Search

Purpose In humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 (PRKD1) and G2/M-phase-specific E3 ubiquitin protein ligase (G2E3) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult G2e3 and Prkd1 knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans. Methods The G2e3 and Prkd1 KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning. Results Body fat was increased in multiple cohorts of G2e3 KO mice relative to their wild-type (WT) littermates. When data from all G2e3 KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight (P

David R Powell,1 Deon D Doree,1 Christopher M DaCosta,1 Kenneth A Platt,2 Robert Brommage,1 Lindsey Buhring,1 Jean-Pierre Revelli,1 Melanie K Shadoan1 1Department of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc, The Woodlands, TX, USA; 2Department of Molecular Biology, Lexicon Pharmaceuticals, Inc., The Woodlands, TX, USACorrespondence: David R PowellDepartment of Pharmaceutical Biology, Lexicon Pharmaceuticals, Inc., 2445 Technology Forest Boulevard, The Woodlands, TX, 77381, USATel +1 713 249 3972Fax +1 281 863 8115Email dpowell@lexpharma.comPurpose: Humans with haploinsufficiency of GPR75, an orphan GPCR, are thin. Gpr75 knockout (KO) mice are also thin with improved glucose homeostasis. We wanted to confirm these findings in Gpr75 KO mice and determine whether decreased energy intake and/or increased energy expenditure contributed to the thin phenotype.Methods: Gpr75 KO mice were generated by homologous recombination. All studies compared female and male Gpr75 KO mice to their wild type (WT) littermates. Body composition was measured by DXA and QMR technologies. Glucose homeostasis was evaluated by measuring glucose and insulin levels during oral glucose tolerance tests (OGTTs). Food intake was measured in group-housed mice. In singly housed mice, energy expenditure was measured in Oxymax indirect calorimetry chambers, and locomotor activity was measured in Oxymax and Photobeam Activity System chambers.Results: In all 12 cohorts of adult female or male mice, Gpr75 KO mice had less body fat; pooled data showed that, compared to WT littermates (n = 103), Gpr75 KO mice (n = 118) had 49% less body fat and 4% less LBM (P < 0.001 for each). KO mice also had 8% less body fat at weaning (P < 0.05), and during the month after weaning as the thin phenotype became more exaggerated, Gpr75 KO mice ate significantly less than, but had energy expenditure and activity levels comparable to, their WT littermates. During OGTTs, Gpr75 KO mice showed improved glucose tolerance (glucose AUC 23% lower in females, P < 0.05, and 26% lower in males, P < 0.001), accompanied by significantly decreased insulin levels and significantly increased insulin sensitivity indices.Conclusion: Gpr75 KO mice are thin at weaning, are hypophagic as the thin phenotype becomes more exaggerated, and exhibit improved glucose tolerance and insulin sensitivity as healthy-appearing adults. These results suggest that inhibiting GPR75 in obese humans may safely decrease energy intake and body fat while improving glucose tolerance and insulin sensitivity.Keywords: obesity, diabetes, druggable, insulin sensitivity, healthy thinness

Purpose Humans with haploinsufficiency of GPR75, an orphan GPCR, are thin. Gpr75 knockout (KO) mice are also thin with improved glucose homeostasis. We wanted to confirm these findings in Gpr75 KO mice and determine whether decreased energy intake and/or increased energy expenditure contributed to the thin phenotype. Methods Gpr75 KO mice were generated by homologous recombination. All studies compared female and male Gpr75 KO mice to their wild type (WT) littermates. Body composition was measured by DXA and QMR technologies. Glucose homeostasis was evaluated by measuring glucose and insulin levels during oral glucose tolerance tests (OGTTs). Food intake was measured in group-housed mice. In singly housed mice, energy expenditure was measured in Oxymax indirect calorimetry chambers, and locomotor activity was measured in Oxymax and Photobeam Activity System chambers. Results In all 12 cohorts of adult female or male mice, Gpr75 KO mice had less body fat; pooled data showed that, compared to WT littermates (n = 103), Gpr75 KO mice (n = 118) had 49% less body fat and 4% less LBM (P < 0.001 for each). KO mice also had 8% less body fat at weaning (P < 0.05), and during the month after weaning as the thin phenotype became more exaggerated, Gpr75 KO mice ate significantly less than, but had energy expenditure and activity levels comparable to, their WT littermates. During OGTTs, Gpr75 KO mice showed improved glucose tolerance (glucose AUC 23% lower in females, P < 0.05, and 26% lower in males, P < 0.001), accompanied by significantly decreased insulin levels and significantly increased insulin sensitivity indices. Conclusion Gpr75 KO mice are thin at weaning, are hypophagic as the thin phenotype becomes more exaggerated, and exhibit improved glucose tolerance and insulin sensitivity as healthy-appearing adults. These results suggest that inhibiting GPR75 in obese humans may safely decrease energy intake and body fat while improving glucose tolerance and insulin sensitivity.

Purpose In humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 (PRKD1) and G2/M-phase-specific E3 ubiquitin protein ligase (G2E3) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult G2e3 and Prkd1 knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans. Methods The G2e3 and Prkd1 KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning. Results Body fat was increased in multiple cohorts of G2e3 KO mice relative to their wild-type (WT) littermates. When data from all G2e3 KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight (P

Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet β-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet α- to β-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90 and ∼3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40–50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1−/−) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5- to 2-fold higher urine glucose/creatinine ratios in Sglt1−/− vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1−/− vs. WT after 24 h (−33 vs. −11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 ± 2% in WT and 17 ± 2% in Sglt1−/−. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ∼10-fold and reduced FGR to 44 ± 3% in WT and to −1 ± 3% in Sglt1−/−. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50–60% of filtered glucose is excreted.

The kinase suppressor of ras 2 (KSR2) gene resides at human chromosome 12q24, a region linked to obesity and type 2 diabetes (T2D). While knocking out and phenotypically screening mouse orthologs of thousands of druggable human genes, we found KSR2 knockout (KSR2(-/-)) mice to be more obese and glucose intolerant than melanocortin 4 receptor(-/-) (MC4R(-/-)) mice. The obesity and T2D of KSR2(-/-) mice resulted from hyperphagia which was unresponsive to leptin and did not originate downstream of MC4R. The kinases AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are each linked to food intake regulation, but only mTOR had increased activity in KSR2(-/-) mouse brain, and the ability of rapamycin to inhibit food intake in KSR2(-/-) mice further implicated mTOR in this process. The metabolic phenotype of KSR2 heterozygous (KSR2(+/minus;)) and KSR2(-/-) mice suggests that human KSR2 variants may contribute to a similar phenotype linked to human chromosome 12q24.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Adult mosquitoes (Diptera: Culicidae) were collected in 2007 and tested for specific viruses, including West Nile virus, as part of the ongoing arbovirus surveillance efforts in the state of Iowa. A subset of these mosquitoes (6,061 individuals in 340 pools) was further tested by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus universal primers. Of the 211 pools of Culex pipiens (L.) tested, 50 were positive. One of 51 pools of Culex tarsalis Coquillet was also positive. The flavivirus minimum infection rates (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) for Cx. pipiens and Cx. tarsalis were 10.3 and 1.2, respectively. Flavivirus RNA was not detected in Aedes triseriatus (Say) (52 pools), Culex erraticus (Dyar & Knab) (25 pools), or Culex territans Walker (one pool). Sequence analysis of all RT-PCR products revealed that the mosquitoes had been infected with Culex flavivirus (CxFV), an insect-specific virus previously isolated in Japan, Indonesia, Texas, Mexico, Guatemala and Trinidad. The complete genome of one isolate was sequenced, as were the envelope protein genes of eight other isolates. Phylogenetic analysis revealed that CxFV isolates from the United States (Iowa and Texas) are more closely related to CxFV isolates from Asia than those from Mexico, Guatemala, and Trinidad.

Vector competence of Aedes vexans (Meigen) and Culex pipiens pipiens L. (Diptera: Culicidae) for West Nile virus (familyFlaviviridae, genusFlavivirus, WNV) was compared. Infection rates of both species were similar 14 d after feeding on chickens, with WNV titers ranging from 10 4.2 to 10 8.7 plaque-forming units (PFU)/ml. Median infectious doses and 95% conÞdence intervals (CI) were 10 6.0(5.8,6.3) and 10 5.7(5.4,5.9) PFU for Ae. vexans and Cx. p. pipiens, respectively. WNV transmission was not observed in Ae. vexans that fed on chickens with WNV titers 10 5.0 PFU/ml, in contrast to a mean (95% CI) transmission rate of 7(2,18)% for Cx. p. pipiens. Mean WNV transmission rates for Ae. vexans and Cx. p. pipiens were 13(7,21)% and 10(5,19)%, respectively, after feeding on chickens with WNV titers of 10 5.3 0.1 and 10 5.7 0.1 PFU/ml, and 31(25,37)% and 41(30,53)% after feeding on chickens with WNV titers 10 6.1 0.1 PFU/ml. Time postinfection (p.i.) signiÞcantly insuenced WNV transmission by Ae. vexans as indicated by a nearly 10-fold increase in transmission rate between days 7 and 14 p.i. Mean WNV load expectorated with saliva of Ae. vexans was 10 2.4(2.1,2.7) PFU, and it was not signiÞcantly affected by the titer of chickens on which they originally fed or time p.i. These data indicate that vector competence of the primarily mammalophilic Ae. vexans, which also feeds on birds, approaches that of Cx. p. pipiens for WNV. Because peridomestic mammals, such as cottontail rabbits, squirrels, and chipmunks, develop WNV titers infective for Ae. vexans, this species may play a signiÞcant role in WNV enzootic cycles.

The West Nile virus (WNV) viremia and shedding profiles of 11 adult fox squirrels (Sciurus niger) infected by needle inoculation or mosquito bite were characterized. Daily mean titers (95% confidence intervals) for all squirrels on days 1 through 6 postexposure (p.e.) were: 10(1.7 (1.32.1)), 10(4.4 (4.04.8)), 10(5.3 (5.05.6)), 10(4.4 (3.94.9)), 10(2.7 (2.03.4)), and 10(1.1 (0.52.1)) plaque-forming units (PFU)/mL. The highest WNV serum titers of individual squirrels infected by needle inoculation or mosquito bite ranged from 10(4.5) to 10(6.1) and from 10(5.1) to 10(5.3) PFU/mL, respectively. Nine (82%) squirrels, including all 4 squirrels infected by mosquito bite, had WNV serum titers > or =10(5.1) PFU/mL that persisted on average for 1.6 +/- 0.3 days. Infection and dissemination rates of Culex pipiens (L.) that fed on squirrels with serum titers of 10(4.4 +/- 0.1) PFU/mL were 56% and 13%, respectively. Both of these rates increased to over 80% when fed on squirrels with a mean WNV titer of 10(5.5 +/- 0.1) PFU/mL. Infection and dissemination also occurred in Aedes triseriatus (Say) but at a much lower rate. WNV was isolated from the oral and rectal cavities of all squirrels and from urine that was opportunistically collected from 5 squirrels. The largest quantity of WNV recovered from swabs of the oral cavity and urine was 10(3.1) PFU. The longest periods after exposure that WNV was isolated from the oral cavity and urine from a squirrel were 22 and 17 days p.e., respectively. WNV RNA was also detected in kidney tissue in 1 squirrel 29 days p.e., suggesting that fox squirrels can be persistently infected. Collectively these observations provide further evidence that squirrels can contribute to the natural history and epidemiology of WNV, especially in peridomestic environments.

To determine if Chlamydia muridarum, or other chlamydiae, are enzootic in rodents, we probed a serum bank of wild Peromyscus spp. mice for immunoglobulin G-antibody reactivity to ultraviolet light-inactivated C. muridarum elementary bodies (EBs) using an enzyme-linked immunoassay. Applying a cut-off for a positive reaction of OD405 nm = 0.1 at a 1:20 dilution, we found titratable antibody reactivity in 190 of 247 specimens surveyed (77%, mean OD405 = 0.33 ± 0.26, range = 0.11–1.81, median = 0.25). In addition, serum samples were obtained from a colony of specific pathogen-free Peromyscus spp. maintained at the University of South Carolina and six of 12 samples were reactive (50%, mean OD405 = 0.19 +/− 0.08, range = 0.1–0.32, median = 0.18). Lastly, 40 additional wild Peromyscus spp. were captured in a disparate region of Midwestern USA and 22 serum specimens were reactive (55%, mean OD405 = 0.22 +/− 0.11, range = 0.1–0.48, median = 0.2). Specificity of selected reactive sera for chlamydial antigen was confirmed on Western blot using resolved purified EBs as the detecting antigen. From tissues removed from several mice at necropsy, the gene for chlamydial 16S ribosomal ribonucleic acid (rRNA) was amplified by polymerase chain reaction (PCR). Positive samples of 16S rRNA were subjected to additional PCR for the major outer membrane protein gene (ompA). The amplicons of three select ompA positive samples were sequenced with ≥99% homology with C. muridarum. Our findings indicate that chlamydial infection is enzootic for Peromyscus spp., and that C. muridarum, or a closely related species or strain, is likely the agent in the tested rodent species.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells' functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1(-/-) mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1(-/-) mice. RPE-rich eyecup cultures from AdipoR1(-/-) reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity., Docosahexaenoic acid is a major and important retinal fatty acid that is recruited and retained in the photoreceptor membrane via an unknown mechanism. Here, Rice et al. show that adiponectin receptor 1 is a key molecular switch for docosahexaenoic acid membrane homeostasis and photoreceptor cell function.

The susceptibility of Aedes triseriatus (Say) (Diptera: Culicidae) to low levels of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was determined and compared with that of Culex pipiens L. to assess the likelihood of its participation in an enzootic cycle involving mammals. Ae. triseriatus and Cx. pipiens were exposed to WNV by feeding on baby chickens with WNV serum titers ranging from 10(4.1 +/- 0.1) to 10(8.6 +/- 0.1) plaque-forming units (PFU)/ml and from 10(4.1 +/- 0.1) to 10(7.0) PFU/ml, respectively. Infection rates and 95% confidence intervals (CIs) of 8% (4, 14) and 25% (15, 38) occurred in Ae. triseriatus and Cx. pipiens after feeding on chickens with WNV titers of 10(4.1 +/- 0.1) PFU/ml and increased to 65% (49, 79) and 100% (72, 100) in Ae. triseriatus and Cx. pipiens after feeding on chickens with titers of 10(7.1 +/- 0.1) PFU/ml. The mean infection rate of Ae. triseriatus ranged from 97% (84, 100) to 100% (79, 100) after feeding on chickens with WNV titers of > or = 10(8.2) PFU/ml. The infectious dose (ID)50 values for Ae. triseriatus and Cx. pipiens were 10(6.5) (6.4, 6.7) and 10(4.9) (4.6, 5.1) PFU/ml, respectively. The combined estimated transmission rate of Ae. triseriatus at 14 and 18 d after feeding on chickens with a mean WNV titer of 10(8.6 +/- 0.1) PFU/ml was 55%. Although Ae. triseriatus is significantly less susceptible to WNV than Cx. pipiens, the susceptibility of Ae. triseriatus to WNV titers < 10(5.0) PFU/ml and its ability to transmit WNV suggest that Ae. triseriatus has the potential to be an enzootic vector among mammalian populations.

Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption (TR4 –/– ). The number of TR4 –/– pups generated by the mating of TR4 +/– mice is well under that predicted by the normal Mendelian ratio, and TR4 –/– mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4 –/– females show defects in reproduction and maternal behavior, with pups of TR4 –/– dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.

Insect peptidoglycan recognition protein-S (PGRP-S), a member of a family of innate immunity pattern recognition molecules conserved from insects to mammals, recognizes bacterial cell wall peptidoglycan and activates 2 antimicrobial defense systems, prophenoloxidase cascade and antimicrobial peptides through Toll receptor. We show that mouse PGRP-S is present in neutrophil tertiary granules and that PGRP-S–deficient (PGRP-S-/-) mice have increased susceptibility to intraperitoneal infection with gram-positive bacteria of low pathogenicity but not with more pathogenic gram-positive or gram-negative bacteria. PGRP-S-/- mice have normal inflammatory responses and production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Neutrophils from PGRP-S-/- mice have normal phagocytic uptake of bacteria but are defective in intracellular killing and digestion of relatively nonpathogenic gram-positive bacteria. Therefore, mammalian PGRP-S functions in intracellular killing of bacteria. Thus, only bacterial recognition by PGRP-S, but not its effector function, is conserved from insects to mammals.

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.

Auto-anti-idiotypic antibodies (Aab-2s) were detected from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The Aab-2s were specific against the idiotypic antibodies (Ab-1s) to the envelope glycoprotein GP5 of PRRSV and were detected from serum samples collected between 21 and 98 days post-infection (DPI). Serological characterization indicated that the Aab-2s recognized the idiotype located within or near the antigen-combining sites of the anti-GP5 antibodies, which was shared by both mouse MAb anti-GP5 and swine polyclonal antibodies. The fact that the Aab-2 inhibited the anti-GP5 antibodies from binding to PRRSV and that they were detected at different time periods in pigs that cleared the infection prior to 98 DPI versus pigs in which virus was detected at 98 DPI suggests that Aab-2 antibodies may play a role in immunity to PRRSV infection.

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2 / male mice we found that -galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles. The nuclear receptors are ligand-inducible transcription factors that regulate the expression of target genes by binding to their specific hormone response elements. They are characterized by a central DNA binding domain, which binds to the hormone response element. The C-terminal half of the receptor encompasses the ligand-binding domain, which mediates ligand binding, dimerization, and transactivation function. Nuclear receptors play roles in various aspects of physiology including metabolism, development, homeostasis, and reproduc

The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.

Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 10(2.3) or 10(3.4) plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 10(5.1) PFU/mL and ranged from 10(4.6) to 10(5.6) PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 10(2.0), 10(2.8), and 10(2) PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.

Eleven epitopes were identified by murine monoclonal antibodies (MAbs) that represented the N, M, GP5 and GP3 proteins of the North American (NA) porcine reproductive and respiratory syndrome (PRRS) virus, KY 35 (NVSL 46907). Three discontinuous epitopes of the N and M proteins were designated EpORF7-Fd through Hd and EpORF6-Ad through Cd. Five continuous epitopes of the GP5 and GP3 proteins were designated EpORF5-A through C and EpORF3-A and B. The MAbs representing EpORF5-C and EpORF6-A and B had neutralizing activity. The MAbs representing the above epitopes, except EpORF7-Gd and Hd, expanded the virus marker system described in a previous study in which a panel of 69 NA viruses and the Lelystad virus were categorized into 5 antigenic groups, I15 through V15 based on the presence or absence of 5 continuous epitopes of the N protein. Antigenic groups I15 and II15, which represented 84.7 and 11.6% of all viruses tested, were categorized further into 9 and 4 subgroups, respectively. The remaining NA viruses and the Lelystad virus were distributed among 4 groups, one of which was represented by 2 subgroups. Significant (P

Background Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. Methods Genomic library λ knock-out shuttle (λKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. Results Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. Conclusion The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases. Copyright © 2000 John Wiley & Sons, Ltd.

Experiments were designed to evaluate the vector competence of Culex tarsalis Coquillet from an area (Sioux City) where Cx. tarsalis is most abundant in Iowa for western equine encephalomyelitis virus (WEE-7738). WEE-7738 was isolated from Aedes trivittatus (Coquillet) collected in Ames, IA, in 1977. Infection rate, dissemination rate, multiplication efficiency, and transmission rate were determined for this virus in the SC strain of Cx. tarsalis. SC strain of Cx. tarsalis was as susceptible to WEE-7738 as Californian strains of Cx. tarsalis were to BFS1703 strain of WEE; OID50 of SC Cx. tarsalis was 2.63 log TCID50 per mosquito and OID50 of Californian strains of Cx. tarsalis were 2.0-4.1 log PFU per mosquito. However, transmission of WEE-7738 (4.2%) by the SC strain of Cx. tarsalis was lower than those (10-60%) reported in other studies.

Induction of the floor plate at the ventral midline of the neural tube is one of the earliest events in the establishment of dorsoventral (d/v) polarity in the vertebrate central nervous system (CNS). The secreted molecule, Sonic hedgehog, has been shown to be both necessary and sufficient for this induction. In vertebrates, several downstream components of this signalling pathway have been identified, including members of the Gli transcription factor family. In this study, we have examined d/v patterning of the CNS in Gli2 mouse mutants. We have found that the floor plate throughout the midbrain, hindbrain and spinal cord does not form in Gli2 homozygotes. Despite this, motoneurons and ventral interneurons form in their normal d/v positions at 9.5 to 12.5 days postcoitum (dpc). However, cells that are generated in the region flanking the floor plate, including dopaminergic and serotonergic neurons, were greatly reduced in number or absent in Gli2 homozygous embryos. These results suggest that early signals derived from the notochord can be sufficient for establishing the basic d/v domains of cell differentiation in the ventral spinal cord and hindbrain. Interestingly, the notochord in Gli2 mutants does not regress ventrally after 10.5 dpc, as in normal embryos. Finally, the spinal cord of Gli1/Gli2 zinc-finger-deletion double homozygous mutants appeared similar to Gli2 homozygotes, indicating that neither gene is required downstream of Shh for the early development of ventral cell fates outside the ventral midline.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post-inoculation (p.i.), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 p.i. at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may plan an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV with prolonged recovery of virus from tonsils of swine.

Seventeen porcine reproductive and respiratory syndrome virus (PRRSV) field isolates, including isolate ISU-P, were evaluated for their susceptibility to antibody dependent enhancement (ADE) of infection mediated by antibodies raised against PRRSV isolate ISU-P. Progeny virus yields of ISU-P and 4 of 16 field isolates in porcine alveolar macrophages (PAM) were reduced following treatment with a concentration of antibody that neutralized ISU-P (p < 0.01). In contrast, the yields of 12 of 17 field isolates were enhanced (p < 0.01). Treatment of all isolates with a 10-fold lower concentration of this antibody significantly (p < 0.01) increased virus yields of all isolates in PAM. However, the degree of enhancement varied among the isolates when compared to the enhancement of the yield of ISU-P. While no differences in enhancement were observed among ISU-P and 9 field isolates, yield enhancement of 6 and 1 isolates were less than and more than the yield enhancement of ISU-P, respectively (p < 0.05). The degree of enhancement mediated by a high concentration of antibody raised against ISU-P was inversely proportional to the ability of the antibody to neutralize the isolates (r = 0.92). In contrast, no direct correlation (r = 0.32) was observed between the degree of enhancement mediated by a low concentration of antibody and the ability of the antibody to neutralize the isolates. These data suggest that the variability in the susceptibility of PRRSV isolates to ADE arise from quantitative and/or qualitative differences in the antigenic determinants associated with virus neutralization and/or ADE. The antigenic diversity and the wide range in the susceptibility to ADE that exists among field isolates indicate that ADE should be taken into consideration in the development of effective immunization strategies for PRRS.

Persistent infection with porcine reproductive and respiratory syndrome virus (PRRSV) was shown in experimentally infected pigs by isolation of virus from oropharyngeal samples for up to 157 days after challenge. Four 4 week old, conventional, PRRSV antibody-negative pigs were intranasally inoculated with PRRSV (ATCC VR-2402). Serum samples were collected every 2 to 3 days until day 42 post inoculation (PI), then approximately every 14 days until day 213 PI. Fecal samples were collected at the time of serum collection through day 35 PI. Oropharyngeal samples were collected at the time of serum collection from 56 to 213 days PI by scraping the oropharyngeal area with a sterile spoon, especially targeting the palatine tonsil. Turbinate, tonsil, lung, parotid salivary gland, spleen, lymph nodes and serum were collected postmortem on day 220 PI. Virus isolation (VI) on porcine alveolar macrophage cultures was attempted on all serum, fecal and oropharyngeal samples, as well as tissues collected postmortem. Postmortem tonsil tissues and selected fecal samples were also assayed for the presence of PRRSV RNA by the polymerase chain reaction (PCR). Serum antibody titers were determined by IFA, ELISA and SVN. Virus was isolated from all serum samples collected on days 2 to 11 PI and intermittently for up to 23 days in two pigs. No PRRSV was isolated from fecal samples, but 3 of 24 samples were PCR positive, suggesting the presence of inactivated virus. Oropharyngeal samples from each pig were VI positive 1 or more times between 56 and 157 days PI. Oropharyngeal samples from 3 of 4 pigs were VI positive on days 56, 70 and 84 PI. Virus was isolated from one pig on day 157 PI, 134 days after the last isolation of virus from serum from this animal. Virus was isolated from oropharyngeal samples for several weeks after the maximum serum antibody response, as measured by IFA, ELISA and SVN tests. All tissues collected postmortem were VI negative and postmortem tonsil samples were also negative by PCR. An important element in the transmission of PRRSV is the duration of virus shedding. The results of this study provided direct evidence of persistent PRRSV infection and explain field observations of long-term herd infection and transmission via purchase of clinically normal, but PRRSV infected, animals. Effective prevention and control strategies will need to be developed in the context of these results.

Infection of porcine alveolar macrophages by the porcine reproductive and respiratory syndrome virus (PRRSV) was significantly enhanced in vitro by antibody raised against the PRRSV isolate ISU-P (p < 0.01). Increased yields and infection rates were highly correlated (r = 0.95) and the ratio of yield to infection rate was greater than 1.4, suggesting that more than one mechanism was responsible for enhanced infection. Antibody-dependent enhancement (ADE) of infection was also demonstrated in vivo using a completely randomized block design (n = 16). The mean level and duration of viremia were greater (p < 0.05) in pigs injected with subneutralizing amounts of PRRSV-specific IgG prior to virus challenge than in control pigs injected with normal IgG. In contrast, virus replication was significantly (p < 0.01) inhibited in pigs with neutralizing antibody titers of 4 log2. The period of time that subneutralizing levels of antibody can persist and contribute to ADE of PRRSV infection was estimated in 4 pigs injected with PRRSV-specific IgG to yield an initial neutralizing antibody titer of 3.8 log2. Neutralizing activity declined to undetectable levels by day 37 postinjection (PI). ADE activity was first detected in undiluted sera on day 20 PI and persisted through day 62 PI. Western immunoblot analysis of sera collected between days 37 and 62 PI detected antibodies specific for the 15-kDa nucleocapsid and 26-kDa glycosylated envelope proteins. These results strongly suggest that ADE has the potential to contribute to the pathogenesis of PRRSV infection and is mediated by antibody specific for the 26-kDa envelope protein.

The dissemination of dengue (DEN) 3 virus in parenterally infected female Aedes aegypti mosquitoes was studied imrnunocytochemically. Antigen was first detected in fat body cells near the thoracic site of virus inoculation. The intussuscepted foregut, salivary glands and nervous tissue were the first major tissues infected. Nervous tissue appeared to be the primary site of amplification. Muscles, tracheae, Malphigian tubules and the posterior midgut did not become infected. The only part of the reproductive system to be infected was the calyx (71% of specimens 16–22 days post-infection) consistent with low rates of vertical transmission. After 7 days post-inoculation the salivary glands of 100% of the specimens examined were infected. Virus dissemination was slow and the most common sequence of infection following intrathoracic inoculation was as follows: thoracic fat body, intussuscepted foregut, salivary glands, cardial epithelium, thoracic ganglion, brain, compound eye, anterior midgut, intermediate midgut/anterior abdominal ganglia, and calyx/hindgut/posterior abdominal ganglia. Fat body and intussuscepted foregut tissues lost infections after 16 days post-inoculation.

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period. These results clearly indicate that the 15-kD protein is the most immunogenic of the 4 viral proteins identified and may provide the antigenic basis for the development of improved diagnostic tests for the detection of PRRS virus antibodies.

Background: The purpose of this study was to elucidate parasympathetic preganglionic neurons in the spinal cord that project axons in pathways to the uterus and to reveal their neurotransmitter phenotype. Methods: “Uterine-related” neurons were identified by using a combination of retrograde axonal tracers: (1) Fluorogold injected into the ganglia of termination of preganglionic fibers, and (2) a transganglionic axonal tracer (pseudorabies virus) injected into the uterus. Immunohistochemistry was used to reveal virus-labeled neurons and their neurotransmitter marker. Results: Double-labeled (Fluorogold + pseudorabies virus) “uterine” preganglionic neurons were identified in the sacral parasympathetic nucleus of the rat lumbosacral spinal cord. Subpopulations of neurons in the sacral parasympathetic nucleus were shown to be immunoreactive for choline acetyltransferase or nitric oxide synthase. Double-staining immunohistochemistry (for pseudorabies virus + neurotransmitter enzyme) revealed that some of the uterine-related preganglionic neurons were cholinergic and some nitric oxide synthase-containing. Conclusions: These results demonstrate a subpopulation of pregauglionic parasympathetic neurons in the sacral parasympathetic nucleus that are involved in uterine innervation. In addition, both acetylcholine and nitric oxide could be used to modify activity in the postganglionic neurons, which directly innervate the uterus. © 1995 Wiley-Liss, Inc.

The Na-glucose cotransporter SGLT2 mediates high-capacity glucose uptake in the early proximal tubule and SGLT2 inhibitors are developed as new antidiabetic drugs. We used gene-targeted Sglt2 knockout (Sglt2−/−) mice to elucidate the contribution of SGLT2 to blood glucose control, glomerular hyperfiltration, kidney growth, and markers of renal growth and injury at 5 wk and 4.5 mo after induction of low-dose streptozotocin (STZ) diabetes. The absence of SGLT2 did not affect renal mRNA expression of glucose transporters SGLT1, NaGLT1, GLUT1, or GLUT2 in response to STZ. Application of STZ increased blood glucose levels to a lesser extent in Sglt2−/−vs. wild-type (WT) mice (∼300 vs. 470 mg/dl) but increased glucosuria and food and fluid intake to similar levels in both genotypes. Lack of SGLT2 prevented STZ-induced glomerular hyperfiltration but not the increase in kidney weight. Knockout of SGLT2 attenuated the STZ-induced renal accumulation of p62/sequestosome, an indicator of impaired autophagy, but did not attenuate the rise in renal expression of markers of kidney growth (p27 and proliferating cell nuclear antigen), oxidative stress (NADPH oxidases 2 and 4 and heme oxygenase-1), inflammation (interleukin-6 and monocyte chemoattractant protein-1), fibrosis (fibronectin and Sirius red-sensitive tubulointerstitial collagen accumulation), or injury (renal/urinary neutrophil gelatinase-associated lipocalin). SGLT2 deficiency did not induce ascending urinary tract infection in nondiabetic or diabetic mice. The results indicate that SGLT2 is a determinant of hyperglycemia and glomerular hyperfiltration in STZ-induced diabetes mellitus but is not critical for the induction of renal growth and markers of renal injury, inflammation, and fibrosis.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.

Transcription of the adipocyte-specific adipsin gene is dramatically reduced in the adipose tissue of a number of genetically and chemically-induced obese rodents. To map the region of the adipsin gene that confers this response to obesity, transgenic mice were made containing -114, -250, -400, -700, and -938 base pairs (bp) to +35 bp of the promoter linked to the bacterial chloramphenicol acetyltransferase gene. Transgenic mice containing as few as 114 bp of the adipsin promoter had high levels of chloramphenicol acetyltransferase activity in adipose tissue. However, only those mice with 938 bp of the adipsin upstream regulatory region showed suppression of expression in adipose tissue in mice that were induced to become obese with monosodium glutamate. Using gel retardation assays, we showed that a 56-bp fragment of DNA mapping between -687 and -743 bp upstream from the start of adipsin expression was bound by protein factors in nuclear extracts prepared from adipose tissue. There was much greater retardation of this fragment with nuclear extracts prepared from adipose tissue of lean versus obese mice. These results indicate that a tissue-specific transcription factor(s) that regulates adipsin expression is less active in the adipose tissue of obese animals.

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers

Mutations in the gene encoding for the Na(+)-glucose co-transporter SGLT2 (SLC5A2) associate with familial renal glucosuria, but the role of SGLT2 in the kidney is incompletely understood. Here, we determined the localization of SGLT2 in the mouse kidney and generated and characterized SGLT2-deficient mice. In wild-type (WT) mice, immunohistochemistry localized SGLT2 to the brush border membrane of the early proximal tubule. Sglt2(-/-) mice had glucosuria, polyuria, and increased food and fluid intake without differences in plasma glucose concentrations, GFR, or urinary excretion of other proximal tubular substrates (including amino acids) compared with WT mice. SGLT2 deficiency did not associate with volume depletion, suggested by similar body weight, BP, and hematocrit; however, plasma renin concentrations were modestly higher and plasma aldosterone levels were lower in Sglt2(-/-) mice. Whole-kidney clearance studies showed that fractional glucose reabsorption was significantly lower in Sglt2(-/-) mice compared with WT mice and varied in Sglt2(-/-) mice between 10 and 60%, inversely with the amount of filtered glucose. Free-flow micropuncture revealed that for early proximal collections, 78 ± 6% of the filtered glucose was reabsorbed in WT mice compared with no reabsorption in Sglt2(-/-) mice. For late proximal collections, fractional glucose reabsorption was 93 ± 1% in WT and 21 ± 6% in Sglt2(-/-) mice, respectively. These results demonstrate that SGLT2 mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. This mouse model mimics and explains the glucosuric phenotype of individuals carrying SLC5A2 mutations.

We used the retrograde transneuronal transport of a neurotropic virus, pseudorabies virus (PRV), to identify the neurons in sympathetic ganglia, spinal cord and brain which regulate renal function and renal circulation. PRV was microinjected into the left kidney of 70, pentobarbital-anesthetized, male rats. After an incubation period of 1-4 days, rats were anesthetized and sacrificed. PRV-infected neurons were located immunocytochemically in pre- and paravertebral sympathetic ganglia, the intermediolateral cell column of the T10-T13 segments and several brainstem cell groups: the medullary raphe nuclei, rostral ventrolateral medulla, rostral ventromedial medulla, A5 cell group, and the paraventricular hypothalamic nucleus. In more heavily infected rats, additional labeling was found in the locus coeruleus, periaqueductal gray matter, lateral hypothalamic area, zona incerta, and anterior hypothalamic area. No infected propriospinal neurons were observed in the lateral spinal nucleus or gray matter of the caudal cervical, lumbosacral or thoracic spinal segments not containing infected putative sympathetic preganglionic neurons. The paucity of infected propriospinal neurons in the presence of infected brainstem neurons, even in lightly infected rats, is discussed in reference to the relative importance of descending vs spinal regulation of the sympathetic outflow to the kidney.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 10 4 PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid- specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (10 2.3 PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs. Pseudorabies or Aujeszky's disease virus is an al- protein-based subunit vaccine using a differential di- phaherpesvirus that causes an economically important agnostic test based on an early excreted PRV disease of swine.