Degree Of Amino Acid Homology Research Articles

Determinants for degradation of SAMHD1, Mus81 and induction of G2 arrest in HIV-1 Vpr and SIVagm Vpr

Vpr and Vpx are a group of highly related accessory proteins from primate lentiviruses. Despite the high degree of amino acid homology within this group, these proteins can be highly divergent in their functions. In this work, we constructed chimeric and mutant proteins between HIV-1 and SIVagm Vpr in order to better understand the structure–function relationships. We tested these constructs for their abilities to induce G2 arrest in human cells and to degrade agmSAMHD1 and Mus81. We found that the C-terminus of HIV-1 Vpr, when transferred onto SIVagm Vpr, provides the latter with the de novo ability to induce G2 arrest in human cells. We confirmed that HIV-1 Vpr induces degradation of Mus81 although, surprisingly, degradation is independent and genetically separable from Vpr׳s ability to induce G2 arrest.

DPOFA, a Cl-/HCO3 -exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium

BackgroundRetinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned drug capable of inhibiting Cl-/HCO3- exchanger activity. We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE.MethodsReverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as Cl-/HCO3- exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl-/HCO3- exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE.ResultsUsing RT-PCR we showed that three isoforms (SLC4A2, SLC4A3, and SLC26A6) are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants.ConclusionsDPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system.

Three tubulin genes of Trichoderma harzianum: alpha, beta and gamma

Three tubulin genes of Trichoderma harzianum were cloned followed genomic walking procedure. The tubulins showed high degree of amino acid homology with other fungal tubulins and were homologous with each other with 32 to 38% amino acid identity. Three measures for the degree of codon usage bias indicated the presence of bias in all the sequences, suggesting high expression levels in all the genes. Protein structures were modeled to provide the basis for understanding the tubulin's properties and its interactions with microtubule-associated proteins. Potential motifs were also postulated.

The Role of ABC Transporters in Protecting Cells from Bilirubin Toxicity

The ATP-binding cassette (ABC) superfamily is the largest transporter family known to translocate a wide variety of exogenous and endogenous substrates across cell membranes. In this chapter we review the potential role of three ABC proteins in the transport of unconjugated bilirubin (UCB). These transporters are MRP1, MRP3 and PGP (MDR1). MRP1 is expressed at high levels in most epithelia, usually at the basolateral membrane. Among a multiplicity of substrates, MRP1 mediates the ATP-dependent cellular export of UCB, and its role has been demonstrated in protecting cells from UCB toxicity. MRP3 is an organic anion transporter whose major substrates are GSH conjugates of organic compounds. Among the MRP family members, MRP3 shares the highest degree of amino acid homology with MRP1. Although the hepatic expression of MRP3 has been reported to be up-regulated by bilirubin and bilirubin glucuronides, it is unknown whether MRP3 is also involved in the transport of UCB. PGP is expressed in organs involved in the elimination of endo- and xenobiotics and UCB is one of these substrates. Since the Km of PGP for UCB is well above pathophysiological levels of Bf, it remains uncertain whether it has a role in protecting against UCB cytotoxicity.

The Yersinia pseudotuberculosis YplA phospholipase differs in its activity, regulation and secretion from that of the Yersinia enterocolitica YplA

Analysis of the Yersinia pseudotuberculosis and Yersinia pestis genomes indicates that both species carry an identical copy of a gene that is predicted to encode a protein which shares 80% similarity to the Yersinia enterocolitica YplA, a secreted phospholipase that has been shown to contribute to virulence. In contrast to well tolerated production of the Y. enterocolitica YplA in Escherichia coli, Y. pseudotuberculosis YplA expression was found to be toxic; however, cell viability could be restored if the Y. pseudotuberculosis YplA was expressed in the presence of its accessory protein YplB. In vitro, Y. pseudotuberculosis YplB was shown to reduce the activity of its cognate phospholipase in a dose-dependent manner. To determine whether the Y. pseudotuberculosis and Y. enterocolitica YplAs were secreted and regulated in a similar manner, secretion and promoter activity assays were performed. Unlike the situation apparent in Y. enterocolitica, expression of the Y. pseudotuberculosis yplA gene did not appear to be controlled by the flagellar regulon, nor did the phospholipase appear to be efficiently exported through the flagellar apparatus. These results indicate that the Yersinia YplAs vary in many of their attributes despite their high degree of amino acid homology.

Byssochlamys nivea with patulin-producing capability has an isoepoxydon dehydrogenase gene ( idh) with sequence homology to Penicillium expansum and P. griseofulvum

Nucleotide sequences of the isoepoxydon dehydrogenase gene ( idh) for eight strains of Byssochlamys nivea were determined by constructing GenomeWalker libraries. A striking finding was that all eight strains of B. nivea examined had identical nucleotide sequences, including those of the two introns present. The length of intron 2 was nearly three times the size of introns in strains of Penicillium expansum and P. griseofulvum, but intron 1 was comparable in size to the number of nucleotides present in introns 1 and 2 of P. expansum and P. griseofulvum. A high degree of amino acid homology (88 %) existed for the idh genes of the strains of B. nivea when compared with sequences of P. expansum and P. griseofulvum. There were many nucleotide differences present, but they did not affect the amino acid sequence because they were present in the third position. The identity of the B. nivea isolates was confirmed by sequencing the ITS/partial LSU (28 S) rDNA genes. Four B. nivea strains were analysed for production of patulin, a mycotoxin found primarily in apple juice and other fruit products. The B. nivea strains produced patulin in amounts comparable to P. expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide.

Both regions 4.1 and 4.2 of E. coli σ 70 are together required for binding to bacteriophage T4 AsiA in vivo

The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing − 10 and − 35 consensus sequence by binding to σ 70 of E. coli. Binding of AsiA to σ 70 in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of σ 70 binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of σ 70 has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4.2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of σ 70 showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in σ 70 fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only σ 70 fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to σ 70, in vivo, requires the presence of both sub-domains of region 4 of σ 70.

Fungal heat-shock proteins in human disease

Heat-shock proteins (hsps) have been identified as molecular chaperones conserved between microbes and man and grouped by their molecular mass and high degree of amino acid homology. This article reviews the major hsps of Saccharomyces cerevisiae, their interactions with trehalose, the effect of fermentation and the role of the heat-shock factor. Information derived from this model, as well as from Neurospora crassa and Achlya ambisexualis, helps in understanding the importance of hsps in the pathogenic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Trichophyton rubrum, Phycomyces blakesleeanus, Fusarium oxysporum, Coccidioides immitis and Pneumocystis jiroveci. This has been matched with proteomic and genomic information examining hsp expression in response to noxious stimuli. Fungal hsp90 has been identified as a target for immunotherapy by a genetically recombinant antibody. The concept of combining this antibody fragment with an antifungal drug for treating life-threatening fungal infection and the potential interactions with human and microbial hsp90 and nitric oxide is discussed.

Na+/Ca2+ Exchanger Activity Modulates Connective Tissue Growth Factor mRNA Expression in Transforming Growth Factor β1- and Des-Arg10-kallidin-stimulated Myofibroblasts

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.

Human Cytomegalovirus UL84 Is a Phosphoprotein That Exhibits UTPase Activity and Is a Putative Member of the DExD/H Box Family of Proteins

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 degrees C. The enzyme preferred Mg2+ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn2+, Ca2+, and Zn2+ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.

Phylogenetic and immunological definition of four lipoylated proteins from Novosphingobium aromaticivorans, implications for primary biliary cirrhosis

Novosphingobium aromaticivorans, a unique ubiquitous bacterium that metabolizes xenobiotics and activates environmental estrogens, has been suggested as a pathogenic factor in the development of primary biliary cirrhosis (PBC). To define the molecular basis of PBC sera reactivity, we investigated the characteristic of the bacterial antigens involved. We cloned and sequenced four genes from N. aromaticivorans coding for immunoreactive proteins, arbitrarily named Novo 1 through Novo 4. We subsequently analyzed these proteins for their homology to known mitochondrial proteins and defined their reactivity using monoclonal antibodies (mAbs), rabbit anti-lipoic acid antibody, and PBC/control sera. Moreover, we studied their phylogenetic relation with the known PBC autoantigens. Novo proteins have an extraordinary degree of amino acid homology with all of the major human mitochondrial autoantigens PDC-E2 (Novo 1 and 2), OGDC-E2 (Novo 3), and BCOADC-E2 (Novo 4). Moreover, Novo 1–4 contain a lipoylated domain, are recognized by AMA-positive sera, and react with specific mAbs to mitochondrial antigens. Interestingly, the phylogenetic relation of the proteins emphasizes the conservation of the lipoylated domain. In conclusion, our data provide a high degree of confidence that N. aromaticivorans may potentiate the breakdown of self tolerance in genetically susceptible individuals.

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival.

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.

CDNA cloning and sequence of European sea bass ( Dicentrarchus labrax) somatolactin

Three cDNA clones encoding for European sea bass somatolactin (SL) were obtained by RT-PCR and 3′ RACE of RNA of pituitary origin. Clone 1 was 582 bp in length, and included a part of the signal peptide and the 5′ end of the mature protein. Clone 2 (1075 bp) included a fragment of the coding sequence and the 3′ untranslated region, which was 888 bp in length and contained two putative polyadenylation signals (AATAAA) at 12–17, and 202–207 nucleotides upstream of the poly (A) tail. Clone 3 was 624 bp in length and its nucleotide sequence encoding for the entire mature protein confirmed the sequence already determined from the first two clones. The size of SL mRNA transcripts was estimated by Northern blot analysis and a single band of approximately 1.6 kb was observed with pituitary RNAs. No band was found with RNAs of brain and liver origin. Alignment of the deduced amino acid sequence revealed that European sea bass SL shared 90–84% identity with perciform, pleuronectiform and scorpaeniform fish SLs, and 77–57% with other SLs of more distant fish orders, with a strict conservation of Cys residues and the N-glycosylation site (Asn–Lys–Thr) at 121–123 amino acid positions. The reconstruction of the phylogenetic tree based on SL nucleotide sequences, and analyzed by maximum likelihood distances, showed the same clustering as the present hierarchy of fish. When comparisons were made among SL, prolactin and growth hormone of European sea bass, the overall amino identity was relatively low (22–23%). However, a high degree of amino acid homology was found at the C-terminus, which contains three of the four Cys residues strictly conserved in all the members of GH/PRL family.

Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors.

Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease: characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M1 receptors

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M1 muscarinic agonists. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M1 muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., El-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230–1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonists to M1 receptors [Huang, X.P., Nagy, P.I., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735–745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M1 receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129–1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M1 receptor affinity, selectivity and activity.

The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens.

The human estrogen receptor alpha (ERalpha) and the recently identified ERbeta share a high degree of amino acid homology; however, there are significant differences in regions of these receptors that would be expected to influence transcriptional activity. Consequently, we compared the mechanism(s) by which these receptors regulate target gene transcription, and evaluated the cellular consequences of coexpression of both ER subtypes. Previously, it has been determined that ERalpha contains two distinct activation domains, ERalpha-AF-1 and ERalpha-AF-2, whose transcriptional activity is influenced by cell and promoter context. We determined that ERbeta, like ERalpha, contains a functional AF-2, however, the ERbeta-AF-2 domain functions independently within the receptor. Of additional significance was the finding that ERbeta does not contain a strong AF-1 within its amino-terminus but, rather, contains a repressor domain that when removed, increases the overall transcriptional activity of the receptor. The importance of these findings was revealed when it was determined that ERbeta functions as a transdominant inhibitor of ERalpha transcriptional activity at subsaturating hormone levels and that ERbeta decreases overall cellular sensitivity to estradiol. Additionally, the partial agonist activity of tamoxifen manifest through ERalpha in some contexts was completely abolished upon coexpression of ERbeta. In probing the mechanisms underlying ERbeta-mediated repression of ERalpha transcriptional activity we have determined that 1) ERalpha and ERbeta can form heterodimers within target cells; and 2) ERbeta interacts with target gene promoters in a ligand-independent manner. Cumulatively, these data indicate that one role of ERbeta is to modulate ERalpha transcriptional activity, and thus the relative expression level of the two isoforms will be a key determinant of cellular responses to agonists and antagonists.

The Estrogen Receptor -Isoform (ER ) of the Human Estrogen Receptor Modulates ER Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens

The human estrogen receptor α (ERα) and the recently identified ERβ share a high degree of amino acid homology; however, there are significant differences in regions of these receptors that would be expected to influence transcriptional activity. Consequently, we compared the mechanism(s) by which these receptors regulate target gene transcription, and evaluated the cellular consequences of coexpression of both ER subtypes. Previously, it has been determined that ERα contains two distinct activation domains, ERα-AF-1 and ERα-AF-2, whose transcriptional activity is influenced by cell and promoter context. We determined that ERβ, like ERα, contains a functional AF-2, however, the ERβ-AF-2 domain functions independently within the receptor. Of additional significance was the finding that ERβ does not contain a strong AF-1 within its amino-terminus but, rather, contains a repressor domain that when removed, increases the overall transcriptional activity of the receptor. The importance of these findings was revealed when it was determined that ERβ functions as a transdominant inhibitor of ERα transcriptional activity at subsaturating hormone levels and that ERβ decreases overall cellular sensitivity to estradiol. Additionally, the partial agonist activity of tamoxifen manifest through ERα in some contexts was completely abolished upon coexpression of ERβ. In probing the mechanisms underlying ERβ-mediated repression of ERα transcriptional activity we have determined that 1) ERα and ERβ can form heterodimers within target cells; and 2) ERβ interacts with target gene promoters in a ligand-independent manner. Cumulatively, these data indicate that one role of ERβ is to modulate ERα transcriptional activity, and thus the relative expression level of the two isoforms will be a key determinant of cellular responses to agonists and antagonists.

Immunocytochemical localization of the Ya, Yb1, Yc, Yf, and Yo subunits of glutathione S-transferases in the cauda epididymidis and vas deferens of adult rats.

Glutathione S-transferases (GSTs) are dimeric proteins grouped into five classes based on the degree of amino acid homology of their subunits. They are involved in cellular detoxification through the catalyzation of the conjugation of reduced glutathione with various electrophilic substances. In the present study, the distribution of Ya and Yc subunits from the alpha family, Yb1 and Yo subunits of the mu class, and the Yf subunit of the pi class were examined with light microscope immunocytochemistry in Bouin-fixed, paraffin-embedded tissue of different regions of the cauda epididymidis and vas deferens. In the cauda, principal cells showed high levels of expression of Ya, Yc, and Yo subunits, while in the vas deferens, staining decreased to moderate levels for the Ya and Yo subunits and to low levels for the Yc subunit. While Yf was maintained at low levels in principal cells of all cauda and vas deferens regions, Yb1 expression was more erratic, presenting a checkerboard-like staining pattern in the proximal vas deferens and showing moderate cytoplasmic but intense nuclear reactivity in all other regions. Basal cells in the cauda were intensely reactive for Yf, while in the vas deferens, they became unreactive. Conversely, basal cells were unreactive for Ya in the cauda and proximal vas deferens, while in the middle and distal vas deferens, they became moderately reactive. In the case of Yb1 and Yo, some basal cells were reactive while others appeared unreactive in all cauda and vas deferens regions. Yc elicited the display of both reactive and unreactive basal cells in the cauda regions, and while the cells were moderately reactive in the proximal vas deferens, they became intensely reactive in the middle and distal vas deferens. In summary, both principal and basal cells show varying degrees of GST expression in the different regions of the cauda and vas deferens, suggesting that these cells are subjected to a complex, changing environment of substrates. Furthermore, while expression often differs from principal to basal cells, the absence of reactivity of a given GST in one cell type is usually compensated for by expression in the other cell type in any given region of the cauda or vas deferens. Taken together, the data suggest that ample protection from harmful circulating electrophiles can be provided for sperm during their storage in the cauda and vas deferens. In addition, since principal cells of the vas deferens are involved in steroid synthesis, the presence of GSTs in these cells may also serve to bind steroids, or this presence may be involved in steroid isomerization.

Complete Nucleotide Sequence of the Japanese Isolate of Barley Yellow Dwarf Virus-PAV Serotype.

The complete nucleotide sequence of the genomic RNA of the Japanese isolate of barley yellow dwarf virus-PAV serotype (BYDV Jpn-PAV) was determined. The genome of BYDV Jpn-PAV was comprised of 5667 nucleotides and had six open reading frames (ORF 1 to ORF 6) on the virus sense strand. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of the Australian (Vic-PAV) and the North American (P-PAV) PAV isolates and the PS1 isolate of MAV serotype (MAV-PS1). Comparisons of the corresponding ORFs showed that the proposed RNA replicase domains (ORF 1 and ORF 2) of all the isolates shared more than 97% amino acid homology. Sequence comparison between Jpn-PAV and MAV-PS1 showed considerable dissimilarity in the ORF 3 to 6. Among the overall genomes of Jpn-PAV and two other PAV isolates, an apparent sequence diversity was found in the ORF 6 domain. The ORF 1 to 5 of Jpn-PAV shared a high degree of amino acid homology with those of Vic- and P-PAV isolates, whereas ORF 6 had only 61% and 78% identity with the respective ORF 6 of Vic-PAV and P-PAV. From sequence comparisons, Jpn-PAV appears to be more closely related to the North American and European PAV isolates than to the Australian isolates.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.