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Human herpes simplex viruses (HSVs) belong to the Herpesviridae family. At present, no vaccine or curative treatment is available for the prevention of HSV infections. Here, we review the cell surface receptors that are recognized by HSV's glycoprotein B, glycoprotein C, glycoprotein D, and the glycoprotein H - glycoprotein L complex to facilitate entry into host cells. These receptors include heparan sulfate (HS), herpesvirus entry mediator (HVEM), and nectin-1/-2, 3-O-sulfated heparan sulfate (3-OS HS)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sheep are known to express the hybrid co-receptor/pattern recognition receptor WC1 on their γδ T cells but details of the ovine WC1 multigenic array and gene expression were unknown. Annotation of the sheep genome assembly (Oar_rambouillet_v1.0) yielded 15 complete and 42 partial WC1 genes predicted to code for six different protein structures. RT-PCR amplification of the most distal scavenger receptor cysteine rich (SRCR) domain known as a1, which serves as the gene signature, from genomic and cDNA templates verified the majority of annotated genes. As for cattle and goats, sheep a1 domain sequences included WC1.1 and WC1.2 types. A unique ovine gene, WC1-16, had multiple SRCR a-pattern domains in tandem similar to one found in goats. Intracytoplasmic domains of WC1 transcripts had splice variants that may affect signal transduction. The larger number of WC1 genes in sheep and differences in structures and splice variants relative to cattle could have implications in expression patterns and engagement of γδ T cells by pathogens or vaccine constructs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide-binding proteins (LBPs) and bactericidal permeability-increasing proteins (BPIs) are effectors of the innate immune response which act in a coordinated manner to bind and neutralize the LPS present in Gram negative bacteria. The structural organization that confers the function of LBPs and BPIs is very similar, however, they are antagonistic to each other. In this work, we characterized two LBP/BPIs from the scallop Argopecten purpuratus, namely ApLBP/BPI1 and ApLBP/BPI2. The molecular and phylogenetic analyses of ApLBP/BPIs indicated that both isoforms display classic characteristics of LBP/BPIs from other invertebrates. Additionally, ApLBP/BPIs are constitutively expressed in scallop tissues and their transcript expression is upregulated in hemocytes and gills in response to an immune challenge. However, some structural characteristics of functional importance for the biological activity of these molecules, such as the net charge differ substantially between ApLBP/BPI1 and ApLBP/BPI2. Furthermore, each isoform displays a specific profile of basal expression among different tissues, as well as specific patterns of expression during the activation of the immune response. Results suggest that functional specialization of ApLBP/BPIs might happen, with potential role as LBP or BPI in this species of scallop. Further research on the biological activities of ApLBP/BPIs are necessary to elucidate their participation in the scallop immune response., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.

Background: Zona pellucida domain-containing proteins (ZP proteins) have been identified as the principle constituents of the egg coat (EC) of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP) structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata., Results: Five ZP proteins (BbZP1-5) were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA) domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates showed that BbZP1-5 form a monophyletic group and share no significant sequence similarities with the ZP proteins of urochordates and the ZP3 subtype of jawed vertebrates. By contrast, small regions of homology were identifiable between the BbZP and ZP proteins of the non-jawed vertebrate, the sea lamprey Petromyzon marinus. The lamprey ZP proteins were highly similar to the ZP1 and ZP2 subtypes of the jawed vertebrates, which suggests that the ZP proteins of basal chordates most likely shared a recent common ancestor with vertebrate ZP1/2 subtypes and lamprey ZP proteins., Conclusions: The results document the spectra of zona pellucida domain-containing proteins of the egg coat of basal chordates. Particularly, the study provides solid evidence for an invertebrate origin of vertebrate ZP proteins and indicates that there are diverse domain architectures in ZP proteins of various metazoan groups.

Zona pellucida (ZP) containing proteins are glycoproteins in teleost chorion and are encoded by several gene subfamilies, mainly including ZPA, ZPB, ZPC and ZPX genes. In teleost species, ZP genes are expressed either in liver under regulation of estrogen or in ovary. In the present study, five ZP gene isoforms were isolated and characterized in Gobiocypris rarus. The putative amino acid sequences of these ZP gene isoforms contain the typical trefoil motif and a ZP domain. These five G. rarus ZP gene isoforms were named as grZPB.1, grZPB.2, grZPB.3, grZPB.4 and grZPB.5. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis indicated that all these ZP mRNA isoforms were exclusively expressed in ovary. G. rarus juveniles at the age of 21 days postfertilization were exposed to 17α-ethinylestradiol (EE2; 0.01, 0.1 and 1 nM), 4-nonylphenol (4-NP; 10, 100 and 1000 nM) or bisphenol A (BPA; 0.1, 1 and 10nM) for 3 days. mRNA expressions of ZPB isoforms following the exposure to xenoestrogen were detected by RT-qPCR. Data were analyzed by the 2(-△△Cq) method. The results indicate that induction by 0.1-1nM EE2 on mRNA expression of the grZPB isoforms is weaker than for vitellogenin. 4-NP exposures at three concentrations had differential effects on the grZPBs. BPA at three concentrations weakly induced mRNA expression of the grZPB isoforms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A novel gene, TMEM114, was annotated as a member of the claudin gene family and was subsequently associated as a cause of autosomal dominant cataract because of a translocation in its putative promoter. Our bioinformatic and molecular analyses of TMEM114, and the closely related TMEM235, demonstrate that these proteins are more closely related to members of the voltage dependent calcium channel gamma subunit family. TMEM114 and TMEM235 differed from claudins in terms of localisation in polarised epithelial cells and by the presence of N-linked glycans. By gene expression knockdown in Xenopus tropicalis we also demonstrate a role for Tmem114 in eye development., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We screened four B. thuringiensis strains whose parasporal inclusions contained the S-layer protein (SLP), and cloned two slp genes from each strain. Phylogenetic analysis indicated these SLPs could be divided into two groups, SLP1s and SLP2s. To confirm whether SLPs were present in the S-layer or as a parasporal inclusion, strains CTC and BMB1152 were chosen for further study. Western blots with whole-cell associated proteins from strains CTC and BMB1152 in the vegetative phase showed that SLP1s and SLP2s were constituents of the S-layer. Immunofluorescence utilizing spore-inclusion mixtures of strains CTC and BMB1152 in the sporulation phase showed that SLP1s and SLP2s were also constituents of parasporal inclusions. When heterogeneously expressed in the crystal negative strain BMB171, four SLPs from strains CTC and BMB1152 could also form parasporal inclusions. This temporal and spatial expression is not an occasional phenomenon but ubiquitous in B. thuringiensis strains.

A fish egg envelope is composed of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among vertebrate species. Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte. We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala. Five major components of the egg envelope were purified and their partial amino acid sequences were determined by sequencing. cDNA cloning revealed that the eel egg envelope was composed of four ZPC homologues and one ZPB homologue. Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins. This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution. Northern blotting and RT-PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver. Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver. Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway to Euteleostei., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Infection with Trypanosoma brucei, which causes African trypanosomiasis, activates microglia, which are constitutively maintained in a quiescent state through CD200-CD200 receptor interactions. C57BL/6 mice have one inhibitory receptor, CD200R and three activating members, CD200 receptor-like (RL)a-c. Infection increased MAC-1 (microglia marker), CD200RLa and CD200RLb, but not CD200, CD200R or CD200RLc, transcript levels in the brains. Minocycline treatment inhibited the infection-induced elevation of MAC-1 and CD200RLa transcripts, but had no significant effect on CD200 or the other receptors. This suggests that CD200RLa might play a role in microglia/macrophage activation during trypanosome infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Tetherin is a recently identified antiviral restriction factor that restricts HIV-1 particle release in the absence of the HIV-1 viral protein U (Vpu). It is reminiscent of APOBEC3G and TRIM5a that also antagonize HIV. APOBEC3G and TRIM5a have been demonstrated to evolve under pervasive positive selection throughout primate evolution, supporting the red-queen hypothesis. Therefore, one naturally presumes that Tetherin also evolves under pervasive positive selection throughout primate evolution and supports the red-queen hypothesis. Here, we performed a detailed evolutionary analysis to address this presumption., Methodology/principal Findings: Results of non-synonymous and synonymous substitution rates reveal that Tetherin as a whole experiences neutral evolution rather than pervasive positive selection throughout primate evolution, as well as in non-primate mammal evolution. Sliding-window analyses show that the regions of the primate Tetherin that interact with viral proteins are under positive selection or relaxed purifying selection. In particular, the sites identified under positive selection generally focus on these regions, indicating that the main selective pressure acting on the primate Tetherin comes from virus infection. The branch-site model detected positive selection acting on the ancestral branch of the New World Monkey lineage, suggesting an episodic adaptive evolution. The positive selection was also found in duplicated Tetherins in ruminants. Moreover, there is no bias in the alterations of amino acids in the evolution of the primate Tetherin, implying that the primate Tetherin may retain broad spectrum of antiviral activity by maintaining structure stability., Conclusions/significance: These results conclude that the molecular evolution of Tetherin may be attributed to the host-virus arms race, supporting the Red Queen hypothesis, and Tetherin may be in an intermediate stage in transition from neutral to pervasive adaptive evolution.

A recent paper reported that a variant (rs2986017) of the calcium homeostasis modulator 1 (CALHM1) gene affects risk for late-onset Alzheimer's disease (AD). This study aims to investigate whether single nucleotide polymorphisms (SNPs) of the CALHM1 gene are associated with AD. SNPs in the genes of two other CALHM subtypes, CALHM2 and CALHM3, were also studied. Our study failed to detect any association between the SNPs of the three genes and AD. Although rs729211 showed marginal association in the APOE4 negative group, the linkage disequilibrium analysis results suggest this to be a false positive.

The liver bile acid-binding proteins, L-BABPs, formerly called the liver "basic" fatty acid-binding proteins, are a subfamily of the fatty acid-binding proteins, FABPs. All the members of this protein group share the same fold: a 10 stranded beta barrel in which two short helices are inserted in between the first and the second strand of antiparallel beta sheet. The barrel encloses the ligand binding cavity of the protein while the two helices are believed to be involved in ligand accessibility to the binding site. The L-BABP subfamily has been found to be present in the liver of several vertebrates: fish, amphibians, reptiles, and birds but not in mammals. The members of the FABP family present in mammals that appear to be more closely related to the L-BABPs are the liver FABPs and the ileal BABPs, both very extensively studied. Several L-BABP X-ray structures are available and chicken L-BABP has also been studied using NMR spectroscopy. The stoichiometry of ligand binding for bile acids, first determined by X-ray crystallography for the chicken liver protein, is of two cholates per protein molecule with the only exception of zebrafish L-BABP which, due to the presence of a disulfide bridge, has a stoichiometry of 1:1. The stoichiometry of ligand binding for fatty acids, determined with several different techniques, is 1:1. An unanswered question of great relevance is the identity of the protein that in mammals performs the function that in other vertebrates is carried out by the L-BABPS.

Protein sequences are treated as stochastic processes on the basis of a reduced amino acid alphabet of 10 types of amino acids. The realization of a stochastic process is described by associated transition probability matrix that corresponds to the process uniquely. Then new distances between transition probability matrices are defined for sequences similarity analysis. Two separate datasets are prepared and tested to identify the validity of the method. The results demonstrate the new method is powerful and efficient.

Background: WC1 co-receptors are group B scavenger receptor cysteine-rich molecules that are found exclusively on gammadeltaT cells and are thought to be encoded by a multi-gene family. Previous studies have shown gammadeltaT cells that respond to a particular stimulus have unique WC1 molecules expressed. Prior to the onset of the studies described here only one full-length WC1 nucleotide sequence was publicly available, though three WC1 molecules had been distinguished based on monoclonal antibody reactivity. Furthermore, the number of WC1 genes found in the bovine genome and their sequences had not yet been resolved., Results: By annotating the bovine genome Btau_3.1 assembly, here we show the existence of 13 members in the WC1 gene family and their organization within two loci on chromosome 5 including three distinct exon-intron gene structures one of which coded for a potentially more primitive and smaller WC1 molecule that is similar to the swine WC1 gene. We also provide cDNA evidence as verification for many of the annotated sequences and show transcripts for isoforms derived by alternative splicing., Conclusion: It is possible that WC1 diversity contributes to functional differences that have been observed between gammadeltaT cell populations. The studies described here demonstrate that WC1 molecules are encoded by a large, multi-gene family whose transcripts undergo extensive alternative splicing. Similar to other non-rearranging immunoreceptors, it is likely that the WC1 gene repertoire underwent expansion in order to keep pace with rapidly changing ligands.

The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nucleotides encoding a polypeptide of 543 amino acid residues. The deduced amino acid sequence of ZP4 revealed high overall homology with rat (82%) and human (78%). Subsequent mass spectrometric analysis of the hamster ZP allowed identification of peptides from all four glycoproteins. The data presented in this study provide evidence, for the first time, that the hamster ZP matrix is composed of four glycoproteins.

Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as "space fillers" to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

Fertilin beta may play an important role in sperm-egg plasma membrane adhesion and fusion. To explore the effects of fertilin beta, the sperm membrane protein subunit, in the sheep fertilization process, we used RACE technique for cloning the full length cDNA of the coding region (CDS) of fertilin beta. The coding region was 2, 217 bp, which consists of 738 amino acids. The fertilin beta in the sperm membrane of sheep shares 79.4%, 66.7%, and 58.1% sequence identity with that in bovine, pig and human, respectively. The phylogenetic analysis of the fertilin beta gene family indicated the fertilin beta was clustered with bovine, and is closest to the one of bovine. This result is consistent with the result of the traditional classification. The protein structure analysis showed the disintegrin domain of sheep fertilin beta contains a TDE. Besides the above tripeptide sequence, the family member of ADAM (A Disintegrin and A Metalloprotease) follow the conserved sequence of ECD of X-D/E-E, and formed the conserved sequence of X-D/E-ECD. The pentapeptide sequence of the sheep fertilin beta is TDECE.

Clostridium difficile isolates (n=149) collected in south-east Scotland between August and October 2005 were typed by four different methods and their susceptibility to seven different antibiotics was determined. The aims were to define the types of strain occurring in this region and to determine whether there were any clonal relationships among them with respect to genotype and antibiotic resistance pattern. Ribotyping revealed that 001 was the most common type (n=113, 75.8 %), followed by ribotype 106 (12 isolates, 8.1 %). The majority of the isolates (96.6 %, n=144) were of toxinotype 0, with two toxinotype V isolates and single isolates of toxinotypes I, IV and XIII. PCR and restriction analysis of the fliC gene from 147 isolates gave two restriction patterns: 145 of pattern VII and two of pattern I. Binary toxin genes were detected in only three isolates: two isolates of ribotype 126, toxinotype V, and one isolate of ribotype 023, toxinotype IV. S-types showed more variation, with 64.5 % (n=40) of the common S-type (4,939) and 21 % (n=13) of S-type 4,741, with six other S-types (one to three isolates each). All ribotype 001 isolates were of the same S-type (4,939), with three isolates of other ribotypes being this S-type. No resistance was found to metronidazole or vancomycin, with resistance to tetracycline only found in 4.3 % of the isolates. A high proportion of isolates were resistant to clindamycin (62.9 %), moxifloxacin, ceftriaxone (both 87.1 %) and erythromycin (94.8 %). Resistance to three antibiotics (erythromycin, clindamycin and ceftriaxone) was seen in 66 isolates, with erythromycin, ceftriaxone and moxifloxacin resistance seen in 96 isolates. Resistance to all four of these antibiotics was found in 62 isolates and resistance to five (the above plus tetracycline) in one isolate: a ribotype 001, toxinotype 0 strain. Whilst ribotype 001 was the most commonly encountered type, there was no evidence of clonal relationships when all other typing and antibiotic resistance patterns were taken into account.

Symbiotic bacterium closely related to the secondary symbiont of tsetse flies, Sodalis glossinidius, has been described from the bloodsucking fly Craterina melbae. Phylogenetic analysis of two genes, 16S rRNA gene and component of type three secretion system, placed the bacterium closer to the Sitophilus-derived branch of Sodalis than to the tsetse symbionts. This indicates that the Craterina-derived lineage of Sodalis originated independent of the tsetse flies symbionts and documents the capability of Sodalis bacteria either to switch between different host groups or to establish the symbiosis by several independent events.

The most important confirmed genetic factor of susceptibility to multiple sclerosis (MS) has been identified in the HLA class II region. The hypothesis that several genes, including HLA class II, may influence the prognosis of patients with MS has been proposed. In a recent study, using low intermediate resolution typing, we found that some HLA alleles may predict disease severity as assessed by magnetic resonance imaging (MRI) measures. The aim of this study was to examine the relationship between high-resolution typing of HLA alleles and disease severity as measured by brain MRI quantitative markers of demyelinating and destructive pathology in patients with MS. In 41 MS patients (27 relapsing-remitting, 7 secondary progressive, and 7 primary progressive), we performed high-resolution typing of alleles HLA-DRB1*04, -DQB1*03, -DRB1*15, -DQB1*06, and of haplotypes -DRB1*04-DQB1*03 and -DRB1*15-DQB1*06. These alleles and haplotypes were associated with higher susceptibility to MS in a recently published case-control study conducted in the Friuli-Venezia-Giulia region, Italy. Of 41 included patients, 13 were men and 28 were women. Mean age was 43.3 (SD 11.4) years, mean disease duration 10.3 (SD 7.8) years, and mean EDSS 2.3. DNA extraction and genomic typing were obtained with the sequence-specific primers method using primer pairs that amplified the HLA alleles. All patients underwent a 1.5-T MRI examination of the brain. Disease severity was assessed by clinical measures [Expanded Disability Status Scale (EDSS)] and MRI measures. T2- and T1-lesion volumes (LVs) and brain atrophy measures [fractions of brain parenchyma (BPF), gray matter (GMF), and white matter (WMF)] were calculated. We used general linear model analysis (GML), controlled for age, disease duration, and treatment status, to compare the MRI measures according to allele and haplotype status. The following significant results were found: HLA-DRB1*1501 positive patients had significantly lower GMF (0.493 vs 0.526, p < 0.001), lower BPF (0.784 vs 0.815, p = 0.018), and higher T1-LV (2.8 vs 0.7ml, p = 0.036); -DQB1*0301 positive patients had significantly higher T2-LV (34.1 vs 0.7 ml, p = 0.041), and showed a trend for lower BPF (0.790 vs 0.846, p = 0.064); -DQB1*0302 positive patients had significantly lower T1-LV (2.4 vs 0.9 ml, p = 0.016); and -DQB1*0602 positive patients had significantly lower GMF (0.492 vs 0.521, p = 0.007) and lower BPF (0.781 vs 0.811, p = 0.023). No differences were found in the indices of MRI disease severity according to HLA haplotype associations. Both in correlation and in regression analyses, we observed significant associations between HLA-DRB1*1501 and lower GMF and BPF and higher T1-LV, between -DQB1*0301 and higher T2-LV and disease duration, between -DQB1*0302 and lower GMF and higher T1- and T2-LV, between -DQB1*0602 and lower GMF and BPF, and between -DQB1*0603 and higher T1-LV and EDSS. High-resolution HLA genotyping analysis revealed a robust relationship between alleles HLA-DRB1*1501, -DQB1*0301, -DQB1*0302, -DQB1*0602, and -DQB1*0603, and more severe damage on inflammatory and neurodegenerative MRI measures.

Background: The syndecans are the major family of transmembrane proteoglycans in animals and are known for multiple roles in cell interactions and growth factor signalling during development, inflammatory response, wound-repair and tumorigenesis. Although syndecans have been cloned from several invertebrate and vertebrate species, the extent of conservation of the family across the animal kingdom is unknown and there are gaps in our knowledge of chordate syndecans. Here, we develop a new level of knowledge for the whole syndecan family, by combining molecular phylogeny of syndecan protein sequences with analysis of the genomic contexts of syndecan genes in multiple vertebrate organisms., Results: We identified syndecan-encoding sequences in representative Cnidaria and throughout the Bilateria. The C1 and C2 regions of the cytoplasmic domain are highly conserved throughout the animal kingdom. We identified in the variable region a universally-conserved leucine residue and a tyrosine residue that is conserved throughout the Bilateria. Of all the genomes examined, only tetrapod and fish genomes encode multiple syndecans. No syndecan-1 was identified in fish. The genomic context of each vertebrate syndecan gene is syntenic between human, mouse and chicken, and this conservation clearly extends to syndecan-2 and -3 in T. nigroviridis. In addition, tetrapod syndecans were found to be encoded from paralogous chromosomal regions that also contain the four members of the matrilin family. Whereas the matrilin-3 and syndecan-1 genes are adjacent in tetrapods, this chromosomal region appears to have undergone extensive lineage-specific rearrangements in fish., Conclusion: Throughout the animal kingdom, syndecan extracellular domains have undergone rapid change and elements of the cytoplasmic domains have been very conserved. The four syndecan genes of vertebrates are syntenic across tetrapods, and synteny of the syndecan-2 and -3 genes is apparent between tetrapods and fish. In vertebrates, each of the four family members are encoded from paralogous genomic regions in which members of the matrilin family are also syntenic between tetrapods and fish. This genomic organization appears to have been set up after the divergence of urochordates (Ciona) and vertebrates. The syndecan-1 gene appears to have been lost relatively early in the fish lineage. These conclusions provide the basis for a new model of syndecan evolution in vertebrates and a new perspective for analyzing the roles of syndecans in cells and whole organisms.

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. In this study, we describe the isolation of the full-length cDNA encoding for CD9 molecule (daCD9) of red stingray, Dasyatis akajei. This 1252 bp cDNA was isolated from leukocyte cDNA library and contains 681 bp open reading frame encoding 226 amino acid residues. Amino acid sequences analysis and structure prediction display approximately 50% identity to higher vertebrates with the presence of conserved structures, including the four transmembrane domains and certain characteristic residues. Southern blot analysis shows that daCD9 exists as a single copy gene. Northern blot analysis reveals that daCD9 is highly expressed in gill and spleen although its expression can be found in other tissues suggesting daCD9 might play an important role in immune defense in this fish.

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F. chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes.

Invasion of tumor cells into the surrounding normal brain tissues is a prominent feature of malignant gliomas. Malignant glioma cells secrete thrombospondin-1 which participates in the motility of glioma cells and binds cell surface heparan sulfate proteoglycan. To clarify the invasion mechanism of tumor cells, expression of the syndecans (syndecan-1, -2, -3, and -4), a major cell surface heparan sulfate proteoglycan family, was analyzed in malignant gliomas. Involvement of nuclear factor-kappaB (NF-kappaB) on syndecan-1 expression was also investigated. Using reverse transcription-PCR, the authors analyzed the expression of syndecan-1, -2, -3, and -4 in 10 malignant glioma cell lines, 2 glioblastoma specimens, and 2 normal brain specimens. All malignant glioma cell lines and glioblastoma specimens expressed all types of syndecan mRNA, except in one glioma cell line that lacked syndecan-3 expression. On the other hand, normal brain specimens expressed syndecan-2, -3, and -4 mRNA, but did not syndecan-1 mRNA. Syndecan-1 protein was localized in the cell surface of all malignant glioma cell lines by flow cytometry. Various levels of active nuclear factor-kappa B (NF-kappaB) was detected in all malignant glioma cell lines using immunoblotting. The expression of active NF-kappaB and syndecan-1 increased in U251 glioma cells after tumor necrosis factor-alpha or interleukin-1beta treatment, which can activate NF-kappaB. The amplification of active NF-kappaB and syndecan-1 by tumor necrosis factor-alpha or interleukin-1beta was suppressed by an inhibitor of NF-kappaB activation (emodin). Emodin also downregulated the expression of syndecan-1 mRNA in U251 cells. These results indicate that malignant glioma cells express all types of syndecans and suggest that NF-kappaB participates in the upregulation of the syndecan-1 expression at the transcriptional level, and increased expression of syndecan-1 could associate with extracellular matrices including thrombospondin-1.

Hedgehog, FGF, VEGF, and Notch signaling pathways network together for vascular remodeling during embryogenesis and carcinogenesis. HHIP1 (HHIP) is an endogenous antagonist for SHH, IHH, and DHH. Here, comparative integromics analyses on HHIP family members were performed by using bioinformatics and human intelligence. HHIP1, HHIP2 (HHIPL1 or KIAA1822) and HHIP3 (HHIPL2 or KIAA1822L) constitute human HHIP gene family. Rat Hhip1, Hhip2, and Hhip3 genes were identified within AC107504.4, AC094820.6, and AC134264.2 genome sequences, respectively. HHIP-homologous (HIPH) domain with conserved 18 Cys residues was identified as the novel domain conserved among mammalian HHIP1, HHIP2, and HHIP3 orthologs. HHIP1 mRNA was expressed in coronary artery endothelial cells, prostate, and rhabdomyosarcoma. HHIP2 mRNA was expressed in trabecular bone cells. HHIP3 mRNA was expressed in testis, thyroid gland, osteoarthritic cartilarge, pancreatic cancer, and lung cancer. Promoters of HHIP family genes were not well conserved between human and rodents. Although GLI-, CSL-, and HES/HEY-binding sites were not identified, eleven bHLH-binding sites were identified within human HHIP1 promoter. Expression of HES/HEY family members, including HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2 and HEYL, in coronary artery endothelial cells was not detected in silico. Up-regulation of HHIP1 due to down-regulation of Notch-CSL-HES/HEY signaling cascade repressing bHLH transcription factors results in down-regulation of the Hedgehog-VEGF-Notch signaling cascade. On the other hand, down-regulation of HHIP1 due to up-regulation of Notch signaling in vascular endothelial cells during angiogenesis results in up-regulation of the Hedgehog-VEGF-Notch signaling cascade. Because HHIP1 is the key molecule for vascular remodeling, HHIP1 is the pharmacogenomics target in the fields of oncology and vascular medicine.

Background Information: The MIPs (major intrinsic proteins) constitute a large family of membrane proteins that facilitate the passive transport of water and small neutral solutes across cell membranes. Since water is the most abundant molecule in all living organisms, the discovery of selective water-transporting channels called AQPs (aquaporins) has led to new knowledge on both the physiological and molecular mechanisms of membrane permeability. The MIPs are identified in Archaea, Bacteria and Eukaryota, and the rapid accumulation of new sequences in the database provides an opportunity for large-scale analysis, to identify functional and/or structural signatures or to infer evolutionary relationships. To help perform such an analysis, we have developed MIPDB (database for MIP proteins), a relational database dedicated to members of the MIP family., Results: MIPDB is a motif-oriented database that integrates data on 785 MIP proteins from more than 200 organisms and contains 230 distinct sequence motifs. MIPDB proposes the classification of MIP proteins into three functional subgroups: AQPs, glycerol-uptake facilitators and aquaglyceroporins. Plant MIPs are classified into three specific subgroups according to their subcellular distribution in the plasma membrane, tonoplast or the symbiosome membrane. Some motifs of the database are highly selective and can be used to predict the transport function or subcellular localization of unknown MIP proteins., Conclusions: MIPDB offers a user-friendly and intuitive interface for a rapid and easy access to MIP resources and to sequence analysis tools. MIPDB is a web application, publicly accessible at http://idefix.univ-rennes1.fr:8080/Prot/index.html.

It is now well established that Toll-like receptors (TLRs) act as primary sensors of microbial compounds. Details of the molecular mechanisms governing TLR responses are emerging steadily and our understanding of the signaling pathways activated these receptors has improved greatly over the last few years. Differences in adaptor usage, cellular localisation and signaling cascades have been elucidated. In this review we will summarize the current understanding of TLR signaling and its regulation.

A functional zona pellucida is critical for both fertilization and the early stages of embryo development. Recent data from genomic and proteomic studies have questioned our simplistic view of the zona as being composed of three proteins whose functions are clearly defined. In the human, for example, the zona pellucida is composed of four proteins, not three. The increased complexity of the zona pellucida in humans and other species across the evolutionary tree now demands that we reconsider our reliance on the mouse model for understanding early fertilization events. Additionally, we are now well placed to examine, for the first time, potential defects in zona genes and their proteins associated with defined pathology.

Functional characterization of Toll-like receptors (TLRs) has established that innate immunity is a skillful system that detects invasion of microbial pathogens. Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune responses and further instruct development of antigen-specific acquired immunity. TLR signaling pathways are finely regulated by TIR domain-containing adaptors, such as MyD88, TIRAP/Mal, TRIF and TRAM. Differential utilization of these TIR domain-containing adaptors provides specificity of individual TLR-mediated signaling pathways. Several mechanisms have been elucidated that negatively control TLR signaling pathways, and thereby prevent overactivation of innate immunity leading to fatal immune disorders. The involvement of TLR-mediated pathways in autoimmune and inflammatory diseases has been proposed. Thus, TLR-mediated activation of innate immunity controls not only host defense against pathogens but also immune disorders.

The structural S-layer proteins of 28 different Corynebacterium glutamicum isolates have been analyzed systematically. Treatment of whole C. glutamicum cells with detergents resulted in the isolation of S-layer proteins with different apparent molecular masses, ranging in size from 55 to 66 kDa. The S-layer genes analyzed were characterized by coding regions ranging from 1,473 to 1,533 nucleotides coding for S-layer proteins with a size of 490-510 amino acids. Using PCR techniques, the corresponding S-layer genes of the 28 C. glutamicum isolates were all cloned and sequenced. The deduced amino acid sequences of the S-layer proteins showed identities between 69 and 98% and could be grouped into five phylogenetic classes. Furthermore, sequence analyses indicated that the S-layer proteins of the analyzed C. glutamicum isolates exhibit a mosaic structure of highly conserved and highly variable regions. Several conserved regions were assumed to play a key role in the formation of the C. glutamicum S-layers. Especially the N-terminal signal peptides and the C-terminal anchor sequences of the S-layer proteins showed a nearly perfect amino acid sequence conservation. Analyses by atomic force microscopy revealed a committed hexagonal structure. Morphological diversity of the C. glutamicum S-layers was observed in a class-specific unit cell dimension (ranging from 15.2 to 17.4 nm), which correlates with the sequence similarity-based classification. It could be demonstrated that differences in the primary structure of the S-layer proteins were reflected by the S-layer morphology.

Interleukin-1 is a key inflammatory cytokine that mediates its effects through a type I receptor and a receptor accessory protein. These two molecules are members of a wider family of proteins that have in common the presence of immunoglobulin domains in the extracellular region of the protein and a TIR domain in the cytoplasmic region. The nature of this family of proteins and their signal transduction pathway is discussed in this review.

Intracellular membrane traffic is governed by a conserved set of proteins, including Syts (synaptotagmins). The mammalian Syt family includes 15 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium-dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison, together with functional studies of rat Syt C2ABs, to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Nearly half of the Syt family does not exhibit calcium/phospholipid binding in comparison to Syt I, the major brain isoform. Syts do, however, possess a more conserved function, namely calcium-independent binding to target SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) heterodimers. All tested isoforms, except Syt XII and Syt XIII, bound the target SNARE heterodimer comprising syntaxin 1 and SNAP-25 (25 kDa synaptosome-associated protein). Our present study suggests that many Syt isoforms can function in membrane trafficking to interact with the target SNARE heterodimer on the pathway to calcium-triggered membrane fusion.

Toll receptors are type I transmembrane proteins that play important roles in development and immunity in animals. Comparison of the genomes of mouse and human on one side and of the fruitfly Drosophila and the mosquito Anopheles (two dipteran insects) on the other, revealed that the four species possess a similar number of Toll receptors (approximately 10). However, phylogenetic analyses indicate that the families of Toll receptors expanded independently in insects and mammals. We review recent results on these receptors, which point to differences in the activation and signaling between Tolls in insects and Toll-like receptors (TLRs) in mammals. Whereas mammalian TLRs appear to be solely dedicated to host-defense, insect Tolls may be predominantly linked to other functions, probably developmental.

PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor 3 (TLR3), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR3 expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR3 was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (Interferon-alpha/beta receptor) suppressed MV-induced TLR3 induction, indicating that type I IFN, IFN-alpha/beta, is critical for MV-mediated TLR3 induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR3 expression. A virus-responsive element that up-regulates TLR3 was identified in the TLR3-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -30bp of TLR3 promoter responded to MV to induce TLR3 expression. This further indicates the importance of type I IFN for TLR3 up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR3 had been induced beforehand. Thus, the MV-induced expression of TLR3 may reflect amplified IFN production that plays a part in host defense to viral infection.

Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.

Purpose of Review: The lung has a unique relationship with the environment. Through evolution the lung has developed strategies to defend itself from microbial invasion. As we encounter increasing multidrug-resistant microorganisms, we need to further our knowledge of innate defense systems in order to design novel strategies to deal with these microbes without inducing over-exuberant inflammation and lung injury., Recent Findings: The development of lung innate immunity requires microbial molecular pattern recognition by the recently described Toll like receptors, the release of early response cytokines that further activate the 'master switch', nuclear factor-kappaB, leading to amplified host defense to invading microbes. A balance of Type 1 and Type 2 cytokines modulates the intensity of innate immunity. Cytokines/chemokines orchestrate the polarization and transition of innate to adaptive immunity., Summary: The elucidation of the pathways involved in innate immunity and factors controlling the transition to adaptive immunity will improve our understanding of the host response to infection and improve our ability to design new therapies for the treatment of infectious disease.

The Na(+) and voltage-dependence of transient rabbit Na(+)/glucose cotransporter (rSGLT1) kinetics was studied with the two-electrode voltage-clamp technique and Xenopus laevis oocytes. Using step changes in membrane potential, in the absence of glucose but with 100 or 10 mM Na(+), transient currents were measured corresponding to binding/debinding of Na(+) and conformational changes of the protein. Previously, only a single time constant has been published for rSGLT1. We, however, observed three decay components; a fast (tau(f), 0.5-1 ms) voltage- and Na(+)-independent decay, and medium (tau(m), 0.5-4 ms) and slow (tau(s), 8-50 ms) voltage- and Na(+)-dependent decays. Transient currents were simulated and fit using a four-state model to obtain kinetic parameters for the system. The four-state model was able to reconstitute an assortment of experimental data.

Purpose of Review: Toll-like receptors (TLRs) are a family of pattern recognition receptors that are activated by specific components of microbes and certain host molecules. They constitute the first line of defense against many pathogens and play a crucial role in the function of the innate immune system. Recently, TLRs were observed to influence the development of adaptive immune responses, presumably by activating antigen-presenting cells. This has important implications for our understanding of how the host tailors its immune response as a function of specific pathogen recognition. The present review discusses the recent studies that demonstrate the role of TLRs in the regulation of adaptive T-helper-1 (Th1) and Th2 responses, and the mechanisms by which the effects are carried out., Recent Findings: Most studies have focused on the role of TLRs and components of their signaling pathways in the control of Th1-type immune responses, and on the implications for their use as antimicrobial agents, such as adjuvants in vaccines, or to treat or prevent the Th2-type dominated immune responses seen in allergies. TLR-deficient mice have been described and used to come to these conclusions. Although controversial, there is also evidence that TLRs may be important for Th2-type responses, possibly by augmenting the overall maturity of dendritic cells., Summary: A greater understanding of the processes by which TLRs regulate adaptive immunity may yield not only improved ways to treat infectious diseases but also new approaches to the treatment and prevention of allergic and certain autoimmune disorders.

Toll receptors in insects, mammals and plants are key players that sense the invasion of pathogens. Toll-like receptors (TLRs) in mammals have been established to detect specific components of bacterial and fungal pathogens. Furthermore, recent evidence indicates that TLRs are involved in the recognition of viral invasion. Signalling pathways via TLRs originate from the conserved Toll/IL-1 receptor (TIR) domain. The TIR domain-containing MyD88 acts as a common adaptor that induces inflammatory cytokines; however, there exists a MyD88-independent pathway that induces type I IFNs in TLR4 and TLR3 signalling. Another TIR domain-containing adaptor, TIRAP/Mal has recently been shown to mediate the MyD88-dependent activation in the TLR4 and TLR2 signalling pathway. Thus, individual TLRs may have their own signalling systems that characterize their specific activities.

Synaptotagmin I is the leading candidate for the calcium sensor that triggers exocytosis at conventional synapses. However, physiological characterization of the calcium sensor for phasic release at the ribbon-style synapses of the goldfish Mb1 bipolar cell demonstrates a lower than predicted affinity for calcium, suggesting that a modified or different sensor triggers exocytosis at this synapse. We examined synaptotagmin immunolabeling in goldfish retina using two different antibodies directed against synaptotagmin epitopes that specifically labeled the expected 65-kDa protein on western blots of goldfish and mouse retinal membranes. The first antiserum strongly labeled conventional synapses in the inner plexiform layer (IPL), but did not label the ribbon-style synapse-containing synaptic terminals of goldfish Mb1 bipolar cells or photoreceptors. The second antibody also specifically labeled the expected 65-kDa protein on western blots but did not label any synapses in the goldfish retina. A third synaptotagmin antibody that performed poorly on western blots selectively labeled goldfish photoreceptor terminals. These results suggest that synaptotagmin may exist in at least three distinct "forms" in goldfish retinal synapses. These forms, which are differentially localized to conventional synapses, bipolar cell, and photoreceptor terminals, may represent differences in isoform, posttranslational modifications, epitope availability, and protein-binding partners. Labeling with these antibodies in the salamander and mouse retina revealed species-specific differences, indicating that synaptotagmin epitopes can vary across species as well as among synapses.

To examine the role of contemporary selection in maintaining significant allele frequency differences at the pantophysin (PanI) locus among populations of the Atlantic cod, Gadus morhua, in northern Norway, we sequenced 127 PanIA alleles sampled from six coastal and two Barents Sea populations. The distributions of variable sites segregating within the PanIA allelic class were then compared among the populations. Significant differences were detected in the overall frequencies of PanIA alleles among populations within coastal and Arctic regions that was similar in magnitude to heterogeneity in the distributions of polymorphic sites segregating within the PanIA allelic class. The differentiation observed at silent sites in the PanIA allelic class contradicts the predicted effects of widescale gene flow and suggests that postsettlement selection acting on cohorts cannot be responsible for the genetic differences described between coastal and Arctic populations. Our results suggest that the marked differences observed between coastal and Arctic populations of G. morhua in northern Norway at the PanI locus reflect the action of recent diversifying selection and that populations throughout the region may be more independent than suggested by previous studies.

Genes with a role in fertilization show a common pattern of rapid evolution. The role played by positive selection versus lack of selective constraints has been more difficult to establish. One problem arises from attempts to detect selection in an overall gene sequence analysis. I have analyzed the pattern of molecular evolution of fertilin, a gene coding for a heterodimeric sperm protein belonging to the ADAM (A disintegrin and A metalloprotease) gene family. A nonsynonymous to synonymous rate ratio (d(N)/d(S)) analysis for different protein domains of fertilin alpha and fertilin beta showed d(N)/d(S) < 1, suggesting that purifying selection has shaped fertilin's evolution. However, an analysis of the distribution of single positively selected codon sites using phylogentic analysis by maximum likelihood (PAML) showed sites within adhesion domains (disintegrin and cysteine-rich) of fertilin beta evolving under positive selection. The region 3' to the EGF-like domain of fertilin alpha, where the transmembrane and cytoplasmic tail regions are supposed to be localized, showed higher d(N) and d(S) than any other fertilin alpha region. However, it was not possible to identify positively selected codon sites due to ambiguous alignments of the carboxy-end region (ClustalX vs. DiAlign2). When this region was excluded from the PAML analysis, most single positively selected codon sites were concentrated within adhesion domains (cysteine-rich and EGF-like). The use of an ancestral sequence prior to a recent duplication event of fertilin alpha among non-Hominidae primates (Macaca, Papio, and Saguinus) revealed that the duplication is partially responsible for masking the detection of positively selected sites within the disintegrin domain. Finally, most ADAM genes with a potential role in sperm maturation and/or fertilization showed significantly higher d(N) estimates than other ADAM genes.

Activity of the taurine transporter (TAUT) is regulated by signal transduction in response to diverse stimuli including tumor promoters such as phobol ester. Regulation of the transcription rate of TAUT appears to play an important role in exerting biological roles of taurine in mammalian tissues in adverse environments. Although cDNA of human TAUT has been cloned and sequenced in placenta, thyroid cells, and retinal pigment epithelial cells, the promoter region of TAUT has never been reported. In order to clone the upstream region of the human TAUT promoter, we have compared TAUT cDNA sequences with the entire human genome sequence. Polymerase chain reaction (PCR) was performed from genomic DNA prepared from a SK-Hep-1 cell line for the amplification of the TAUT promoter region including the partial exon (150 bp) and the 5' untranslated region (UTR, 380 bp). The PCR product of the promoter region, which was 1800 bp long, was ligated into the pGEM-T vector, and sequenced. The 5' flanking region of the TAUT promoter was analysed for the identification of enhancer and regulation motifs. Surprisingly we found the consensus TPA responsive element (TGAGTCAG) which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway. The well known fact that proto-oncogene AP1 (cFos/cJun heterodimer or cJun/cJun homodimer) binds to TRE implies that TAUT expression might be closely linked to tumor promotion. Since AP1 activity is also tightly regulated in nerve cells, AP1-regulated TAUT transcription might be an important step in nerve cell function. Furthermore, the TFIID binding site, cap signal for transcription initiation, PEA3 motif, heat shock factor binding motif, and many other motifs were found in the TAUT promoter region, and require characterization.

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.