Aberrant 11beta-hydroxysteroid dehydrogenase-1 activity in the cpk mouse: implications for regulation by the Ke 6 gene

Glucocorticoids have been used to create experimental polycystic kidney disease in rodents and to induce cysts in embryonic kidneys cultures. In addition, the plasma corticosterone levels are higher in a heritable murine model of polycystic kidney disease, cpk mice, in the first postnatal week. Previously, we had shown that the 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) gene is down-regulated in the cpk mice in a coordinated pattern with the Ke 6 gene. In this study, we measured the level of 11betaHSD-1 activity in kidney and liver tissues of cpk homozygote mice and found a reduction in its activity only in the kidney, not in the liver. The activity of the 11betaHSD-1 enzyme appears to be tightly correlated to the level of Ke 6 protein in these tissues. We discuss the possibility that the activity of the 11betaHSD-1 enzyme may be regulated by the Ke 6 enzyme. Ke 6 gene expression has been located to the outer stripe region of rodent kidneys, which is the same region of expression as that for the 11betaHSD-1 gene. These results suggest that down-regulation of the Ke 6 gene may lead to elevated corticosterone levels, mediated through an inhibition of 11betaHSD-1 activity.     

G-protein beta gamma subunits mediate specific phosphorylation of the protein-tyrosine phosphatase SH-PTP1 induced by lysophosphatidic acid

SH-PTP1 is a protein-tyrosine phosphatase preferentially expressed in hematopoietic cells and bearing two SH2 (src homology-2) domains. In the human megakaryocytic cell line Dami, lysophosphatidic acid (LPA) promoted a rapid increase in SH-PTP1 phosphorylation on both serine and tyrosine residues. Only tyrosine phosphorylation was significantly inhibited by pertussis toxin and by the protein kinase C inhibitor GF109203X. Moreover, SH-PTP1 was phosphorylated upon challenge with other agonists acting via G-protein-coupled receptors such as alpha-thrombin, epinephrine, and ADP, whereas the closely related protein-tyrosine phosphatase SH-PTP2 failed to share such a regulation in Dami cells. We developed an in vitro assay that reproduced LPA-dependent phosphorylation of SH-PTP1 in a cell-free system. The fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1-(495-689) or the transducin subunit Galphat-GDP, which act as specific antagonists of Gbetagamma, inhibited SH-PTP1 phosphorylation. Moreover, purified transducin Gbetagamma subunits mimicked the effect of LPA. Finally, stable expression of beta-adrenergic receptor kinase 1-(495-689) in Dami cells resulted in the inhibition of SH-PTP1 as a specific target of protein kinases linked to G-protein-coupled receptors via Gbetagamma subunits.     

Characterization of SNARE protein expression in beta cell lines and pancreatic islets

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.     

Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism

Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive Gi-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.     

Effects of the estrous cycle on local humoral immune responses and protection of intranasally immunized female mice against herpes simplex virus type 2 infection in the genital tract

This study demonstrates that the levels of gB-specific IgG and IgA in vaginal washes of mice immunized intranasally (i.n.) with a recombinant adenovirus vector expressing herpes simplex virus (HSV) glycoprotein B (AdgB8) vary inversely with each other and are dependent on the stage of the estrous cycle. Anti-gB IgA titers in vaginal washes were significantly higher during estrus than diestrus or proestrus, whereas specific IgG titers were significantly higher during diestrus than estrus. This was further demonstrated in hormone-treated mice, where progesterone administration induced a diestrus-like state that resulted in elevated specific IgG-to-IgA ratios. Interestingly, unimmunized mice were only susceptible to intravaginal (ivag) infection with HSV-2 during diestrus. Mice immunized i.n. with AdgB8 and given progesterone were protected from a lethal intravaginal HSV-2 challenge, despite the fact that virus replication was present for 4 days postchallenge. Further, high numbers of gB-specific IgA and IgG antibody-secreting cells were present in both the genital tracts and the draining iliac lymph nodes of i.n.-immunized, but not unimmunized, mice 6 days following ivag HSV-2 challenge. These results demonstrate that the levels of specific antibodies in the female genital tract are dependent on the stage of the estrous cycle. Furthermore, i.n. AdgB8 immunization provided a significant level of protection and specific IgA and IgG antibody-secreting cells in the genital tissues during resolution of an ivag infection with HSV-2.     

Measles vaccine efficacy in Masvingo district, Zimbabwe

OBJECTIVE: To carry out a survey in Masvingo District to determine the efficacy of measles vaccine. DESIGN: A retrospective study, using existing health care facility records and interviews with care givers. SETTING: Using the standard WHO-EPI cluster sampling methodology, 30 clusters were randomly selected in Masvingo District. SUBJECTS: 14 or more children in each of the 30 clusters were selected. MAIN OUTCOME MEASURES: Occurrence of measles or lack of it among the children aged 12 to 23 months, age at vaccination, status, the age at which the child had measles and availability of a health card. RESULTS: In Masvingo District from 1987 to 1994, measles incidence remained very high, though mortality drastically declined. Using field survey data measles vaccine efficacy was estimated at 78.3pc (95pc CI 54.1; 89.8). Vaccine coverage was estimated to be 75pc. CONCLUSION: The efficacy results fall at the lower end, but within the normal limits, of those expected for measles vaccine as used in Zimbabwe. Steps to increase vaccine coverage are of the highest priority.     

Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 A. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an alpha-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.     

Assignment of absolute stereochemistry to an insect pheromone by chiral amplification

We assessed the replicability of reliability and validity of a brief self-report disability scale, adapted from the Medical Outcomes Survey (short form), in a 15-center, cross-national, multilingual study of psychological illness among primary care patients (n = 5438). Across all 15 centers in the World Health Organization Collaborative Study of Psychological Problems in General Health Care, the reliability of the disability scale was high and individual items were responsive at similar levels of disability. Self-report disability was consistently correlated with disability in work role (including housework) as rated by interviewers according to the Groningen Social Disability Schedule, a semistructured method taking local norms into account. Disability as measured by the self-report questionnaire was also consistently correlated with depressive symptoms as measured by the General Health Questionnaire. At each center, the disability items formed a moderately to strongly hierarchical (Guttman-like) scale. These findings support the feasibility of using self-report disability scales in cross-national primary care research.     

An Accurate Technique for Discrimination between Transient and Permanent Faults in Transmission Networks

This paper presents an assessment study for adaptive single-pole automatic reclosure (ASPAR) schemes to select correctly the best scheme at responsible indices. Suggested solutions for the drawbacks that were noticed with the previous methods are investigated. In addition, this paper presents a proposed technique for ASPAR on transmission lines using wavelet packet transform (WPT). The ASPAR aims to discriminate between the faults' natures and to detect the instant of arc extinguished. The proposed technique uses an adaptive threshold level, and therefore, no adjustment is needed for various transmission systems. The proposed technique is examined at different fault scenarios that involve different fault locations, inception angles, and representation to secondary arc characteristics with various arc models. In addition, the behavior of ASPAR is well studied under power swing situation. The effects of shunt reactor compensators on the proposed technique are checked. The proposed scheme is tested for different network configurations, single and double circuits, to realize the robustness and the capability of the proposed scheme. The ATP/EMTP package is developed to emulate different fault signals to validate the proposed scheme. The proposed adaptive threshold scheme discriminates correctly between permanent and transient faults, thereby enhancing power system stability for compensated and uncompensated networks.