particular morphogenesis of the AM fungal feeding structures, the arbuscules, was shown to depend on the novel VAPYRIN protein.^9–11VAPYRINs are plant-specific proteins consisting of two protein-protein interaction domains, an N-terminal major sperm protein (MSP) domain and a C-terminal ankyrin (ANK) domain. MSP of C. elegans forms a cytoskeletal network required for the motility of the ameboidal sperm.¹² MSP domains also occur in VAP proteins that are involved in membrane fusion processes in various eukaryotes.¹³ The ANK domain, on the other hand, closely resembles animal ankyrins which serve to connect integral membrane proteins to elements of the spectrin cytoskeleton,¹⁴ thereby facilitating the assembly of functional membrane microdomains in diverse animal cells.¹⁵ Ankyrin repeats exhibit features of nano-springs, opening the possibility that ankyrin domains may be involved in mechanosensing.¹⁶ Based on these structural similarities, VAPYRIN may promote intracellular accommodation of endosymbionts by interacting with membranes and/or with the cytoskeleton. Indeed, VAPYRIN protein associates with small subcellular compartments in petunia and in Medicago truncatula.^9,10Ankyrin repeats typically consist of 33 amino acids, of which 30–40% are highly conserved across most taxa. These residues confer to the repeats their basic helix-turn-helix structure.¹⁷ Ankyrin domains often consist of arrays of several repeats that form a solenoid with a characteristic crescent shape.¹⁷ Besides the ankyrin-specific motiv-associated amino acids there is little conservation between the ankyrin domains of different proteins, or between the individual repeats of a given ankyrin domain,¹⁷ a feature that was also observed in petunia VAPYRIN (Fig. 1A).⁹ However, sequence comparison of VAPYRINs from eight dicots, three monocots and the moss Physcomitrella patens revealed a high degree of sequence conservation beyond the ankyrin-specific residues (Fig. 1B and Sup. Fig. S1). When the degree of conservation was determined for the individual ankyrin repeats among all the 12 species, it appeared that repeats 7, 9 and 10 exhibited particularly high conservation (Fig. 1C).Open in a separate windowFigure 1Sequence analysis and phylogeny of VAPYRIN from diverse plants. (A) Predicted amino acid sequence of the petunia VAPYRIN protein PAM1. The 11 repeats of the ankyrin domain are aligned, and the ankyrin consensus sequence is shown below the eleventh ankyrin repeat (line c). Conserved residues that are characteristic for ankyrin repeats (Mosavi et al. 2004)¹⁷ are depicted in bold face. (B) Unrooted phylogenetic tree representing the VAPYRINs of eight dicot species (Petunia hybrida, Solanum lycopersicon, Solanum tuberosum, Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula and Glycine max) three monocot species (Sorghum bicolor, Zea mays and Oryza sativa), and the moss Physcomitrella patens. (C) Degree of conservation of the individual ankyrin repeats of VAPYRIN. Schematic representation of the MSP domain as N-terminal barrel-shaped structure, and of the individual ankyrin repeats as pairs of alpha-helices. An additional loop occurring only in monocots (grass-loop) is inserted above repeat 4, and the deletion between repeat 7 and 8 is indicated (gap). This latter feature is common to all VAPYRIN proteins. The percentage of amino acid residues that are identical in at least 11 of the 12 VAPYRINS is given below the MSP domain and the eleven ankyrin repeats. The box highlights repeats 7–10 which contribute to the predicted binding site (compare with Figs. 3 and ​and44).Sequence comparison of the eleven repeats of all the twelve plant species revealed that the individual repeats clustered according to their position in the domain, rather than according to their origin (plant species) (Fig. 2). This shows that the repeats each are well conserved across species, but show little similarity among each other within a given VAPYRIN protein. The higher conservation of repeats 9 and 10 was reflected by the compact appearance of the respective branches, in which the monocot and moss sequences were nested closely with the dicot sequences, compared to other repeats, where the branches appeared fragmented between monocots and dicots, and where the P. patens sequence fell out of the branch as in the case of repeats 4–6 (Fig. 2). Taken together, this points to an old evolutionary origin of the entire ankyrin domain in lower land plants, with no subsequent rearrangement of ankyrin repeats.Open in a separate windowFigure 2Phylogenetic analysis of the individual ankyrin repeats of VAPYRIN. Phylogenetic representation of an alignment of all the 11 repeats of the 12 VAPYRINs compared in Figure 1B and C. The repeats cluster according to their position within the domain, rather than to their origin (plant species). Numbers indicate the position of the repeats within the domain (compare with Fig. 1C). P. patens repeats are highlighted (small circles) for clarity. The monocot repeat 4 sequences (boxed) are remote from the remaining repeat 4 sequences because of the grass loop (compare with Fig. 1C).Ankyrin domains function as protein-protein interaction domains,¹⁷ in which the residues on the surface are involved in the binding of their protein partners.¹⁴ The fact that repeats 9 and 10 exhibited particularly high levels of conservation across species from moss to angiosperms indicated that this region may contain functionally important residues. Within repeat 10, sixteen amino acid positions were identical in >90% of the analyzed species (Fig. 3A and grey bars). Nine of those represent residues that are characteristic for ankyrin repeats (red letters) and determine their typical 3D shape.¹⁷ These residues are considered ankyrin-specific, and are unlikely to be involved in a VAPYRIN-specific function. The remaining seven highly conserved residues in repeat 10, however, are VAPYRIN-specific, since they have been under positive selection, without being essential for the basic structure of the ankyrin repeat. Ankyrin-specific and VAPYRIN-specific residues where identified throughout the entire ankyrin domain (Sup. Fig. 1), and subsequently mapped on a 3-dimensional model of petunia VAPYRIN to reveal their position in the protein (Fig. 3B–G). The ankyrin-specific residues were found to be localized primarily to the interior of the ankyrin domain, with the characteristic glycines (brown) marking the turns between helices and loops (Fig. 3B, D and F, compare with A). In contrast, the VAPYRIN-specific residues were localized primarily on the surface of the ankyrin domain (Fig. 3C, E and G). A prominent clustering of VAPYRIN-specific residues was identified on the concave side of the crescent-shaped ankyrin domain comprising repeats 7–10 close to the gap (Figs. 3G and ​and44). This highly conserved VAPYRIN-specific region contains several negatively and positively charged residues (D, E and K, R, respectively) and aromatic residues (W, Y, F), which may together form a conserved binding site for an interacting protein.Open in a separate windowFigure 33D-Mapping of conserved positions within the ankyrin domain of VAPYRIN. (A) Conserved amino acid residues were evaluated for ankyrin repeat 10 of petunia VAPYRIN as an example. The degree of conservation between the 12 VAPYRINs analyzed in Figures 1B and ​and22 is depicted with grey bars. Average conservation between all the 132 ankyrin repeats of the 12 VAPYRIN sequences is shown with black bars. Residues that are conserved in all 132 repeats (red letters) define the ankyrin consensus sequence, which confers to the repeats their characteristic basic structure.¹⁷ Residues that are >90% conserved but are not part of the basic ankyrin sequence (highlighted with asterisks) are VAPYRIN-specific and may therefore have been conserved because of their specific function in VAPYRIN. Arrows indicate the characteristic antiparallel helices, the turns are marked by conserved glycine residues (underlined; compare with B, D and F). (B–G) 3D-models of the petunia VAPYRIN PAM1. Conserved amino acid residues were color-coded according to their physico-chemical properties (http://life.nthu.edu.tw/∼fmhsu/rasframe/SHAPELY.HTM) with minor modification (see below). In (B, D and F) the ankyrin-specific residues are highlighted (corresponding to the bold letters in Fig. 1A). In (C, E and G), the VAPYRIN-specific residues are highlighted. Note the patch of high conservation on the concave side of the crescent-shaped ankyrin domain between repeats 7–10 next to the gap. (B–E) represent respective side views of the ankyrin domain, (F and G) exhibit the concave inner side of the domain. Color code: Bright red: aspartic acid (D), glutamic acid (E); Yellow: cysteine (C); Blue: lysine (K), arginine (R); Orange: serine (S), threonine (T); Dark blue: phenylalanine (F), tyrosin (Y); Brown: glycine (G); Green: leucin (L), valine (V), isoleucin (I), alanine (A); Lilac: tryptophane (W); Purple: histidine (H); Pink: proline (P).Open in a separate windowFigure 4The highly conserved surface area in domain 8–10 of the ankyrin domain of petunia VAPYRIN. Close-up of the highly conserved region of petunia PAM1 as shown in Figure 3G. Amino acids were color-coded as in Figure 3 and their position in the amino acid sequence is indicated (compare with Sup. Fig. 1).In this context, it is interesting to note that human ankyrin R also contains a binding surface on the concave side of the D34 domain for the interaction with the CBD3 protein.¹⁴ Consistent with an essential function of the C-terminal third of the ankyrin domain, mutations that abolish this relatively short portion of VAPYRIN, have a strong phenotype, indicating that they may represent null alleles.⁹ Based on this collective evidence, we hypothesize that repeats 7–10 are involved in the formation of a protein complex that is essential for intracellular accommodation of rhizobia and AM fungi. Biochemical and genetic studies are now required to identify the binding partners of VAPYRINs, and to elucidate their role in plant endosymbioses.     

Identification of a nicotine transporter in leaf vacuoles of Nicotiana tabacum

Alkaloids are, in some plant species, transported from the source organ after their biosynthesis and moved to sink organs via long distance transport. One representative is nicotine, which is biosynthesized in roots, then translocated to the leaves, and finally accumulated in the leaf vacuoles in Nicotiana species. Although the nicotine translocation was identified more than 10 years ago, no transport protein has been characterized concerning the inter-organ movement of this alkaloid.We characterized a novel multidrug and toxic compound extrusion (MATE)-type transporter, Nt-JAT1 (Nicotiana tabacum jasmonate-inducible alkaloid transporter 1). Nt-JAT1 was co-regulated with biosynthetic genes following methyl jasmonate treatment. This MATE gene was expressed in the leaves, stems, and roots in tobacco plants. Biochemical analyses suggested that Nt-JAT1 transported nicotine. The location of Nt-JAT1 was shown to be the tonoplast. These data suggested that Nt-JAT1 plays an important role in the nicotine translocation by acting as a transporter responsible for the unloading of nicotine in the aerial parts of the plant and its deposition in the vacuoles (Fig. 1). To our knowledge, this is the first identification of a vacuolar transporter for alkaloids in plant. A possible application of this transporter for the production of valuable alkaloids is also discussed.Open in a separate windowFigure 1A model of nicotine accumulation in leaf cells. Nt-JAT1 transports nicotine to the vacuole via the H⁺ gradient at tonoplast.Key words: Nicotiana tabacum, nicotine, alkaloid, MATE, transporter, leaf vacuole     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Negative effects of desiccation on the protein sorting and post-translational modification

Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress.¹ In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.Key words: AP-1 protein, cell ultrastructure, desiccation, golgi bodies, physcomitrella, proteomeThe plant Golgi apparatus is composed of many small stacks of cisternae, sometimes known as dictyosomes. The Golgi is a complex polarized organelle consisting of both a cis and trans side, containing compartments with functionally different capacities for directing cellular components. The plant Golgi apparatus synthesis a wide range of cell wall polysaccharides and proteoglycans, and also carries out O-linked glycosylation and N-linked glycan processing.^2–5 Moreover, the Golgi is involved in returning escaped proteins back to the endoplasmic reticulum, sorting of proteins and polysaccharides to the cell wall or vacuoles, and in organizing the compartmentation of its own enzymes by retention or retrieval mechanisms.⁶ In conclusion, The Golgi apparatus is central to the growth and division of the plant cell through its roles in protein glycosylation, protein sorting and cell wall synthesis.The transit of proteins and lipids from the trans-Golgi network (TGN) and the plasma membrane to endosomes within eucaryotic cells occurs via the budding and fusion of clathrin-coated vesicles (CCVs).^7,8 At the TGN, this process is mediated by the heterotetrameric AP-1 adaptor complex, which consists of two large subunits, β and γ1; a medium subunit, µ1; and a small σ1 subunit. Recruitment of AP-1 to the TGN membrane is regulated by a small GTPase, ADP-ribosylation factor 1 (ARF1), which cycles between an inactive GDP-bound form in cytosol and an active GTP-bound form that associates with the membrane like other small GTPase.⁹ There is also evidence that phosphorylation/dephosphorylation events are involved in the regulation of the function of AP-1. Ghosh and Kornfeld demostrated that AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated µ1. Once on the membrane, the µ1 subunit undergoes phosphorylation, which results in a conformation change. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of µ1 (and µ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.¹⁰Plants experience desiccation stress either as part of a developmental programme, such as during seed maturation, or because of reductions in air humid and water availability in the soil. Underlying the ability of bryophytes to withstand periods of desiccation are morphological and biochemical adaptations. Plants respond to stress as individual cells and synergistically as a whole organism. Scanning electron microscopy observation showed that the P. patens gametophore cells were shrunk upon the treatment of desiccation, and the shrinking started from the edge of the leaves (Fig. 1). We could clearly observe some dark granula in the untreated cells, but these granula disappeared post-desiccation treatment (Fig. 1). Transmission electron microscopy also revealed that the large stacks of Golgi bodies and numerous coated vesicles are typically visible in the hydrated cells (Fig. 2), but these are absent in the desiccative cells (data not shown). The plant Golgi apparatus plays an important role in protein glycosylation and sorting. Therefore, this event means that the protein sorting and the cargo transporting are disrupted by desiccation stress. During desiccation, the absentness of Golgi bodies reduce the leaf activities of cell, and this is expected to similar to plant dormancy which is a phenomenon in resurrection plants and some drought-tolerant plants. In addition, through two-dimensional gel electrophoresis (2-DE) and LC-MS/MS analysis, AP-1 large subunit was identified as downregulated protein during the course of dehydration (Fig. 3). AP-1 is ubiquitously expressed and participates in the budding of clathrin-coated vesicles from the trans-Golgi network (TGN) and endosomes. AP-1 also recognizes sorting motifs in cargo molecules. Our results suggested that desiccation led to a marked disrupt in protein posttranslational modification, protein sorting and cell walls synthesis.Open in a separate windowFigure 1Scanning Electron microscopy images of normal and dehydrated P. patens gametophores. (A) the fresh leaf; (B) enlargement of the rectangle area of (A); (C) dehydrated gametophores of P. patens. Bar = 5 µm.Open in a separate windowFigure 2Transmission electron microscopy images of cell in fresh game-tophores. The arrows indicate Golgi body, Bar = 2 µm.Open in a separate windowFigure 3Part protein profile of the control and desiccation plants. The arrows indicate the AP-1 large subunit.     

Manufacturing Therapeutic Exosomes: from Bench to Industry

​Exosome,Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinical-grade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.Open in a separate window     

Dynamic protein trafficking to the cell wall

Recently we have studied the secretion pattern of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2) in tobacco protoplast using the protein fusions, secGFP-PMEI1 and PGIP2-GFP. Both chimeras reach the cell wall by passing through the endomembrane system but using distinct mechanisms and through a pathway distinguishable from the default sorting of a secreted GFP. After reaching the apoplast, sec-GFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP undergoes endocytic trafficking. Here we describe the final localization of PGIP2-GFP in the vacuole, evidenced by co-localization with the marker Aleu-RFP, and show a graphic elaboration of its sorting pattern. A working model taking into consideration the presence of a regulated apoplast-targeted secretion pathway is proposed.Key words: cell wall trafficking, endocytosis, GPI-anchor, PGIP2, PMEI1, secretion pathway, vacuole fluorescent markerCell wall biogenesis, growth, differentiation and remodeling, as well as wall-related signaling and defense responses depend on the functionality of the secretory pathway. Matrix polysaccharides, synthesized in the Golgi stacks, and cell wall proteins, synthesized in the ER, are packaged into secretory vesicles that fuse with the plasma membrane (PM) releasing their cargo into the cell wall. Also the synthesis and deposition of cellulose itself are driven by the endomembrane system which controls the assembly, within the Golgi, and the export to the plasma membrane of rosette complexes of cellulose synthase.¹ Secretion to the cell wall has always been considered a default pathway² but recent studies have evidenced a complex regulation of wall component trafficking that does not seem to follow the default secretion model. Recent evidence that several cell wall proteins are retained in the Golgi stacks until specific signals at the N-terminal domain are proteolitically removed is a case in point.^3–5 Moreover, it has previously been reported that secretion of exogenous marker proteins (secGFP and secRGUS) and cell wall polysaccharides reach the PM through different pathways.⁶ More recently, we have reported that cell wall protein trafficking also occurs through mechanisms distinguishable from that of a secreted GFP suggesting that more complex events than the mechanisms of bulk flow control cell wall growth and differentiation.⁷ To follow cell wall protein trafficking we used a Phaseolus vulgaris polygalacturonase inhibitor protein (PGIP2) and an Arabidopsis pectin methylesterase inhibitor protein (PMEI1) fused to GFP (PGIP2-GFP and secGFP-PMEI1). Both apoplastic proteins are involved in the remodeling of pectin network with different mechanisms. PGIP2 specifically inhibits exogenous fungal polygalacturonases (PGs) and is involved in the plant defense mechanisms against pathogenic fungi.^8,9 PMEI1 counteracts endogenous PME and takes part in the physiological synthesis and remodeling of the cell wall during growth and differentiation.^10,11 The specific functions of the two apoplastic proteins seem to be strictly related to the distinct mechanisms that control their secretion and stability in the cell wall. In fact, while secGFP-PMEI1 moves through ER and Golgi stacks linked to a glycosyl phosphatidylinositol (GPI)-anchor, PGIP2-GFP moves as a cargo soluble protein. Furthermore, secGFP-PMEI1 is stably accumulated in the cell wall, while PGIP2-GFP, over the time, is internalized into endosomes and targeted to vacuole, likely for degradation. After reaching the cell wall, the different fate of the two proteins seems to be strictly related to the presence/absence of their physiological counteractors. PMEI regulates the demethylesterification of homogalacturonan by inhibiting pectin methyl esterase (PME) activity through the formation of a reversible 1:1 complex which is stable in the acidic cell wall environment.¹² Stable wall localization of PMEI1 is likely related to its interaction with endogenous PME, always present in the wall. Unlike PMEs, fungal polygalacturonases (PGs), the physiological interactors of PGIP2, are present in the cell wall only during a pathogen attack. The absence of PGs may determine PGIP2 internalization. Internalization events have been already reported for PM proteins,^13–16 while cell wall protein internalization is surely a less well-known event. To date, only internalization of an Arabidopsis pollen-specific PME^4,5,17 and PGIP2 ⁷ has been reported.To further confirm the internalization of PGIP2-GFP and its final localization into the vacuole, we constructed a red fluorescent variant (RFP) of the green fluorescent marker protein that accumulates in lytic or acidic vacuole because of the barley aleurain sorting determinants (Aleu-RFP).¹⁸ The localization of PGIP2-GFP was compared to that of Aleu-RFP by confocal microscopy in tobacco protoplasts transiently expressing both fusions. Sixty hours after transformation, PGIP2-GFP labeled the central vacuole as indicated by complete co-localization with the vacuolar marker (Fig. 1A–D). Instead, at the same time point, secGFP-PMEI1 still labeled the cell wall (Fig. 1E–H) and never reached the vacuolar compartment. To summarize PGIP2-GFP secretion pattern, a graphic elaboration of confocal images is reported describing the sorting of PGIP2GFP in tobacco protoplast (Fig. 1I). The protein transits through the endomembrane system (green) and reaches the cell wall which is rapidly regenerating as evidenced by immunostaining with the red monoclonal antibody JIM7 that binds to methylesterified pectins.¹⁹ PGIP2-GFP is then internalized in endosomes, labeled in yellow because of the co-localization with the styryl dye FM4-64, a red marker of the endocytic pathway.Open in a separate windowFigure 1PGIP2-GFP, but not secGFP-PMEI1, is internalized and reaches the vacuole in tobacco leaf protoplasts. (A) Approximately 60 h after transformation, PGIP2-GFP labeled the central vacuole as indicated by co-localization with the vacuole marker Aleu-RFP (B). (C) Merged image of (A and B). (D) Differential interference contrast (DIC) image of (A–C). On the contrary, secGFP-PMEI1 still labeled cell wall (E). (F) No co-localization is present in the vacuole labeled by Aleu-RFP. (G) Merged image of (E and F). (H) DIC image of (E–G). (I) Graphic elaboration of confocal images describing the sorting of PGIP2. The protein is sorted by the endomembrane system (green) to the cell wall (red) that is regenerated by the protoplast. Lacking the specific ligand, it is then internalized in endosome (yellow). Details are reported in the text.In Figure 2 we propose a model of the mechanism of secGFP-PMEI1 and PGIP2-GFP secretion derived from the different lines of evidence previously reported in reference ⁷. SecGFPPMEI1 (Fig. 2-1), but not PGIP2-GFP (Fig. 2-2), carries a GPI-anchor, required for its secretion to the cell wall. When the anchorage of GPI is inhibited by mannosamine (Fig. 2-a) or by the fusion of GFP to the C-terminus of PMEI1 (Fig. 2-b), the two non-anchored proteins accumulate in the Golgi stacks. Evidence of retention in Golgi stacks has already been reported for other two cell wall proteins.^3–5 Unlike secGFP-PMEI1, PGIP2-GFP is not stably accumulated in the cell wall and undergoes endocytic trafficking (Fig. 2-3). PGIP2-GFP internalization, likely due to the absence of PGs, might also be related with its ability to interact with homogalacturonan and oligogalacturonides,²⁰ which have been reported to internalize^21,22 (Fig. 2-4). Since SYP 121, a Qa-SNARE, is involved in the default secretion of secGFP,²³ but not in secretion of PGIP2-GFP and secGFP-PMEI1, trafficking mechanisms underlying secretion into the apoplast are likely different from those underlying the default route (Figs. 2-5). Taken as a whole, evidence suggests the existence of currently undefined signals that control apoplast-targeted secretion.Open in a separate windowFigure 2Schematic illustration for secGFP-PMEI1 and PGIP2-GFP trafficking. See text for details.     

Gain weight by “going diet?” Artificial sweeteners and the neurobiology of sugar cravings: Neuroscience 2010

America’s obesity epidemic has gathered much media attention recently. A rise in the percent of the population who are obese coincides with an increase in the widespread use of non-caloric artificial sweeteners, such as aspartame (e.g., Diet Coke) and sucralose (e.g., Pepsi One), in food products (Figure 1). Both forward and reverse causalities have been proposed [1,2]. While people often choose “diet” or “light” products to lose weight, research studies suggest that artificial sweeteners may contribute to weight gain. In this mini-review, inspired by a discussion with Dr. Dana Small at Yale’s Neuroscience 2010 conference in April, I first examine the development of artificial sweeteners in a historic context. I then summarize the epidemiological and experimental evidence concerning their effects on weight. Finally, I attempt to explain those effects in light of the neurobiology of food reward.Open in a separate windowFigure 1Time line of artificial sweetener use and obesity trends in the United States. Blue line: changes in the percentage of the population who are obese (BMI >30) from 1961 to 2006. Source: National Health and Nutrition Examination Survey [57]. Orange line: changes in the percentage of the population who are regular artificial sweetener users from 1965 to 2004. Source: National Household Survey [2]. Purple line: changes in the number of new artificial sweetener containing food products introduced to the American market from 1999 to 2004. Source: Mintel Market Analysis [14]. Bars below the time axis indicates the type and availability of artificial sweeteners in the United States over time. Source: Kroger et al [9].     

Erratum to: Singh R.K. and Gunjan A. Histone tyrosine phosphorylation comes of age. Epigenetics 2011; 6:153-60.

We recently noticed that there is a major error in Figure 1 of our review published in Epignetics 2010, Volume 6, Issue 2. During the preparation of the figure, the human and yeast H2B tyrosines were numbered the same, making the human numbering incorrect. The correct Figure 1 with proper numbering of human tyrosines is below.Erratum to:Singh R.K. and Gunjan A. Histone tyrosine phosphorylation comes of age.Epigenetics 2011; 6:153-60.We recently noticed that there is a major error in Figure 1 of our review published in Epignetics 2010, Volume 6, Issue 2. During the preparation of the figure, the human and yeast H2B tyrosines were numbered the same, making the human numbering incorrect. The correct Figure 1 with proper numbering of human tyrosines is below.Open in a separate windowFigure 1. Tyrosine residues are highly conserved between budding yeast and mammalian core histones. The four canonical core histone proteins from the budding yeast Saccharomyces cerevisiae are indicated by the prefix “Sc” and denoted in blue. The mammalian core histones and the mammalian variant histone H2A.X are shown in black. The number of amino acid (aa) residues in each core histone is indicated on the right. The location of the a-helices in the secondary structure of the histone proteins is indicated by cylinders. Tyrosine residues are shown as balloons and the tyrosine residues essential for viability in budding yeast histones are indicated by red balloons. Tyrosines in mammalian histones have not yet been evaluated to determine the residues essential for viability. Note the high degree of conservation of the location as well as the spacing of all but one tyrosine residue between budding yeast and mammalian core histones (H3 Y54 being the exception). Tyrosine residues that have recently been shown to be phosphorylated in vivo are marked by yellow “explosion” signs and the letter “P.” Additional tyrosine residues that are predicted to be reasonably accessible in the nucleosomal context under certain conditions and can be potentially phosphorylated in vivo are indicated by a yellow halo only on the mammalian histones for clarity, but are likely to be just as applicable to the yeast histones. Solid yellow halo indicates higher probability of phosphorylation, while a dashed yellow halo indicates lower probability of phosphorylation.