Evidence that an L-fucose-containing component in the β-cell plasma membrane is involved in the regulation of glucose-induced insulin release

The effect of the L-fucose-selective lectin Ulex Europeus I (UEA I), a blocker of the Na⁺, K⁺, CIF co-transport system in the kidney, was tested on insulin secretion from isolated β-cell-rich pancreatic islets. UEA I at doses from 50 to 100 μg ml^-1 significantly reduced the glucose-induced (20 mmol l^-1) insulin release whereas the basal (3 mmol l^-1) release was unaffected. The inhibitory effect of 100 μg ml^-1 UEA I was completely abolished by 10 mmol 1-^l L-fucose. The data suggest that an I -fucose-containing structure in the β-cell plasma membrane participates in the regulation of glucose-induced insulin release. This structure may be similar to the L-fucose-containing glycoprotein in the kidney tubules that is believed to be the Na⁺, K⁺, Cl^- co-transporter.     

Evidence that an l-fucose-containing component in the β-cell plasma membrane is involved in the regulation of glucose-induced insulin release

The effect of the l -fucose-selective lectin Ulex Europeus I (UEA I), a blocker of the Na⁺, K⁺, Cl^? co-transport system in the kidney, was tested on insulin secretion from isolated β-cell-rich pancreatic islets. UEA I at doses from 50 to 100 μg ml^?1 significantly reduced the glucose-induced (20 mmol l^?1) insulin release whereas the basal (3 mmol l^?1) release was unaffected. The inhibitory effect of 100 μg ml l^?1 UEA I was completely abolished by 10 mmol l^?1 l -fucose. The data suggest that an l -fucose-containing structure in the β-cell plasma membrane participates in the regulation of glucose-induced insulin release. This structure may be similar to the l -fucose-containing glycoprotein in the kidney tubules that is believed to be the Na⁺, K⁺, Cl^? cotransporter.     

Evidence that oxytocin is a physiological component of LH regulation in non-pregnant women

BACKGROUND: Regulation of the LH surge is central to the functioning of the female ovulatory cycle. In animal models, oxytocin has been shown to alter LH activity. Oxytocin advanced the LH surge and, conversely, oxytocin receptor antagonists inhibited full production of the LH surge in rats. Few data exist on the possibility that oxytocin modulates LH in women. METHODS: Ten non-pregnant women participated in this study over two menstrual cycles. One cycle was a control cycle, and the other a trial cycle; the two were separated by at least one cycle. When the diameter of an ovarian follicle was >15 mm, a subject was allocated at random into either a control or treatment group. In a control cycle, volunteers received normal saline; in a treatment cycle, volunteers received an oxytocin antagonist (atosiban). RESULTS: For treatment cycles, the maximum LH concentration was significantly less than that in control cycles (42.1 +/- 6.2 versus 60.3 +/- 8.3 IU/l respectively; P < 0.05). Maximum FSH and estradiol concentrations were not significantly different between the two groups. CONCLUSIONS: The results indicated that inhibition of endogenous oxytocin affects the endocrinology of the ovulatory cycle in women, and strongly suggest that oxytocin has a role in the physiological processes of LH regulation.     

Evidence that axoplasmic transport of trophic factors is involved in the regulation of peripheral nerve fields in salamanders

1. We have compared the effects of partial denervation with those of colchicine-induced block of axoplasmic flow, on the peripheral fields of nerves innervating the hind limb of salamanders.2. Acute application of colchicine solution (0.03-0.10 M) to spinal nerve 16 results in a dose-dependent increase in skin and muscle fields of the adjacent nerves (15 and 17). The time course, magnitude and distribution of the response to 0.10 M colchicine is not distinguishable from that of the compensatory sprouting of nerves 15 and 17 that occurs after section of nerve 16.3. In contrast to the situation with nerve section, sprouting of adjacent nerves occurred after colchicine applications which produced no behavioural deficit, no change in the peripheral field of the treated nerve, and no interference with impulse conduction in it; nor was there subsequent degeneration in the nerve.4. The same concentrations of colchicine reduced the axoplasmic flow of catecholamines and cholinesterase; treated nerves contained fewer microtubules than untreated controls.5. A similar application of colchicine solution to nerve 15 prevented it from sprouting in response to the stimulus provided by section of nerve 16.6. We conclude that nerve terminals are continuously supplied by axoplasmic flow with a trophic factor concerned with the regulation of nerve fields. When the supply of this factor is reduced, adjacent nerves sprout and invade the territory of the treated nerve. In addition, the ability of nerves to sprout is itself dependent upon the maintenance of axoplasmic flow.     

The stimulus secretion coupling of glucose-induced insulin release

The effect of glucose upon the handling of K⁺ by islets of Langerhans removed from normal rats was investigated by measuring both the net uptake of⁸⁶Rb⁺ and its efflux from prelabelled islets. The inflow of K⁺ into islet cells is mediated, in part at least, by an ouabain-sensitive pump. Glucose fails to affect the inflow rate of K⁺, but it apparently decreases the permeability of islet cells plasma membrane to effluent K⁺. The glucose-induced change in permeability is a rapid and rapidly reversible phenomenon. Under steady-state conditions, it leads to an increase in the islet cells K⁺ pool and a decrease of its fractional turnover rate.     

Genetic regulation of the kinetics of glucose-induced insulin release in man Studies in families with diabetic and non-diabetic probands

Insulin release and sensitivity were estimated from glucose and insulin curves obtained at a glucose infusion test performed on altogether 601 subjects belonging to 155 nuclear families. Ascertainment was through one of the parents, and 96 of the probands had diabetes with clinical onset after the age of 30 years, while 59 were healthy subjects. Three variables obtained by a computer model were analysed, i.e. the glucose regulation of insulin release by a direct stimulatory event (KI) and time-dependent modulatory events (KP) as well as insulin sensitivity (KG). Complex segregation analysis revealed that the variables are genetically regulated, but there was no evidence for a major locus. The children of the diabetics did not differ from those of the non-diabetics as far as insulin release is concerned.     

RBMX is a component of the centromere noncoding RNP complex involved in cohesion regulation

Satellite I RNA, a noncoding (nc)RNA transcribed from repetitive regions in human centromeres, binds to Aurora kinase B and forms a ncRNP complex required for chromosome segregation. To examine its function in this process, we purified satellite I ncRNP complex from nuclear extracts prepared from asynchronized or mitotic (M) phase‐arrested HeLa cells and then carried out LC/MS to identify proteins bound to satellite I RNA. RBMX (RNA‐binding motif protein, X‐linked), which was isolated from M phase‐arrested cells, was selected for further characterization. We found that RBMX associates with satellite I RNA only during M phase. Knockdown of RBMX induced premature separation of sister chromatid cohesion and abnormal nuclear division. Likewise, knockdown of satellite I RNA also caused premature separation of sister chromatids during M phase. The amounts of RBMX and Sororin, a cohesion regulator, were reduced in satellite I RNA‐depleted cells. These results suggest that satellite I RNA plays a role in stabilizing RBMX and Sororin in the ncRNP complex to maintain proper sister chromatid cohesion.     

Evidence that TRPC1 is involved in hippocampal glutamate-induced cell death

Massive neuronal activation by glutamate can result in an excessive rise in cytoplasmic calcium, a process ultimately leading to neuronal death. We have investigated the role of the transient receptor potential channel 1 (TRPC1) in mediating glutamate-induced neuron death. We show that 2-APB (a blocker of store-operated Ca2+ entry) dramatically reduces glutamate-induced cell death in hippocampal organotypic slice cultures and that glutamate-induced toxicity is accompanied by an increase in TRPC1 expression. RNAi mediated knock-down ofTRPC1 in slice cultures prevented glutamate-induced cell death, indicating that TRPC1 plays a prominent role in calcium entry following exposure to glutamate. Thus, TRPC1 may represent a promising target for pharmacological interventions to prevent or reduce glutamate-induced neuronal damage.     

Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, alpha-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with beta-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus.     

Evidence that hyaluronidase is not involved in tissue invasion of the protozoan parasite Entamoeba histolytica

As previous reports suggested that a hyaluronidase is involved in tissue invasion of Entamoeba histolytica, we searched for such an activity in trophozoite extracts. A hyaluronidase activity was not detectable in long-term cultures or in amoebae freshly passaged through a gerbil liver, as evidenced by four different techniques.     

Evidence that post-tetanic potentiation is mediated by neuropeptide release in Aplysia.

Many neuromuscular and central synapses exhibit activity-dependent plasticity. The sustained high-frequency firing needed to elicit some forms of plasticity are similar to those often required to release neuropeptides. We wanted to determine if neuropeptide release could contribute to post-tetanic potentiation (PTP) and chose neuromuscular synapses in buccal muscle I3a to explore this issue. This muscle is innervated by two motor neurons (termed B3 and B38) that show PTP in response to tetanic stimulation. B3 and B38 use glutamate as their fast transmitter but express different modulatory neuropeptides. B3 expresses FMRFamide, a neuropeptide that only slightly increases its own excitatory junction potentials (EJPs). B38 expresses the small cardioactive peptide (SCP), a neuropeptide that dramatically increases its own EJPs. It was our hypothesis that SCP released from B38's terminals during tetanic stimulation mediated a component of PTP for B38. Because no antagonist to SCP currently exists, we used several indirect approaches to test this hypothesis. First, we studied the effects of increasing stimulation frequency during the tetanus or lowering temperature on PTP. Both of these changes are known to dramatically increase SCP release. We found that increasing the frequency of stimulation increased PTP for both neurons; however, the effects were larger for B38. Decreasing the temperature tended to reduce PTP for B3, while increasing PTP for B38. These results were consistent with known properties of SCP release from B38. Next we selectively superfused the neuromuscular synapses with exogenous SCP to determine if this would occlude the effects of SCP released from B38 during a tetanus. We found that exogenous SCP dramatically reduced PTP for B38 but had little effect on PTP for B3. Thus our results support the hypothesis that physiological stimulation of B38 elicits PTP that is predominantly dependent on the release of SCP from its own terminals. They also demonstrate that the mechanisms underlying PTP can be very different for two motor neurons innervating the same target muscle.     

The effect of secretin on the glucose-induced insulin release from isolated pancreatic islets in mice

A simple and rapid method for isolation of pancreatic islets from mice using Percoll as a separation medium is described. Increasing concentrations of secretin from 10(-11) to 10(-6) M were without effect on the insulin release from the islets at 5 X 5 mM glucose, whereas significant increases were found at 12 mM glucose. Secretin did not elicit additional increases of the insulin release at 5 X 5 and 12 mM glucose with 5 mM theophylline. The mechanism by which secretin acts on the pancreatic islets is discussed. Based upon the above observations it is suggested that secretin apparently represents a modulator of the insulin release from the pancreatic islets.     

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.     

Evidence that the src gene product of Rous sarcoma virus is membrane associated

We have investigated the intracellular localization of the src phosphotransferase and of pp60^src by cellular fractionation. Fractionation of cell lysates by differential centrifugation showed that both the phosphotransferase activity, measured using an immune complex assay, and pp60^src cosedimented with particulate fractions enriched in cellular membranes. Upon further fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients the src phosphotransferase and pp60^src fractionated in parallel with plasma membranes. The phosphotransferase activity remained associated with cellular membranes under conditions of high ionic strength or of high concentrations of divalent cation chelators further supporting a direct membrane association. The src phosphotransferase activity is higher in buffer containing only Triton X-100 detergent as compared to a more complicated detergent mixture, but under these assay conditions the pp60^scr appears to undergo proteolysis to a 52K-dalton polypeptide (pp52^src) which is active as a phosphotransferase. Pyrimidine triphosphates were shown to act as donors in the immune complex assay of src phosphotransferase activity. [γ-³²P]CTP was about one-third as efficient as [γ-³²P]ATP in serving as a donor. Both CTP and UTP were effective inhibitors of src phosphotransferase activity when either radiolabeled ATP or GTP were used as substrates. The ability of src to interact efficiently with both purine and pyrimidine triphosphates contrasts markedly with most well-studied protein kinases which have been shown to use almost exclusively purine triphosphates.     

Evidence that blood group A antigen on lymphocytes is derived from the plasma

Serum from group O volunteers, who had been injected with porcine A blood group substance, was used in lymphocytotoxicity tests. Positive reactions were obtained only with lymphocytes of group A secretors; the strongest reactors were Le(a--b--). The same group O sera reacted with group O lymphocytes which had been exposed to a glycosphingolipid fraction prepared from the plasma of A,Le(a--b--) secretors. These reactions were specifically inhibited by A substance. It is suggested that, unlike the A antigen on red cells, the A antigen detected in lymphocytotoxicity tests is entirely derived from the plasma.     

Monoclonal antibody 4C7 recognizes an endothelial basement membrane component that is selectively expressed in capillaries of lymphoid follicles

In order to define compartment-related structures within the extracellular matrix of human lymphoid organs, monoclonal antibodies (MAbs) were generated by immunizing mice with stromal fragments of human tonsils. One MAb (4C7) was selected which recognized an endothelial basal membrane component that is selectively expressed in capillaries of lymphoid follicles. The epitope was also present in follicles within chronically inflamed synovial membrane and in a hyperplastic thymus of a patient with myasthenia gravis. B-cell non-Hodgkin's lymphomas with a follicular growth pattern expressed the antigen in neoplastic follicles, whereas diffuse growing lymphomas lacked the antigen. The restricted distribution pattern suggests involvement of the 4C7-defined antigen in the organization of the follicular compartment within human lymphoid tissue.     

Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar typhimurium systemic infection

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.     

Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.