Identification of the LH and TSH-secreting cells in the pituitary gland of the rhesus monkey

Summary Pituitary glands from juvenile (pre-pubertal) and adult male and female rhesus monkeys were examined following immunocytochemical staining with antisera to the beta subunits of ovine luteinizing hormone (LH) and of human thyroid stimulating hormone (TSH). The LH antiserum reacts with a cell that is PAS-positive, occurs singly and is randomly distributed throughout the pars distalis. The diameter of these cells is approximately 11.5 m. They do not seem to vary in number in either juveniles (pre-pubertals) or adults, or in males or females. There appears to be fewer LH cells in the pituitary glands of pregnant and lactating females. In addition to staining cells in the pars distalis, the antiserum also reacts with a population of cells located in the pars tuberalis.The cells that stain with the anti-TSH serum are confined primarily to the pars distalis. They are approximately 15.8 m in diameter and are generally found in groups or clusters located in the anterior and medial regions of the gland. The TSH cells vary in number from one animal to another; however, this variability is unrelated to the age or the sex of the animals. No demonstrable changes occur in the number of TSH cells during pregnancy or lactation.Supported by NIH General Research Support Grant RR05654The author wishes to express appreciation to the Hormone Distribution Program of NIAMDD for the preparations of ovine FSH, TSH and human TSH, and to Drs. H. Papkoff for the ovine LH and LH, L. Reichert for the human FSH, J. Vaitukaitis for the anti-human TSH, and L.A. Sternberger for the PAP complex     

Effects of the removal of olfactory bulbs on the appearance and then on the latency of the "mouse-killing reaction" in the rat of the Wistar SM strain

The 20% of male 5-6 month-old Wistar rats SM prove to be natural killers. A four-month long insulation of "surely non-killer" rats has not allowed to notice a mouse-killing behaviour in any animal. The total bilateral removal of olfactory bulbs in "non-killer rats" causes a "mouse-killing behaviour" in all the animal 24 hours after the operation, but with a latency of response that has proved different in the various rats. Precisely, while 24 hours and 15 days after the bulb operation the rats can be distinguished into "rapid killers" and "slow killers", after 30 days all the animals become "rapid killers" with a remarkably shortened period of mouse-killing latency (1 - 60 seconds).     

The ultrastructure of the corpus luteum of the guinea-pig

Summary An electron-microscopic investigation, based on the suggestion that differences seen in progesterone levels under differing hormonal conditions might be reflected in the ultrastructural organisation of the lutein cells of the guinea-pig was undertaken. Comparisons were made between corpora lutea taken from animals during the normal oestrous cycle, pregnancy and lactation, and after hysterectomy or hypophysectomy.The lutein cells from the oestrous cycle corpus luteum appeared to be of two types, light and dark. The former were more numerous. The main difference between them lay in the arrangement of the endoplasmic reticulum. Lutein cells from corpora lutea (with the exception of the old degenerating corpora lutea) all contained well-developed agranular endoplasmic reticulum, little granular endoplasmic reticulum, several electron-dense lipid granules, lysosomal bodies which ranged from small spherical bodies to large autophagic vesicles and mitochondria. The mitochondria were numerous, and in the corpus luteum of pregnancy, they were closely associated with the parallel arrays of granular endoplasmic reticulum.With minor exceptions, the lutein cells of the guinea-pig present a strikingly uniform picture despite their hormonal condition.The manner in which this uniformity of ultrastructure may be related to observed differences in progesterone levels in the corpus luteum of the guinea-pig is discussed.Meat and Livestock Commission (MLC) Scholar.The authors wish to thank Dr. J. S. Perry for doing the surgery involved in this work and for the specimens of corpora lutea of hysterectomy. They are also grateful to him for his helpful discussions and interest throughout.     

Histochemical method for the demonstration of the activity of phosphoglucomutase

Summary A method for the demonstration of the activity of phosphoglucomutase in tissues is described. In the histochemical system the enzyme converts the substrate -D-glucose-1-phosphate to -D-glucose-6-phosphate. The resulting -D-glucose-6-phosphate is oxidized by exogenous glucose-6-phosphate dehydrogenase to D-glucono--lactone-6-phosphate, whereby the activity of endogenous NADPH-tetrazolium oxidoreductase reduces Nitro-BT to the slightly soluble diformazan. The problems involved in the histochemical demonstration of phosphoglucomutase are discussed.     

Separation of the pineal vesicle from the wall of the third ventricle during the post-hatching development of the chicken

Summary According to light- and electron-microscopic observations the pineal organ of the 3-day-old chicken consists of a prominent end vesicle and a tapering parenchymal stalk. During this stage the pineal lumen is in open communication with the third ventricle. However, in the 40-day-old chicken, which still possesses a well-developed end vesicle, the proximal portion of the pineal stalk displays regressive changes leading to local fragmentation. At this stage the pineal stalk is reduced, and the pineal lumen is missing. In 1-year-old chickens the parenchyma of the proximal portion of the stalk is further diminished, and in 3-year-old domestic fowl is completely displaced by bundles of collagenous fibers, only some nerve fibers being present. This post-hatching pineal development may reflect the sequence of changes leading from pineal sense organs to pineal glands.This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Functional Consequences of the Subdomain Organization of the Sulfs

Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine 6-O-sulfate modifications within heparan sulfate proteoglycans and regulate their interactions with various signaling molecules, including Wnt ligands. Although the Sulfs are multidomain proteins, there is limited information available about how the subdomains contribute to their enzymatic and signaling activities. In this study, we found that both human Sulfs were synthesized as prepro-enzymes and cleaved by a furin-type proteinase to form disulfide-bond linked heterodimers of 75- and 50-kDa subunits. The mature Sulfs were secreted into conditioned medium, as well as retained on the cell membrane. Although the catalytic center resides in the N-terminal 75-kDa subunit, the C-terminal 50-kDa subunit was indispensable for both arylsufatase and glucosamine 6-O-sulfate-endosulfatase activity. We found that the hydrophilic regions of the Sulfs were essential for endosulfatase activity but not for arylsulfatase activity. Using Edman sequencing, we identified furin-type proteinase cleavage sites in Sulf-1 and Sulf-2. Deletion of these sequences resulted in uncleavable forms of Sulfs. The uncleavable Sulfs retained enzymatic activity. However, they were unable to potentiate Wnt signaling, which may be due to their defective localization into lipid rafts on the plasma membrane.Heparan sulfate proteoglycans (HSPGs)² are major components of the extracellular matrix/cell surface and regulate a variety of biological phenomena, including cell proliferation, cell migration, and differentiation (1). These effects are mediated through the ability of HSPGs to bind to a diverse repertoire of protein ligands. Among these are morphogens, growth factors, chemokines, and other classes of molecules (2, 3).HSPGs consist of multiple heparan sulfate (HS) chains covalently linked to a limited set of core proteins. The HS chains contain repeating uronic acid and glucosamine disaccharide units. The binding functions of HSPGs depend on the fine structure of the attached heparan sulfate chains where sulfation modifications occur in four positions (N-, 3-O, and 6-O of glucosamine and 2-O of uronic acid) in highly variegated, yet highly regulated patterns (3, 4). 6-O-Sulfation of glucosamine is established to be critical for certain HSPG functions in organisms from Drosophila through mammals (5, 6).Several years ago, we cloned cDNAs encoding two novel extracellular sulfatases (Sulf-1 and Sulf-2) in human and mouse (7), initiated by the identification of the Sulf-1 ortholog in the quail embryo (QSulf-1) (8). We and others showed that both Sulfs are neutral pH endosulfatases, which remove glucosamine-6-O-sulfate from internal glucosamine residues of highly sulfated subregions within heparin/HSPGs (7, 9, 10). The ability of these enzymes to modulate the heparin/HSPG interactions of a number of growth factors, morphogens, and chemokines has been confirmed in direct binding assays (9, 11–13). In some cellular contexts, the Sulfs act to promote signaling pathways (Wnts, bone morphogenetic protein, and glial cell-derived neurotrophic factor) (9–11, 14), whereas in others the Sulfs are inhibitory (fibroblast growth factor-2 and transforming growth factor-β) (15–17). The importance of the Sulfs in development has been revealed by gene knockdown (8) and knock-out studies (11, 18–20). The phenotypes in single and double null mice include abnormalities in general growth, muscle innervation, muscle regeneration, skeletal tissue, and lung development. The Sulfs have been extensively investigated in cancer with some studies consistent with tumor suppression activity (15, 21, 22) and others with a pro-oncogenic role (23–25).As is the case for the prototypic QSulf-1 (8), HSulf-1 and HSulf-2 consist of four domains from the N to C terminus: a signal peptide, a catalytic domain of 374 amino acids, a basic hydrophilic domain of 346/366 amino acids, and a C-terminal domain of 109/127 amino acids (7, 8). In the 17-member sulfatase family (26), the Sulfs share the most extensive sequence homology with lysosomal glucosamine-6-sulfatase in the catalytic and C-terminal domains, although the centrally inserted hydrophilic domain is absent from this enzyme and other sulfatases. Limited information has been available about the proteolytic processing of the Sulfs during synthesis. In the present study, we show that the mature form of each human Sulf consists of a heterodimer of 75- and 50-kDa subunits, which is formed through the action of a furin-type proteinase on a proprotein of 125 kDa. We investigate the structural requirements for the enzymatic and signaling activities of these proteins.     

Influence of the antimitotic naphthoquinone on the cleavage of the eggs of the mollusc,Mytilus edulis L.

Summary 1. A brief account is given of the cleavage ofMytilus edulis L.2.Naphthoquinone in concentrations above 1 million produces cytolysis of the newly fertilised eggs. It causes some blockage of cleavage at the single cell stage in concentrations down to 140 M. The main area of its antimitotic activity is in the range between 1 M and 16 M. Resistance to the substance increases as cleavage proceeds. 3. Some abnormalities of cleavage are found at all antimitotic concentrations. The types of abnormality are described. They do not appear to fit easily into the hypothesis of Huber (1947) that the effect of this substance is to enhance the contraction and inhibit the expansion of the cell cortex.     

The fiber composition of the semitendinosus muscle of the rabbit

Summary The semitendinosus muscle of the rabbit is composed of a homogeneous fiber population. Fiber typing was carried out by means of light and electron microscopy according to which this muscle exhibits structural features that are either characteristic for both red and white fibers, or that do not allow for any classification according to the A-, B-, C-fiber system. Hence the long-held assumption that the semitendinosus muscle of the rabbit represents a classical paradigm of a red muscle should be revised in light of the present results.     

An investigation of the role of solutes in the xylem sap and in the xylem parenchyma as the source of root pressure

Summary Solute osmotic potentials (_x) in the vessels of hydroponically grown maize roots were measured to assess the osmotic-xylem-sap mechanism for generating root pressure (indicated by guttation). Solutes in vessels were measured in situ by X-ray microanalysis of plants frozen intact while guttating. Osmotic potentials outside the roots (_o) were changed by adding polyethylene glycol to the nutrient solution. Guttation rate fell when _o was decreased, but recovered towards the control value during 3–5 days when _o was greater than or equal to –0.3 MPa, but not when _o was equal to –0.4 MPa. In roots stressed to _o = –0.3 MPa, _x, was always more positive than _o, and _x changed only slightly (ca. 0.05 MPa). Thus the adjustment in the roots which increased root pressure cannot be ascribed to _x, contradicting the osmotic-xylem-sap mechanism. An alternative driving force was sought in the osmotic potentials of the vacuoles of the living cells (_v), which were analysed by microanalysis and estimated by plasmolysis. _v showed larger responses to osmotic stress (0.1 MPa). Some plants were pretreated with abundant KNO₃ in the nutrient solution. These plants showed very large adjustments in _v (0.4 MPa) but little change in _x (0.08 MPa). They guttated by 4 h after _o was lowered to –0.4 MPa. It is argued that turgor pressure of the living cells is a likely alternative source of root pressure. Published evidence for high solute concentrations in the xylem sap is critically assessed.Abbreviations _o external water potential - _x osmotic potential of xylem sap - _v osmotic potential of vacuolar sap - EDX energy dispersive X-ray microanalysis - CSEM cryo-scanning electron microscope - LN₂ liquid nitrogen - PEG polyethylene glycol     

Speculations on the origin of the genetic code

The most primitive code is assumed to be a GC code: GG coding for glycine, CC coding for proline, GC coding for alanine, CG coding for arginine. The genetic code is assumed to have originated with the coupling of glycine to its anticodon CC mediated by a copper-montmorillonite. The polymerization of polyproline followed when it was coupled to its anticodon GG. In this case the aminoacyl-tRNA synthetase was a copper-montmorillonite. The first membrane is considered to be a sheet formed from polyglycine. As the code grew more complicated, the alternative hydrophobic-hydrophilic polypeptide (alanine-arginine) was coded for by the alternating CG copolymer. This alternating polypeptide (ala-arg) began to function as both a primitive membrane and as an aminoacyl-tRNA synthetase. The evolution of protein structure is tightly coupled to the evolution of the membrane. The a helix was evolved as lipids became part of the structure of biological membranes. The membrane finally became the fluid mosaic structure that is now universal.Based on a presentation made at a workshop-Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

An analysis of the meaning of the concepts of "commitment" and "determination" in the study of patterns of development

The use of the term "commitment" by different authors was compared and the term itself was compared with the term "determination". Different authors understand the term "commitment" in different ways. It is proposed to preserve the terms "competence", "determination" (labile and stable) and "differentiation" in studies of normal development at stages preceding the appearance of organ rudiments in order to facilitate the use of the knowledge acquired by experimental embryology and to decipher these concepts at the molecular level. The meaning of the term "commitment" should be made more precise when describing the experimental results and also when assessing the results obtained by various authors and published in numerous papers and reviews.     

Formation of the Interaction between the Wave Components of the Main EEG Rhythms in Children up to Five Years of Age

Using the analysis of the interaction between EEG components [1], the authors studied the regularities of the formation of the EEG wave structure in 36 children aged 4 to 7 months, 2 to 3 years, and 4 to 5 years. The EEG of 4- to 7-month-old children had a relatively organized temporal structure, whose components were connected mainly with those of the slow-wave range. This structure is reproduced in most of the leads and is more stable in the anterior cortical areas than in the posterior ones. The waking 2- to 5-year-old children had two functional nuclei in the relation structure of the EEG components characterized by statistically significant enhancement in the probability of the consecutive appearance of waves of certain ranges. These nuclei include a slow-wave nucleus in the range of the ₂–₁-frequencies and an alpha-nucleus in the ₁- and ₂-ranges (₂ and ₁ 2- to 3-year-old children) of the EEG frequencies. The relations between the components of the slow-wave nucleus begin to form in infants, prevail in 2- to 3-year-old children, and weaken by the age of 4 to 5 years. A trend is observed in 2- to 5-year-old children towards an increase in the average frequency of the waves that form the slow-wave nucleus. The temporal (strengthening interaction between the waves) and spatial organization of the interaction between the wave components of the alpha-nucleus is accelerated at the age of 4 to 5 years. The average frequency of the components forming this nucleus gradually increases with the children's age from 4 to 7 months to 4 to 5 years. Competitive relations exist between the two nuclei, characterized by a significant decrease in the probability of transitions between the components of different nuclei. The ₂-component may play a special role of the connecting link between the nuclei at the age of 4 to 5 years, the interwave transition from the - to -frequencies and from the latter to the -range being effected by a leaplike rather than smooth frequency increase. The character of local and spatial rearrangements of the EEG temporal structure detected in this study reflects a gradual morphofunctional maturation of the brain as a system that maintains the specific features of self-regulation mechanisms and coordination of the intersystem interactions at various stages of a child's development.     

Quantification of the Impact of Feature Selection on the Variance of Cross-Validation Error Estimation

Given the relatively small number of microarrays typically used in gene-expression-based classification, all of the data must be used to train a classifier and therefore the same training data is used for error estimation. The key issue regarding the quality of an error estimator in the context of small samples is its accuracy, and this is most directly analyzed via the deviation distribution of the estimator, this being the distribution of the difference between the estimated and true errors. Past studies indicate that given a prior set of features, cross-validation does not perform as well in this regard as some other training-data-based error estimators. The purpose of this study is to quantify the degree to which feature selection increases the variation of the deviation distribution in addition to the variation in the absence of feature selection. To this end, we propose the coefficient of relative increase in deviation dispersion (CRIDD), which gives the relative increase in the deviation-distribution variance using feature selection as opposed to using an optimal feature set without feature selection. The contribution of feature selection to the variance of the deviation distribution can be significant, contributing to over half of the variance in many of the cases studied. We consider linear-discriminant analysis, 3-nearest-neighbor, and linear support vector machines for classification; sequential forward selection, sequential forward floating selection, and the -test for feature selection; and -fold and leave-one-out cross-validation for error estimation. We apply these to three feature-label models and patient data from a breast cancer study. In sum, the cross-validation deviation distribution is significantly flatter when there is feature selection, compared with the case when cross-validation is performed on a given feature set. This is reflected by the observed positive values of the CRIDD, which is defined to quantify the contribution of feature selection towards the deviation variance.[1,2,3,4,5,6,7,8,9,10,11,12,13]     

Photoreduction of the Folate Cofactor in Members of the Photolyase Family

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH⁻* to the DNA lesion and electron back-transfer to semireduced FADH^o after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.DNA photolyases and cryptochromes (cry)² form a large family of related flavoproteins with DNA repair activity and photoreceptor function, respectively. Members of this protein family were identified in all kingdoms of life and can be grouped in at least nine subclades (1). DNA photolyases repair cytotoxic and mutagenic DNA lesions that are formed during exposure of DNA to UV-B. These DNA lesions are cyclobutane pyrimidine dimers (CPDs) or pyrimidine-pyrimidone (6-4) photoproducts. According to their substrate specificity, DNA photolyases are designated as CPD photolyases or (6-4) photolyases (2). The repair of both types of DNA lesions by photolyase requires the catalytic fully reduced and anionic flavin cofactor FADH⁻ that, when photoexcited, injects an electron directly into the DNA lesion (1) as shown in Fig. 1A (electron transfer pathway 1). During extraction from the cell and purification under aerobic conditions the flavin cofactor is usually oxidized to the semireduced and eventually to the fully oxidized form. Reduction of these flavin species to FADH⁻ in vitro can be achieved by illumination of the enzyme in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol. This process is named photoactivation (1). Photoactivation in vitro requires photoexcitation of the flavin and a triad of redox-active residues in the protein moiety that is highly conserved in DNA photolyases (3, 4) as shown in Fig. 1A (electron transfer pathway 2). These residues are generally tryptophans that allow transport of an electron from the protein surface to the U-shaped flavin cofactor, which is buried within the C-terminal α-helical domain (5–9). Whether the same mechanism is used by photolyase to photoreduce FAD in vivo is a matter of debate (10). Photoreduction of the flavin cofactor was also observed in cryptochrome blue/UV-A photoreceptors. However, instead of fully reduced flavin, semireduced flavin species (either anionic flavin semiquinone radical or neutral semiquinone radical) accumulate. This form of the photoreceptor is considered as the signaling state (11–14).Open in a separate windowFIGURE 1.Electron transfer pathways in cry3 and structures of folates. A, indicated are the distances of the tryptophans in the tryptophan triad (Trp-356, -409, -432) of Trp-432 to FADH⁻ and of FADH⁻ to the 5,10-methenylTHF (MTHF) cofactor in cry3. Shown are also the two established routes of electrons from FADH⁻ to the DNA lesion (Route 1) and within the tryptophan triad to FAD (Route 2). The third electron transfer pathway from FADH⁻ to 5,10-methenylTHF (Route 3) is the subject of this study. B, chemical structures of folates and their molecular masses. Folypolyglutamate molecules have a pteridin and a p-aminobenzoate moiety linked with a glutamate chain with a variable number of glutamic acids. The various THF species differ in their oxidation state of the C1 unit that is attached at the N-5 or N-10 position or form a bridge between both.A recently discovered subclade of the DNA photolyase/cryptochrome family are DASH cryptochromes, which have members in plants, bacteria, and aquatic animals (6, 15–17). Because DASH cryptochromes were found to lack repair activity for CPDs in double-stranded DNA, they were considered as cryptochrome-type photoreceptors (6, 16). However, it was recently shown that DASH cryptochromes repair CPDs in single-stranded DNA (18) and loop structures of double-stranded DNA (19) and, thus, belong to the CPD photolyase group. In contrast to conventional CPD photolyases, DASH cryptochromes are unable to flip the CPD lesion out of the DNA duplex (7).Besides the flavin cofactor that is essential for enzymatic activity, DNA photolyases and most likely all cryptochromes contain a second chromophore (1). Like the catalytic flavin, the second chromophore is non-covalently attached to the protein moiety. The majority of DNA photolyases and, as far as studied, the cryptochromes including the DASH-type like cry3 from Arabidopsis thaliana contain polyglutamated 5,10-methenyltetrahydrofolate (5,10-methenylTHF) as the second chromophore (1, 12, 17, 20, 21) (see Fig. 1B for folate structures). Several organisms like the cyanobacterium Anacystis nidulans (Synechococcus elongatus) produce deazariboflavins (7,8-didemethyl-8-hydroxy-5-deazariboflavin) and utilize them as second cofactor (22). In photolyases of thermophilic bacteria and Archaea of the genus Sulfolobus, FMN and FAD, respectively, were found as second cofactors (23, 24). The sole function of the second cofactors demonstrated at present is transfer of excitation energy to the catalytic flavin cofactor via a Förster-type mechanism. The crystal structures of DNA photolyases and DASH cryptochromes revealed that the second chromophores are located in a cleft between the N-terminal α/β domain and the C-terminal α domain (7–9). The centroid distances between the catalytic FAD and the second chomophore are in the range of 15–18 Å. The close distances and the angles between the transition dipole moments of the two cofactors are favorable for efficient energy transfer. Indeed, energy transfer efficiencies are about 70% for Escherichia coli photolyase (25), close to 100% for A. nidulans photolyase (26), and between 78% (dark-adapted) and 87% (light-adapted) for Arabidopsis cry3 (27). Although the second cofactors are not essential for catalysis (28, 29), they increase the efficiency of repair and possibly of photoactivation by having higher extinction coefficients than FADH⁻ in the near UV and blue region (30). The spectral overlap between 5,10-methenylTHF emission and the absorption of the different flavin redox states is on the order FADH^o > FAD_ox > FADH⁻ (31).Illumination in vitro of photolyase that contains fully oxidized or semireduced flavin results in light-induced absorbance changes. The decrease in absorption in the 450–470-nm region reflects a decrease in the amount of fully oxidized FAD concomitant with transient increase in absorption above 500 nm, which indicates the formation of a neutral semiquinone radical. Excitation of the 5,10-methenylTHF antenna chromophore at its absorption peak at 380 nm causes a likewise photoreduction of the catalytic FAD (1, 27, 28, 30, 31). However, irreversible bleaching of the 380-nm peak is observed under high irradiance UV-A or camera flash illumination (28, 30). This irreversible bleaching goes along with release of the folate cofactor from the protein moiety (30) and was named photodecomposition of 5,10-methenylTHF (28). However, the identity of the formed folate species remained unknown (30). In our previous spectroscopic characterization of Arabidopsis cry3, a similar bleaching of the 380-nm peak was observed (27).Here we show that a third electron transfer pathway exists in photolyase and DASH cryptochome, where the 5,10-methenylTHF cofactor is photoreduced to 5,10-methyleneTHF. Thus, bleaching at 380 nm does not simply reflect destruction but is a specific chemical conversion of the second chromophore.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Ultrastructural characteristics of the salivary glands of of the tick Ixodes persulcatus. II. Type III alveoli of the sucking female

Electron microscope observations were conducted to study changes in the cellular structure of IIId type alveoli of feeding females of I. persulcatus. Within the first 1-2 days of feeding granulosecreting glandular cells "d" and "e" get free of secretory inclusions. After that cells "d" degenerate completely and cells "e" undergo morphological transformations and start to fulfil osmoregulatory functions. From the third day of feeding cytoplasm of cells "e" and basal intersticial cells form a complex labyrinth from a system of branching processes limiting intercellular space from the basal to apical surfaces of alveolar wall. Cells "e" of I. persulcatus in their structure and functional activity closely resemble the earlier described cells "f" in alveoli III of the tick Rhipicephalus appendiculatus.     

A general equation for the evaluation of the error that affects the value of the maximum specific growth rate

A general equation is proposed to evaluate the absolute error that affects the maximum specific growth rate calculated from batch or continuous experiments. This error depends on the relative errors of the cell concentration measurements and on the duration of the test.Nomenclature X ₁ Cell concentration at the beginning of the exponential growth phase or of the washing-out period - X ₁ Lowest value of X ₁ due to experimental errors - X ₁ Highest value of X ₁ due to experimental errors - X ₂ Cell concentration at the end of the exponential growth phse or of the washing-out period - X ₂ Lowest value of X ₂ due to experimental errors - X ₂ Highest value of X ₂ due to experiment errors - Relative error that affects X ₁ - Relative error that affects X ₂ - t Exponential growth stage or washing-out duration - Asolute error that affects - Maximum specific growth rate - ₁ Lowest value of due to experimental errors - ₂ Highest value of due to experimental errors The author is with the Instituto Mauá de Technologia, Estrada das Lágrimas 2035, 09580-900, São Caetano do Sul, SP, Brazil