Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

An Improved Algorithm for the Piecewise-Smooth Mumford and Shah Model in Image Segmentation

An improved algorithm for the piecewise-smooth Mumford and Shah functional is presented. Compared to the previous work of Chan and Vese, and Choi et al., extensions of the key functions are replaced by updating the level set function based on an artificial image that is composed of the diffused image and the original image. The low convergence problem of the classical algorithm is efficiently solved in the proposed approach. The resulting algorithm has also been demonstrated by several cases.[1,2,3,4,5,6,7,8,9,10,11]     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 μg of protein) for mass spectrometry and immunological analyses. High levels of interferon-γ (IFN-γ) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-γ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-α response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-γ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy “contact-specific fractions” revealed 16 proteins that are key candidates as vaccine or diagnostic targets.Tuberculosis (TB)1 is a major health problem throughout the world. A recent World Health Organization report shows that TB has been increasing at a rate of 1% per year, and an estimated 9.2 million new cases arise each year (1). Although TB is preventable, there has been an increase in its incidence in recent years. Re-emergence of TB is mainly due to its association with human immunodeficiency virus infection (2) and also due to the occurrence of multidrug-resistant strains of the causative agent, Mycobacterium tuberculosis (3).Vaccination of general population is cost effective and represents one of the best biological measures for disease control. The current vaccine against tuberculosis, Bacille Calmette-Guérin (BCG), has been administered to more people than any other vaccine. The side effects of BCG are tolerable, and it prevents miliary and meningeal tuberculosis in young children. In striking contrast, it affords limited and highly variable protection (0–80%) against pulmonary TB (4). Thus, BCG does not seem to be a satisfactory vaccine (5, 6) and necessitates exploration of newer strategies to improve BCG or to develop a more effective vaccine.One of the potential strategies for the development of an improved TB vaccine involves the use of the proteins secreted by M. tuberculosis during growth. There is evidence that proteins actively secreted by M. tuberculosis during growth induce cell-mediated immune responses by causing expansion of specific interferon-γ (IFN-γ)-producing T lymphocytes that are capable of recognizing and exerting antimicrobial effects against infected macrophages (7). The importance of IFN-γ pathways in host defense against M. tuberculosis was clarified by experimental studies on IFN-γ knock-out mice as well as the identification and characterization of humans with mutations in IFN-γ receptor (8, 9).Several studies have been carried out to define the secreted proteome of M. tuberculosis. The earliest study aimed at the identification of mycobacterial culture filtrate proteins, using chromatography and N-terminal sequencing to identify eight culture filtrate proteins (10). Later, many studies used two-dimensional (2D) PAGE combined with sensitive mass spectrometric methods for identification of proteins. The above mentioned approaches have identified nearly 300 culture filtrate proteins (11–13).Identification of T cell antigens in a complex mixture was first done by a T cell Western blot method (14). Later, two-dimensional separation methods were used that involved protein separation by either IEF (15) or chromatography (16) in the first dimension and preparative SDS-PAGE followed by whole gel elution (17) in the second dimension. Mouse T cell antigens of M. tuberculosis were identified using this method (15). Mycobacterial antigens that induce an immune response in healthy household contacts and treated TB patients were also mapped using this approach (16).In the present study, 2D liquid phase electrophoresis (LPE) along with an in vitro IFN-γ assay and LC-MS/MS were used to identify potential human T cell antigens. Systematic screening of the M. tuberculosis culture filtrate (CF) proteome and comparative evaluation of cellular immune responses between TB patients and healthy contacts led to the identification of 59 proteins in the most immunogenic 2D LPE fractions. Twenty-four potentially novel T cell antigens were identified, and 16 proteins were identified in 10 2D LPE fractions that differentiated healthy contacts from TB patients based on IFN-γ responses.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Size-Selective Predation on Groundwater Bacteria by Nanoflagellates in an Organic-Contaminated Aquifer

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily Spumella guttula (Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate⁻¹ · h⁻¹ and 1.4 nl · flagellate⁻¹ · h⁻¹, respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm³ average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.While heterotrophic protists have been found in pristine and contaminated aquifers (3, 29, 34, 37, 54–57), very little research has been performed to elucidate their role in the subsurface. In other environments (e.g., surface and marine waters, topsoil, and wastewater treatment plants), it is well documented that they typically consume bacteria (2, 11, 15, 41, 42, 47), although some have been observed to consume high-molecular-weight organics (48, 59) and even viruses (20, 39). Protists typically graze selectively, depending upon the size (1, 9, 17, 25, 52), growth condition (18, 53), species (16, 17, 35), and motility (18) of their prey. In carbon-limited environments, protists decrease bacterial competition, resulting in a greater bacterial uptake rate for organic substrate per unit of bacterial biomass (27). Based upon indirect field observations, it is also hypothesized that this may be the role they play in organically contaminated aquifers (31). In nutrient-limited environments, protists may release nitrogen or phosphorus needed by bacteria (10, 28, 44, 61).Studies at the U.S. Geological Survey’s (USGS) Toxic Substances Hydrology Program research site at the Massachusetts Military Reservation (MMR) on Cape Cod, Mass., have shown that sandy aquifer sediments can harbor large protistan populations even at relatively low levels (≤2 mg/liter) of dissolved organic matter (30). Protistan abundances in the MMR aquifer plume range from 1 × 10⁴ to 7 × 10⁴ g (dry weight)⁻¹ (30) and consist primarily of nanoflagellates (2 to 3 μm in length) (29) that belong to the genera Bodo, Cercomonas, Cryptaulax, Cyanthomonas, Goniomonas, and Spumella, along with some undescribed species (37). A few amoebae (63) and no ciliates (29) have been observed.Results of a principal-component factor analysis of protistan and bacterial abundances and chemical constituents in the MMR plume suggested that the flagellates were preying upon unattached bacteria (30). Additional evidence of predation was obtained from flowthrough columns of aquifer sediment from which fluorescently labeled unattached bacteria eluted at much lower rates than they did from sterile (protist-free) controls (31). However, these results provide only indirect evidence of predation because no enumerations of the bacteria within the flagellates were performed.The purpose of the research reported in this paper was to directly determine whether the MMR nanoflagellates can consume unattached bacteria in the plume and the extent to which they engage in size-selective grazing. Rates of bacterivory (grazing and clearance rates) were estimated in the laboratory by using fluorescently labeled, monodispersed bacteria (FLB) and nanoflagellates that had been isolated from the MMR aquifer plume. Although other methods exist (19, 26, 38, 43, 45, 62, 64–66), we chose to use fluorescent labeling to study flagellate bacterivory because this procedure requires shorter incubation times and relies upon direct visual observation of the prey within the predator. In addition, experiments could be designed with different sizes of FLB to determine if the nanoflagellates can discriminate between prey. This involved using FLB with cell lengths of 0.1 to 0.5, 0.5 to 0.8, 0.8 to 1.5, and >1.5 μm (average cell volumes of 0.06, 0.14, 0.21, and 0.87 μm³, respectively) in the grazing experiments.     

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

47,XYY Syndrome and Male Infertility

Men with 47,XYY syndrome present with varying physical attributes and degrees of infertility. A retrospective chart review was performed on a male infertility and genetic anomaly database. Three patients with 47,XYY were found. Each presented with > 2 years of infertility. All were tall with elevated body mass indices. Scrotal findings ranged from normal to atrophic testicles. Semen analyses demonstrated oligospermia and varying endocrine profiles. Because of the diverse phenotype and potential lack of symptoms, identification and diagnosis of men with 47,XYY syndrome may be difficult. We recommend careful screening of 47,XYY patients and referral to primary physicians for long-term follow-up for increased incidence of health-related comorbidities.Key words: Infertility syndromesThe 47,XYY sex chromosome variation is the most common sex chromosome anomaly after Klinefelter syndrome (47,XXY),^1–3 occurring in approximately 1 out of 1000 live male births.^4,5 Parental nondisjunction at meiosis II resulting in an extra Y chromosome produces a 47,XYY karyotype in the affected offspring.^6–8 46,XY/47,XYY mosaics from parental nondisjunction during cell division after postzygotic mitosis can result in addition of the extra Y chromosome in early embryonic development.^6,8Most patients with 47,XYY have a delayed diagnosis, with a median age of 17.1 years at diagnosis, as was shown in a Danish cohort study.⁹ Although most have no phenotypic abnormalities, XYY boys are at greater risk for behavioral problems, mild learning disability, delayed speech and language development, and tall stature.¹⁰ Studies have increasingly reported an association between 47,XYY and fertility problems, noting an increased incidence of chromosomally abnormal spermatozoa in the semen of men with 47,XXY syndrome.^7,11–15 This greater prevalence of hyperhaploid sperm results in an increased risk of passing the extra Y chromosome to offspring.¹⁴ Men with 47,XXY syndrome can have variable sperm counts, ranging from normal to azoospermia.^{3,8,14,16–18}Here we review pertinent findings on physical examination and laboratory evaluation in three men with 47,XXY syndrome diagnosed during infertility evaluation as well as review the available literature on the subject, with special emphasis on male fertility effects.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]