Characterization of Tetratricopeptide Repeat-Containing Proteins Critical for Cilia Formation and Function

Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC) proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs) into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.     

Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.     

Formation of Peptide Bonds from Metastable versus Crystalline Phase: Implications for the Origin of Life

Formation of peptide bonds was attempted bythermal activation of dry amino acids from aqueous solutionthat simulated prebiotic evaporative environments. Theevaporation trend of amino acids solutions shows abifurcation and can lead to either a crystalline phase(near equilibrium) or a metastable non-crystalline phase(far from equilibrium). Only amino acids in this metastablephase are able to form peptide bonds by thermal activationat temperatures that are generated by solar radiationtoday. We suggest that this metastable phase is the idealinitial material to trigger amino acid assemblage withprotein-like structure because provide the driving force(supersaturation) for an intense interaction betweenmonomers of different amino acids and allows activation ofthese monomers in plausible prebiotic conditions.     

Marginal Zinc Deficiency in Pregnant Rats Impairs Bone Matrix Formation and Bone Mineralization in Their Neonates

Zinc (Zn) deficiency during pregnancy may result in a variety of defects in the offspring. We evaluated the influence of marginal Zn deficiency during pregnancy on neonatal bone status. Nine-week-old male Sprague-Dawley rats were divided into two groups and fed AIN-93G-based experimental diets containing 35 mg Zn/kg (Zn adequately supplied, N) or 7 mg Zn/kg (low level of Zn, L) from 14-day preconception to 20 days of gestation, that is, 1 day before normal delivery. Neonates were delivered by cesarean section. Litter size and neonate weight were not different between the two groups. However, in the L-diet-fed dam group, bone matrix formation in isolated neonatal calvaria culture was clearly impaired and was not recovered by the addition of Zn into the culture media. Additionally, serum concentration of osteocalcin, as a bone formation parameter, was lower in neonates from the L-diet-fed dam group. Impaired bone mineralization was observed with a significantly lower content of phosphorus in neonate femurs from L-diet-fed dams compared with those from N-diet-fed dams. Moreover, Zn content in the femur and calvaria of neonates from the L-diet group was lower than that of the N-diet-fed group. In the marginally Zn-deficient dams, femoral Zn content, serum concentrations of Zn, and osteocalcin were reduced when compared with control dams. We conclude that maternal Zn deficiency causes impairment of bone matrix formation and bone mineralization in neonates, implying the importance of Zn intake during pregnancy for proper bone development of offspring.     

Abnormal movement of polymorphonuclear neutrophils in the Immotile Cilia Syndrome : Cinemicrographic analysis

Polymorphonuclear leukocytes (PMN) moving on a flat substrate in vitro tend to make fewer turns and persist in a particular direction during periods of rapid movement. It has been suggested that this persistence of unidirectional movement is modulated by the cellular cytoskeleton, particularly the arrangement of microtubules and associated structures. A cinemicrographic analysis of the movement of PMN from five subjects with the Immotile Cilia Syndrome (ICS) showed that there was an abnormality of movement characterized by an increased tendency to turn, resulting in a more tortuous path of movement than seen with controls. Since subjects with ICS are known to have an abnormality of microtubule-associated structures and function of their cilia, it is possible that the abnormalities of ciliated cells and PMN may be the result of the effects of the underlying genetic defect upon microtubule-associated functions in both cell types.     

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE^−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE^−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1^−/−ApoE^−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.     

Copper Excess Impairs Mobilization of Storage Proteins in Bean Cotyledons

Germination represents a limiting stage of plant life cycle. One of the underlying metabolic activities following imbibition of seed is the reserve mobilization. Seeds of bean (Phaseolus vulgaris L. var. soisson nain hatif) were germinated by soaking in distilled water or 200?μM CuCl(2). Storage proteins breakdown and amino acids freeing from reserve tissues were investigated. Compared to the control, Cu caused a reduction in germination rate, embryo growth, and in mobilization of cotyledonary biomass. The failure in albumin and globulin hydrolysis after the exposure to the pollutant was argued by (1) higher contents of remaining proteins than control ones, (2) persistence of some polypeptide bands resolved by polyacrylamide gel electrophoresis of albumin and globulin-rich fractions, and (3) decrease in the availability of amino acids. Nitrogen starvation in embryonic axis should be associated with the Cu-imposed delay in growth.     

Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe

Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm’s canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer’s vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.     

Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation

Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.The Henipavirus genus of the paramyxovirus family comprises two recently emerged, zoonotic pathogens. Hendra virus, first identified in Australia in 1994, caused respiratory illness in and the subsequent death of over one dozen horses and two of the three humans infected (12, 21, 25). Nipah virus led to an outbreak of respiratory and encephalitic illnesses in Malaysia in 1999, affecting both swine and humans and leading to fatality in 105 of the 265 human cases (11). Additional periodic outbreaks of infections with these viruses have occurred (11), and evidence indicates human-to-human transmission of Nipah virus in at least one outbreak (16). Henipavirus, like many paramyxoviruses, requires the presence of two surface glycoproteins for virus-cell and cell-cell fusion (8, 9, 36): the fusion protein, F, which mediates the membrane fusion event, and the attachment protein, G, which binds cellular receptors ephrin B2 (6, 22) and ephrin B3 (23) and which is required for F-mediated membrane fusion. Interactions between the fusion and attachment proteins of a number of paramyxoviruses have been observed previously (30, 33, 35), and interactions between the henipavirus F and G proteins have been demonstrated by coimmunoprecipitation (1-5, 18). However, important questions remain concerning the timing of these interactions and the mechanism by which the attachment protein regulates F-mediated fusion. Results from studies of measles virus (30), Newcastle disease virus (33), and human parainfluenza virus (35) have suggested that the initial interaction between the two glycoproteins occurs within the endoplasmic reticulum (ER) at the time of synthesis, potentially allowing the attachment protein to hold the F protein in its prefusion conformation. In contrast, the retention of the parainfluenza virus type 5 (PIV5) F protein in the ER does not lead to the retention of the PIV5 attachment protein (29), suggesting that interaction between these glycoproteins does not occur soon after synthesis. Recently, henipavirus F proteins have been shown to undergo processing through a complex intracellular trafficking pathway, with expression on the cell surface in a nonfusogenic precursor form (F₀), subsequent endocytosis, cleavage by cathepsin L into the fusogenic (F₁+F₂) form, and retrafficking to the plasma membrane (10, 19, 26-28). While the half-life (t_1/2) of F protein uncleaved by cathepsin L is approximately 2 h (28), results from initial studies of Hendra virus G trafficking indicate much slower trafficking of this protein through the secretory pathway (37), a result inconsistent with the formation of an F-G complex in the ER.To more closely examine the trafficking of the henipavirus glycoproteins, endoglycosidase H (endo H) analysis was used as a marker for trafficking time to the medial-Golgi compartment. We previously reported that wild-type (wt) Hendra virus G protein becomes endo H resistant, with a t_1/2 of between 2 and 3 h (37), suggesting slow trafficking through the secretory pathway. Porotto et al. (31) described a mutant G protein lacking the first 32 residues of the cytoplasmic tail (G-Δ32), which showed enhanced fusion promotion. This Hendra virus G-Δ32 mutant also exhibited higher overall expression than the wt Hendra virus G (S. D. Whitman and R. E. Dutch, unpublished results). To determine if this mutant exhibited altered trafficking kinetics, Vero cells transfected with pCAGGS-Hendra G-Δ32 were examined by pulse-chase analysis followed by endo H treatment as described previously (37). Trafficking kinetics similar to those of wt G were observed, as G-Δ32 became endo H resistant, with a t_1/2 of approximately 2 to 3 h (Fig. ​(Fig.1A).1A). In contrast, endo H analysis of Hendra virus F revealed a resistant population of F₀ within 30 min (Fig. ​(Fig.1B),1B), with the majority of F converted to either an F₀ endo H-resistant form or to the F₁ cleaved form by 2 h. The cleavage of F₀ was observed concomitantly with the appearance of two partially endo H-resistant F₁ bands, suggestive of differential complex sugar additions within the Golgi compartment. These data confirm that Hendra virus F and G traffic through the secretory pathway at different rates, with Hendra virus G trafficking unaffected by the 32-amino-acid deletion. Endo H analysis of the Nipah virus G dimeric form (Fig. ​(Fig.1C),1C), for which the mobility shift after endo H treatment was most apparent, mirrors that of wt Hendra virus G, suggesting that slow trafficking through the secretory pathway is a property of both henipavirus G proteins.Open in a separate windowFIG. 1.Endo H digestion indicates differential rates of trafficking for the henipavirus F and G proteins. (A) Vero cells were transfected with pCAGGS-Hendra G-Δ32, and 24 h posttransfection, the cells were labeled for 30 min and chased for various times and Hendra virus G was immunoprecipitated using an antibody directed to a soluble form of G (7). Endo H digestion was performed as described previously (37). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized using the Typhoon imaging system. (B) Vero cells were transfected with pCAGGS-Hendra F. Twenty-four hours later, the cells were labeled for 30 min. Hendra virus F was immunoprecipitated with a Hendra virus F-specific antibody (28) and subjected to endo H treatment. (C) Pulse-chase analysis of Vero cells transfected with pCAGGS-Nipah G was performed. Immunoprecipitation with antibody to a soluble form of G (7) and endo H analysis were performed as described previously. R and S denote the endo H-resistant and -sensitive species, respectively. +, present; −, absent.Slow trafficking of Hendra virus G through the secretory pathway may be caused by interactions with other viral proteins or with its receptor ephrin B2. Chinese hamster ovary (CHO) cells do not express ephrin B2 (39) and thus were utilized to examine Hendra virus G trafficking in the absence of its cognate receptor. When Hendra virus G-Δ32 was expressed in CHO cells, an endo H-resistant population appeared at 2 h (Fig. ​(Fig.2A)2A) and was found to have increased at subsequent time points, consistent with results obtained using Vero cells. These data suggest that the low trafficking rate of Hendra virus G is not due to association with ephrin B2 and is not cell type specific. Coexpression of either the Hendra virus matrix (M) protein or the F protein with G-Δ32 did not alter trafficking kinetics, indicating that an interaction with either M or F is not responsible for the low rate of G-Δ32 trafficking (Whitman and Dutch, unpublished).Open in a separate windowFIG. 2.Endo H analysis indicates that slow trafficking through the secretory pathway is not dependent on ephrin B2 or on sequences present in the Hendra virus G cytoplasmic tail. (A) CHO cells expressing Hendra virus G-Δ32 were metabolically labeled with ³⁵S for 30 min and chased for the times indicated. Following immunoprecipitation and endo H analysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized via the Typhoon imaging system. (B) Pulse-chase analysis of Vero cells expressing the Hendra virus G-Δ41 tail was performed, followed by immunoprecipitation and endo H analysis. R and S denote the endo H-resistant and -sensitive species, respectively. +, present; −, absent.Lysine-rich motifs (KKXX or KXKXX) have been shown previously to be involved in the retention of type I glycoproteins in the ER (17), while motifs containing multiple arginines have been implicated in the retention of type II integral membrane proteins (20, 32). The type II Hendra and Nipah virus G proteins contain no and one arginine residue in their cytoplasmic domains, respectively, but do have several lysine-rich motifs. To determine if these motifs facilitated ER retention of Hendra virus G, thus resulting in a lowered trafficking rate, an additional mutant (Hendra virus G-Δ41) with the removal of the first 41 amino acids of the cytoplasmic tail, deleting both KKXX and KXKXX motifs and the majority of the cytoplasmic tail, was constructed. Consistent with the results from endo H analyses of wt Hendra virus G and Hendra virus G-Δ32, an endo H-resistant form of Hendra virus G-Δ41 appeared after only 2 h (Fig. ​(Fig.2B),2B), suggesting that Hendra virus G is not retained in the ER by the putative ER retention motifs KKXX and KXKXX or by any other sequence in the cytoplasmic tail.Endo H data strongly indicated differential rates of trafficking through the secretory pathway for Hendra virus G and Hendra virus F, suggesting that interactions between these two proteins likely occur subsequent to transport to the cell surface. To verify the different trafficking kinetics of these two proteins, the rates at which Hendra virus F and G appear on the cell surface were determined by using surface biotinylation, with the more highly expressed mutant Hendra virus G-Δ32 utilized in these experiments to facilitate visualization of the surface population. Hendra virus F was present on the cell surface as F₀ at the completion of the 30-min labeling (Fig. ​(Fig.3A)3A) (time zero), with F₁+F₂ appearing on the surface at approximately 2 h (Fig. ​(Fig.3A),3A), consistent with the complex trafficking pathway observed for F. In contrast, the majority of Hendra virus G-Δ32 arrived on the cell surface after the 2-h time point (Fig. ​(Fig.3B).3B). Similar cell surface profiles were observed when the proteins were coexpressed (Fig. ​(Fig.3C).3C). These data confirm the differential trafficking rates and suggest that Hendra virus F and G do not interact within the secretory pathway but traffic independently to the cell surface.Open in a separate windowFIG. 3.Analysis of cell surface populations confirms the differential trafficking rates of the Hendra virus F and G proteins. (A) Vero cells were transfected with pCAGGS-Hendra F. Twenty-four hours later, the cells were metabolically labeled with ³⁵S for 30 min and chased for the indicated times. Analysis of biotinylated surface proteins was performed as described previously (37). (B) Surface biotinylation of Vero cells expressing Hendra virus G-Δ32 was performed, and samples were analyzed as described previously (37). (C) Biotinylation analyses of Vero cells expressing both Hendra virus F and Hendra virus G-Δ32 were performed as described previously.Exit out of the ER is a carefully controlled process, allowing only properly folded proteins to be transported to the Golgi compartment (15). Thus, slow folding kinetics for Hendra virus G may explain the delayed appearance of an endo H-resistant population. Unlike Hendra virus F, which folds as a trimer (13, 14), Hendra virus G is a tetramer composed of two disulfide-linked dimers (7). To examine folding kinetics, cross-linking analysis (13) was performed at various time points post-metabolic labeling. By the end of the 30-min labeling, the majority of Hendra virus F was folded into a trimeric state which could be stabilized by the addition of a cross-linker (Fig. ​(Fig.4A),4A), and little of the F protein was present in the monomeric form after the addition of the cross-linker. No further increases in trimers were subsequently observed, suggesting very rapid oligomerization of Hendra virus F. Rapid oligomerization of paramyxovirus fusion proteins is consistent with the prefusion structure (38), in which monomers are tightly folded together to form the trimeric unit. The presence of a dimeric species with the addition of a cross-linker is due likely to incomplete cross-linking and does not represent an intermediate conformational population. The folding of Hendra virus G into a tetramer, however, occurs at a lower rate than that of F. In the absence of a cross-linker, monomeric G is present until 2 h postlabeling, suggesting that the formation of the disulfide-linked dimer is intrinsically slow (Fig. ​(Fig.4B).4B). The formation of the disulfide-linked dimer occurs at a rate similar to that of tetramer formation, as the majority of G can be cross-linked to a tetramer by the 2-h time point. Tetramerization of other paramyxovirus attachment proteins, such as PIV5 hemagglutinin-neuraminidase, occurs more rapidly, with t_1/2s for these proteins of 25 to 30 min (24), contrasting greatly with the t_1/2 of 1 to 2 h observed for Hendra virus G. These results suggest that, unlike other paramyxovirus attachment proteins, Hendra virus G undergoes very slow tetramerization and that the slow trafficking of this protein through the secretory pathway is likely a direct reflection of the low intrinsic rate of folding and oligomerization.Open in a separate windowFIG. 4.Cross-linking analysis of Hendra virus glycoproteins indicates slow tetramerization of the Hendra virus G protein. (A) Vero cells expressing Hendra virus F were metabolically labeled for 30 min and chased for the indicated times. Cross-linking with DTSSP [3,3′-dithiobis(sulfosuccinimidyl propionate)] was performed as described previously (14). (B) Vero cells expressing Hendra virus G-Δ32 were labeled for 30 min and chased for the indicated times, and cross-linking analysis with DTSSP was performed as described previously (14). +, present; −, absent.While the majority of paramyxovirus F proteins require their homotypic attachment protein for fusogenic activity, the role of the attachment protein in controlling F protein function remains unclear. The attachment protein has been proposed to hold the F protein in its prefusion conformation until receptor binding occurs (34), and research from several systems has suggested that this F protein-attachment protein interaction occurs in the ER during initial protein folding (30, 33, 35). Data from experiments presented here demonstrating differential rates of oligomerization and secretory pathway transport for Hendra virus F and G strongly indicate that the association of the newly synthesized proteins does not occur in the ER but that they instead traffic independently through the secretory pathway. Thus, at least for the henipaviruses, F-G interactions are unlikely to play a role in preventing premature triggering of the newly synthesized F protein. An alternative model for paramyxovirus fusion has suggested that attachment protein-fusion protein interactions occur only after the attachment protein binds the receptor. However, analysis of henipavirus G and F mutants suggests that F-G avidity inversely correlates with fusion (2, 3, 5), and Hendra virus G protein mutants deficient in receptor binding also lose the ability to coimmunoprecipitate Hendra virus F (4). These data support a model in which F-G interactions occur prior to receptor binding, with the subsequent G-ephrin B2 interaction leading to the release of the F-G interaction and the triggering of fusion. Taken together, these results support a model in which the henipavirus F and G associate only after trafficking to the cell surface. The mechanisms by which this interaction is promoted and/or regulated represent an exciting area of future research.     

Formation of Sindbis Virus Proteins: Identification of a Precursor for One of the Envelope Proteins

Exposure of Sindbis virus-infected chicken embryo cells to a short pulse of radioactive amino acids revealed the formation of primarily three proteins: the nucleocapsid (C) of the virus, one of the viral envelope proteins (E1), and a glycoprotein that did not appear in the virion. This third protein (PE2) has now been identified as a precursor of the other viral envelope protein (E2) on the basis of two observations: (i) the simultaneous disappearance of radioactive PE2 and appearance of labeled E2 in pulse-chase experiments, and (ii) the identity of (14)C-arginine tryptic peptides in fingerprints of the two proteins. The nucleocapsid was the most heavily labeled protein in the cell and appeared in the virus during the short pulse. The two (14)C-labeled envelope proteins, although having different kinetics of labeling in the cell, appeared simultaneously in the virus only after the chase. Addition of pactamycin, a drug inhibiting initiation of protein synthesis, preferentially inhibited the formation of capsid protein Assuming that Sindbis virus proteins are formed initially as a single polypeptide, our studies locate the nucleocapsid at the amino-terminal end of the polypeptide chain.     

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.     

Possible Role of Malaria in the Aetiology of the Nephrotic Syndrome in Nairobi

In a study in Nairobi, Kenya, 30 young adults with nephrotic syndrome were investigated in detail and a further 18 were studied less completely. In no case was evidence found to support a possible role for malaria in the aetiology of the syndrome.     

Cep97 Is Required for Centriole Structural Integrity and Cilia Formation in Drosophila

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Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

Pannexin1 (Panx1) subunits oligomerize to form large-pore channels between the intracellular and extracellular milieu that have been shown to regulate proliferation, differentiation and cell death mechanisms. These key cellular responses are ultimately necessary for normal tissue development and function but the role of Panx1 in development, differentiation and function in many tissues remains unexplored, including that of the breast. Panx1 was identified to be expressed in the mammary gland through western blot and immunofluorescent analysis and is dynamically upregulated during pregnancy and lactation. In order to evaluate the role of Panx1 in the context of mammary gland development and function, Panx1^-/- mice were evaluated in comparison to wild-type mice in the mammary glands of virgin, lactating and involuting mice. Our results revealed that Panx1 ablation did not affect virgin or involuting mammary glands following histological and whole mount analysis. Panx1 was necessary for timely alveolar development during early lactation based on a decreased number of alveolar lumen following histological analysis and reduced proliferation following Ki67 immunofluorescent labelling. Importantly, the loss of Panx1 in lactating mammary glands did not overtly affect epithelial or secretory differentiation of the mammary gland suggesting that Panx1 is not critical in normal mammary gland function. In addition, PANX1 mRNA expression was correlated with negative clinical outcomes in patients with breast cancer using in silico arrays. Together, our results suggest that Panx1 is necessary for timely alveolar development following the transition from pregnancy to lactation, which may have implications extending to patients with breast cancer.     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Poor Cerebral Inflammatory Response in eIF2B Knock-In Mice: Implications for the Aetiology of Vanishing White Matter Disease

Background

Mutations in any of the five subunits of eukaryotic translation initiation factor 2B (eIF2B) can lead to an inherited chronic-progressive fatal brain disease of unknown aetiology termed leucoencephalopathy with vanishing white matter (VWM). VWM is one of the most prevalent childhood white matter disorders, which markedly deteriorates after inflammation or exposure to other stressors. eIF2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. A previous study demonstrated that Eif2b5^R132H/R132H mice suffer delayed white matter development and fail to recover from cuprizone-induced demyelination, although eIF2B enzymatic activity in the mutant brain is reduced by merely 20%.

Principal Findings

Poor astrogliosis was observed in Eif2b5^R132H/R132H mice brain in response to systemic stress induced by peripheral injections of lipopolysaccharide (LPS). Even with normal rates of protein synthesis under normal conditions, primary astrocytes and microglia isolated from mutant brains fail to adequately synthesise and secrete cytokines in response to LPS treatment despite proper induction of cytokine mRNAs.

Conclusions

The mild reduction in eIF2B activity prevents the appropriate increase in translation rates upon exposure to the inflammatory stressor LPS. The data underscore the importance of fully-functional translation machinery for efficient cerebral inflammatory response upon insults. It highlights the magnitude of proficient translation rates in restoration of brain homeostasis via microglia-astrocyte crosstalk. This study is the first to suggest the involvement of microglia in the pathology of VWM disease. Importantly, it rationalises the deterioration of clinical symptoms upon exposure of VWM patients to physiological stressors and provides possible explanation for their high phenotypic variability.