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Pancreatic β-cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the β cell, and here, we characterize its role in the regulation of β-cell function. Pim3 expression in the β-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the β cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3-/- mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3-/- mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3-/- mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3-/- islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating β-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3-/- islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the β cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell, 72, 971-983]. Despite identification of the gene in 1993, the underlying life-long disease process and effective treatments to prevent or delay it remain elusive. In an effort to fast-track treatment strategies for HD into clinical trials, we have developed a new large-animal HD transgenic ovine model. Sheep, Ovis aries L., were selected because the developmental pattern of the ovine basal ganglia and cortex (the regions primarily affected in HD) is similar to the analogous regions of the human brain. Microinjection of a full-length human HTT cDNA containing 73 polyglutamine repeats under the control of the human promotor resulted in six transgenic founders varying in copy number of the transgene. Analysis of offspring (at 1 and 7 months of age) from one of the founders showed robust expression of the full-length human HTT protein in both CNS and non-CNS tissue. Further, preliminary immunohistochemical analysis demonstrated the organization of the caudate nucleus and putamen and revealed decreased expression of medium size spiny neuron marker DARPP-32 at 7 months of age. It is anticipated that this novel transgenic animal will represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation. New sequence accession number for ovine HTT mRNA: FJ457100.

Type 2 diabetes (T2D) is characterized by elevated blood glucose levels owing to insufficient secretion and/or activity of the glucose-lowering hormone insulin. Glucagon-like peptide-1 (GLP-1) has received much attention as a new treatment for diabetes because of its multiple blood glucose-lowering effects, including glucose-dependent enhancement of insulin secretion, inhibition of gastric emptying, and promotion of the survival and growth of insulin-producing beta-cells. GLP-1, along with GLP-2 and oxyntomodulin, is produced in the intestinal L-cell via processing of proglucagon by prohormone convertase 1/3 (PC1/3), while in the pancreatic alpha-cell, coexpression of proglucagon and the alternate enzyme PC2 typically results in differential processing of proglucagon to yield glucagon. We used alginate-encapsulated alpha-cells as a model to evaluate continuous delivery of PC1/3- or PC2-derived proglucagon products. In high fat-fed and db/db mice, PC1/3-, but not PC2-expressing alpha-cells improved glucose handling and transiently lowered fasting glucose levels, suggesting that continuous delivery of PC1/3-derived proglucagon products via cell therapy may be useful for diabetes treatment. In addition, we show that long-term treatment with PC1/3-expressing, but not PC2-expressing, alpha-cells improved cold-induced thermogenesis in db/db mice, demonstrating a previously unappreciated effect of one or more PC1/3-derived alpha-cell products.

Objective: Glucagon, which raises blood glucose levels by stimulating hepatic glucose production, is produced in alpha-cells via cleavage of proglucagon by prohormone convertase (PC)-2. In the enteroendocrine L-cell, proglucagon is differentially processed by the alternate enzyme PC1/3 to yield glucagon-like peptide (GLP)-1, GLP-2, and oxyntomodulin, which have blood glucose-lowering effects. We hypothesized that alteration of PC expression in alpha-cells might convert the alpha-cell from a hyperglycemia-promoting cell to one that would improve glucose homeostasis., Research Design and Methods: We compared the effect of transplanting encapsulated PC2-expressing alpha TC-1 cells with PC1/3-expressing alpha TCDeltaPC2 cells in normal mice and low-dose streptozotocin (STZ)-treated mice., Results: Transplantation of PC2-expressing alpha-cells increased plasma glucagon levels and caused mild fasting hyperglycemia, impaired glucose tolerance, and alpha-cell hypoplasia. In contrast, PC1/3-expressing alpha-cells increased plasma GLP-1/GLP-2 levels, improved glucose tolerance, and promoted beta-cell proliferation. In GLP-1R(-/-) mice, the ability of PC1/3-expressing alpha-cells to improve glucose tolerance was attenuated. Transplantation of PC1/3-expressing alpha-cells prevented STZ-induced hyperglycemia by preserving beta-cell area and islet morphology, possibly via stimulating beta-cell replication. However, PC2-expressing alpha-cells neither prevented STZ-induced hyperglycemia nor increased beta-cell proliferation. Transplantation of alpha TCDeltaPC2, but not alpha TC-1 cells, also increased intestinal epithelial proliferation., Conclusions: Expression of PC1/3 rather than PC2 in alpha-cells induces GLP-1 and GLP-2 production and converts the alpha-cell from a hyperglycemia-promoting cell to one that lowers blood glucose levels and promotes islet survival. This suggests that alteration of proglucagon processing in the alpha-cell may be therapeutically useful in the context of diabetes.

For microarrays, the transition from research to clinical and diagnostic applications is well underway. Microarrays use a range of specific probes that are immobilized in known locations on a support matrix; this technique can measure levels of specific DNA, RNA and proteins, as well as carbohydrates and lipids. It is anticipated that analysis of these levels will lead to identification of biomarkers for the diagnosis, treatment and prognosis of a wide range of diseases. So far, this type of analysis has been particularly useful in clinical oncology, but the technology is being actively and successfully explored for diseases such as diabetes, endocrine tumors and endocrine modulators of tumors. There are now many commercial sources of microarrays, which have robust quality-control procedures in place. Progress will be enhanced when biomarkers can be established, statistical approaches can be refined and when we better understand the interactions of genes and of particular gene loci in disease progression.

Study Design: Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties., Objectives: To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner., Summary of Background Data: Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process., Methods: Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed., Results: Transfection was achieved successfully with 340% +/- 7% (mean +/- SD) relative telomerase activity in hTERT-transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions., Conclusions: Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency, or G1-cell cycle checkpoint control was evident.

RWJ-416457, an investigational pyrrolopyrazolyl-substituted oxazolidinone, inhibited the growth of linezolid-susceptible staphylococci, enterococci, and streptococci at concentrations of < or =4 microg/ml, generally exhibiting two- to fourfold-greater potency than that of linezolid. Time-kill studies demonstrated bacteriostatic effects for both RWJ-416457 and linezolid.

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.

The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic alpha-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel alpha-cell line (alphaTC-DeltaPC2) derived from PC2 homozygous null animals. The alphaTC-DeltaPC2 cells are shown to be similar to the well characterized alphaTC1-6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in alphaTC-DeltaPC2 leads to a complete block in the production of mature glucagon. Surprisingly, alphaTC-DeltaPC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70-71). Further analysis reveals that alphaTC-DeltaPC2 cells, unlike alphaTC1-6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that alphaTC-DeltaPC2 cells provide additional evidence for PC2 as the major convertase in alpha-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases.

Since porcine endogenous retroviruses (PERVs) can infect cultured human cells, they are a potential hazard to xenotransplantation. For this reason, endogenous retroviruses from the Westran (Westmead Hospital transplantation) inbred line of pigs were analyzed by using consensus primers for the type A and type B viruses to amplify 1.8-kb envelope gene fragments. After preliminary analysis with restriction enzymes KpnI and MboI, 31 clones were sequenced. Between types A and B, five recombinant clones were identified. Fifty-five percent of clones (17 of 31) had premature stop codons within the envelope protein-encoding region. Endogenous retroviruses in Westran pigs were physically mapped by fluorescence in situ hybridization (FISH) using PERV-A and PERV-B envelope clones as probes to identify at least 32 integration sites (19 PERV-A sites and 13 PERV-B sites). The chromosomal sites of integration in the Westran strain are quite different from those in the European Large White pig. The recombinant clones suggest that defective PERVs could become infective through recombination and further that PERVs might recombine with human endogenous retroviruses in xenotransplants.

Prohormone convertase 2 (PC2) plays an essential role in the processing of proglucagon to mature active glucagon in pancreatic alpha-cells (J Biol Chem 276:27197-27202, 2001). Mice lacking PC2 demonstrate multiple defects, including chronic mild hypoglycemia and dramatic hyperplasia of the pancreatic alpha-cells. To define the contribution of mature glucagon deficiency to the hypoglycemia and alpha-cell hyperplasia, we have attempted to correct the defects by delivery of exogenous glucagon by micro-osmotic pumps. Intraperitoneal delivery of 0.5 microg glucagon/h in PC2(-/-) mice resulted in the normalization of blood glucose concentrations. Islet remodeling through the loss of hyperplastic alpha-cells was evident by day 11 after pump implantation; by 25 days postimplantation, PC2(-/-) islets were indistinguishable from wild-type islets. These rapid changes were brought about by induction of apoptosis in the alpha-cell population. Morphological normalization of islets was also accompanied by marked downregulation of endogenous preproglucagon gene expression, but with little or no change in the level of preproinsulin gene expression. Exogenous glucagon delivery also normalized hepatic expression of the gluconeogenic enzyme PEPCK. These results demonstrate that the lack of mature glucagon in PC2(-/-) mice is responsible for the aberrant blood glucose levels, islet morphology, and gene expression, and they confirm the role of glucagon as a tonic insulin antagonist in regulating glycemia.

The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. From high-throughput screening of a chemical library, three novel inhibitors of the Escherichia coli MurA enzyme were identified: the cyclic disulfide RWJ-3981, the purine analog RWJ-140998, and the pyrazolopyrimidine RWJ-110192. When MurA was preincubated with inhibitor, followed by addition of UNAG and PEP, the 50% inhibitory concentrations (IC(50)s) were 0.2 to 0.9 microM, compared to 8.8 microM for the known MurA inhibitor, fosfomycin. The three compounds exhibited MICs of 4 to 32 microg/ml against Staphylococcus aureus; however, the inhibition of DNA, RNA, and protein synthesis in addition to peptidoglycan synthesis by all three inhibitors indicated that antibacterial activity was not due specifically to MurA inhibition. The presence of UNAG during the MurA and inhibitor preincubation lowered the IC(50) at least fivefold, suggesting that, like fosfomycin, the three compounds may interact with the enzyme in a specific fashion that is enhanced by UNAG. Ultrafiltration and mass spectrometry experiments suggested that the compounds were tightly, but not covalently, associated with MurA. Molecular modeling studies demonstrated that the compounds could fit into the site occupied by fosfomycin; exposure of MurA to each compound reduced the labeling of MurA by tritiated fosfomycin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to the MurA enzyme, at or near the site where fosfomycin binds.

Amidino benzimidazoles have been identified as inhibitors of the bacterial KinA/Spo0F two-component system (TCS). Many of these inhibitors exhibit good in vitro antibacterial activity against a variety of susceptible and resistant Gram-positive organisms. The moiety at the 2-position of the benzimidazole was extensively modified. In addition, the regioisomeric benzoxazoles, heterocyclic replacements for the benzimidazole, have been synthesized and their activity against the TCS evaluated.

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans approximately 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at approximately day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.

Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5' to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases.

Mammals possess multiple, closely linked beta-globin genes that differ in the timing of their expression during development. These genes have been thought to be derived from a single ancestral gene, by duplication events that occurred after the separation of the mammals and birds. We report the isolation and characterization of an atypical beta-like globin gene (omega-globin) in marsupials that appears to be more closely related to avian beta-globin genes than to other mammalian beta-globin genes, including those previously identified in marsupials. Phylogenetic analyses indicate that omega-globin evolved from an ancient gene duplication event that occurred before the divergence of mammals and birds. Furthermore, we show that omega-globin is unlinked to the previously characterized beta-globin gene cluster of marsupials, making this the first report of an orphaned beta-like globin gene expressed in a vertebrate.

The Eph family of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and "native" mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

Pancreatic beta cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of beta cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the alpha subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the beta cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased urea production under low-glucose conditions. The above results demonstrate genome-wide integration of beta-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the beta-cell glucose response.

The Drosophila melanogaster small optic lobes gene (sol) is required for normal development of the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein products of sol and its human homologue SOLH contain zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal domain of unknown function. Long-distance PCR was used to amplify genomic DNA for Solh, the mouse homologue of sol, following the identification of mouse Solh expressed sequence tags. The nucleotide sequence of the Solh coding region (6.0 kb) was determined. The predicted Solh protein of 1095 amino acid residues shows 89% identity (93% similarity) to the human homologue. Solh was localized by in situ hybridization to band A3.3 on mouse Chromosome 17, in a region of maintained homology with human 16p13.3. Antipeptide antibodies were prepared and verified by demonstration of specific reactivity with recombinant human SOLH protein prepared by in vitro transcription/translation and expression in insect cells using the baculovirus system. The antibodies were used to show that the Solh protein localizes to the olfactory bulb in mouse and rat brain, suggesting that it could have an analogous role in development of sensory system neurons in Drosophila and in mammals., (Copyright 2000 Academic Press.)

We have isolated and sequenced full-length cDNA clones for leptin in the dasyurid marsupial Sminthopsis crassicaudata (fat-tailed dunnart). Southern and in situ hybridisation data indicated a single leptin gene in the S. crassicauda- ta genome, localised to arbitrary chromosome bands 5q24--> q31 on the long arm of chromosome 5, the short-arm terminus of which bears the only nucleolar organising region. The nucleotide sequence of the cDNAs revealed that the primary translation product of S. crassicaudata leptin is composed of 167 amino acid residues, with a potential signal peptide of 21 residues. The mature protein of 146 amino acids is 82% similar to both the mouse and human proteins and is predicted to have a molecular weight of 16.26 kDa. Northern blot analysis revealed that the corresponding mRNA is approximately 3.9 kb in size and is expressed only in white adipose tissue of this marsupial species. Evolutionary analyses indicate that S. crassicaudata leptin cDNA has evolved at a significantly faster rate than cDNAs from eutherian mammals., (Copyright 2000 S. Karger AG, Basel)

Arginine vasopressin interacts with the vasopressin type 1a receptor (V1aR) to initiate physiological effects such as vasoconstriction of blood vessels and glycogenolysis. AVP is also involved in central nervous effects such as body homeostasis and blood pressure control. The complete genomic organization of the sheep V1aR gene has been determined, including the presence of one major and two minor transcriptional start sites at -321, -206 and -91bp respectively, relative to the ATG codon. Another more distal minor transcriptional start site was also localized between nucleotides -997 and -892 relative to the ATG codon. One intron exists in the sheep V1aR gene and potential cis- and trans- acting sites were identified in the sheep V1aR promoter. The promoter was also compared to the rat V1aR promoter. The sheep V1aR promoter displays features typical of housekeeping genes, although tissue-specific expression does not support this. V1aR mRNA is absent in the adult sheep liver but not the kidney. One copy of the V1aR gene exists in the sheep genome, which was localized to chromosome 3q23-24, and to the homoeologous position, 5q23-24 in cattle.

A new class of inhibitors of the two-component regulatory systems (TCS) of bacteria was discovered based on the salicylanilide screening hits, closantel (1) and tetrachlorosalicylanilide (9). A systematic SAR study versus a model TCS, KinA/Spo0F, demonstrated the importance of electron-attracting substituents in the salicyloyl ring and hydrophobic groups in the anilide moiety for optimal activity. In addition, derivatives 8 and 16, containing the 2, 3-dihydroxybenzanilide structural motif, were potent inhibitors of the autophosphorylation of the KinA kinase, with IC50s of 2.8 and 6. 3 &microM, respectively. Compound 8 also inhibited the TCS mediating vancomycin resistance (VanS/VanR) in a genetically engineered Enterococcus faecalis cell line at concentrations subinhibitory for growth. Closantel (1), tetrachlorosalicylanilide (9), and several related derivatives (2, 7, 10, 11, 20) had antibacterial activity against the drug-resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

This SAR study has shown that the salicylanilide is the pharmacophore for inhibition of the bacterial two-component system. Hydrophobic substituents improve the potency of inhibitors in this series; however, hydrophobicity is not the sole determinant for inhibition; structural and electronic requirements also exist. Closantel (1) was found to inhibit a two-component system and to have antibacterial activity against drug resistant S. aureus and E. faecium.

A series of diaryltriazole analogs was discovered to inhibit bacterial two-component regulatory systems in our primary assays, KinA/Spo0F and NRII/NRI. They also showed inhibitory activity in whole cell mechanism-based assays, and they possessed potent activities against several strains of Gram-positive pathogenic bacteria in the standard MIC broth assay.

Mutations in the Drosophila melanogaster small optic lobes (sol) gene cause a sever reduction in the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein product of sol contains zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal region of unknown function. We have isolated human brain cDNA for SOLH, a human homologue of sol. The human SOLH gene consists of 14 exons distributed over more than 45 kb of genomic DNA. The encoded SOLH protein of 1086 amino acids has strong similarity to the D. melanogaster protein. The calpain-like domain and C-terminal region are highly conserved (58% identity), and similar Cys2-Cys2 zinc fingers are present in the N-terminal region. A reported Caenorhabditis elegans homologue contains the calpain domain and C-terminal region, but appears to lack the zinc finger region. A single copy of the zinc finger sequence is present in adjacent C. elegans genomic cosmid DNA sequence, and we show that it is part of the C. elegans sol-like transcript. Northern analysis of human tissues revealed a SOLH transcript of approximately 5 kb that was strongest in human brain. We have mapped the SOLH gene to chromosome 16p13.3 by in situ hybridization. SOLH is a candidate gene for CATM (hereditary cataracts with microphthalmia), which maps in this region.

The Gbx2 homeodomain is widely conserved in metazoans. We investigated the mouse Gbx2 locus by isolation and characterization of genomic clones and by physical localization to the genome. The Gbx2 gene contained a single intron that separated the proposed functional protein domains. This organization was conserved with human GBX2. Physical localization of Gbx2 to Chromosome 1C5-E1 indicated that the genomic relationship between the linked Gbx2 and En1 genes differs between mouse and human, making it unlikely to be of functional significance. We also extended the known expression pattern of Gbx2 beyond the gastrulation stage embryo and the developing CNS to pluripotent cells in vitro and in vivo. Gbx2 expression was demonstrated in undifferentiated embryonic stem cells but was downregulated in differentiated cell populations. In the embryo, Gbx2 expression was detected before primitive streak formation, in the inner cell mass of the preimplantation embryo. Gbx2 is therefore a candidate control gene for cell pluripotency and differentiation in the embryo.

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein., (Copyright 1997 Academic Press.)

We have cloned the complete coding region for a human homologue of the Drosophila melanogaster sluggish-A and yeast PUT1 genes, previously shown to encode proline oxidase activity in these organisms. The predicted 516-residue human protein shows strong homology (51% amino acid sequence identity) to the D. melanogaster protein, indicating that this new human gene may encode proline oxidase. Northern analysis shows that the gene is expressed in human lung, skeletal muscle and brain, to a lesser extent in heart and kidney, and weakly in liver, placenta and pancreas. The gene was mapped by fluorescence in situ hybridization and by in situ hybridization with a [3H]-labelled DNA probe to chromosome 22q11.2, a region previously implicated in type-I hyperprolinaemia in a case of CATCH 22 syndrome, a contiguous gene deletion syndrome involving 22q11. Taken together, the evidence indicates that this new human gene is a good candidate gene for type-I hyperprolinaemia. In view of the neurological phenotype of the D. melanogaster sluggish-A mutant, it is of interest that schizophrenia and bipolar disorder susceptibility genes also map in this region.

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

The human PIN1 gene encodes an essential nuclear peptidyl-prolyl cis/trans isomerase involved in the regulation of mitosis. PIN1 is a member of a new class of peptidyl-prolyl cis/trans isomerases that includes the Escherichia coli parvulin, yeast ESS1, and Drosophila melanogaster dodo gene products. Analysis of human ESTs showed that there are two different but closely related human transcripts, one of which corresponds to PIN1. Gene localization, using both FISH and tritium-labeled probes, showed that each of the human transcripts hybridized to 1p31 and 19p13. Primers were designed to discriminate between the two transcripts, and PCR on DNA from hamster/human somatic cell hybrids retaining chromosomes 1 or 19 was used to map the human PIN1 gene to chromosome 19, and PIN1L, a closely related gene, to chromosome 1. The results establish that PIN1 is at 19p13 and PIN1L at 1p31. PCR was used to clone the coding region for PIN1L. The PIN1L cDNA is 89% identical at the nucleotide level to the PIN1 transcript, but contains a shift in the reading frame. It encodes a 100-amino-acid variant protein consisting of 63 amino acids homologous (90% identical) to PIN1 and containing the entire WW domain, fused to a 37-amino-acid tail. The protein encoded by PIN1L may have some functional role or alternatively PIN1L may be a transcribed pseudogene., (Copyright 1997 Academic Press.)

Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome.

The TI1/UPK1b gene codes for a protein of the "tetraspan" family and is expressed as a differentiation product of the mammalian urothelium. A partial genomic clone of the human homologue of the TI1/UPK1b gene was isolated and used as probe to localize the human gene to chromosome 3q13.3-q21 by in situ hybridization. Using the same probe, a TaqI restriction fragment length polymorphism, with 29% heterozygosity, was identified by Southern analysis.

In a search for new protein tyrosine kinases (PTKs) in early hemopoietic cells, we have identified a sequence closely related to the Fibroblast Growth Factor Receptor (FGFR) family. A cDNA isolated from a mouse embryo library was 89% identical to FGFR-3 in both its coding and 3' untranslated regions. However, the region homologous to exons 5 to 9 of FGFR-3 was missing. In addition, the ORF was interrupted by several stop codons and frame shifts, indicating that this sequence is not functional. These transcripts were therefore copied from a novel FGFR-3 pseudogene, that we called psiFGFR-3. Partial analysis of this gene showed the absence of introns, which is a characteristic feature of a processed pseudogene. psiFGFR-3 gene was localized on Chromosome 1H4-6. Its transcription was shown to be antisense and its expression was restricted to fetal tissues. These results indicate that psiFGFR-3 has been inserted in Chromosome 1 in antisense orientation close to a heterologous promoter.

We have cloned and characterized the mouse gene encoding Kiz1/Limk1, a new member of the zinc-finger LIM family that also has a kinase domain. The gene encompasses 25 kb of the mouse genome, and the organization of its 16 exons does not correlate with its functional domains. The promoter region of Kiz1/Limk1 was identified by cloning a 1.06-kb genomic fragment upstream from the first ATG in a promotorless CAT vector. This construct was demonstrated to drive CAT expression in Jurkat cells. The promoter sequence lacks conventional TATA and CAAT motifs but contains consensus binding sequences for several transcriptional regulators implicated in control of transcription in many different cell types, including Sp1, Ets, and E2A. Analysis of the chromosomal localization of KIZ1/LIMK1 indicates that it lies on human chromosome 17 in the region 17q25 and on mouse Chromosome 5, band G2.

cDNA clones corresponding to the sequence for human early pregnancy factor were isolated from a human melanoma library and hybridized to DNA digested with four restriction enzymes obtained from twelve different subjects. Up to 20 cross hybridizing bands were observed. When hybridized to metaphase spreads from four different humans, significant signals were present in nine locations, on eight different chromosome arms. These results suggest that the early pregnancy factor gene is a member of a large gene family. The coding sequence for early pregnancy factor has a high degree of homology with the sequence for human chaperonin 10, and the gene family described here should contain the genes for both of these proteins.

In this study, we have isolated and characterized a gene and cDNA encoding a mouse Theta class GST. The gene, mGSTT2, spans approximately 3.1 kb and is composed of five exons interrupted by four introns. The gene was localized to Chromosome 10B5-C1 by in situ hybridization. Southern blot analysis of mouse genomic DNA suggests that there is only one copy of mGSTT2 in the mouse genome. The cDNA derived from mGSTT2 was isolated from a mouse liver cDNA library and has an open reading frame of 732 bp encoding a peptide of 244 amino acids with a calculated molecular weight of 26,676 Da. The encoded protein shares amino acid sequence identities of 92, 77, 51, and 55% with rat subunit Yrs, human subunit GSTT2, rat subunit 5, and human subunit GSTT1, respectively.

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.

Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxo-prolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization.

Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. (Biochem J. 300: 271-276, 1994) reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a lambda gt11 human liver 5'-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment.