Reduced calcium and inhibition of protein kinase C mimic the enhancement of ornithine decarboxylase activity of prolactin in Ambystoma tigrinum tissues

We previously reported that prolactin (PRL) could increase the activity of ornithine decarboxylase (ODC) in liver slices taken from larval tiger salamanders (Ambystoma tigrinum). This action of the hormone was inhibited by oxytocin (OT), the calcium ionophore A23187, and diacyglycerol (DG) and was duplicated by 10 microM verapamil (VML), a calcium channel blocker. Here, we expand these results to show that 1) a higher dose of VML (50 microM) produces an additive effect with PRL; 2) addition of small amounts of calcium (0.1 mM) to the liver culture medium blocks PRL action; 3) neither nifedipine (NIF), a different type of calcium channel blocker, nor EDTA alter PRL action; and 4) gossypol, a reported inhibitor of protein kinase C, mimics PRL action. Additionally, we show that PRL increases ODC activity in tiger salamander tail skin in vitro, a tissue previously demonstrated to be a PRL target tissue in this species. The same set of treatments which we have shown to modify PRL effects on ODC in liver slices affects PRL action in the tail skin in a parallel manner. Thus, the mechanism whereby PRL enhances ODC activity appears to be the same in both these tissues. These results are discussed in conjunction with the findings from similar studies using mammalian tissues in an attempt to assess the current picture of the mechanism of PRL action and the possible role of inositol phospholipid turnover, calcium, and protein kinase C in the action of this hormone.     

Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells.

The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane.     

Molecular cloning and characterization of form I antigen genes of Shigella sonnei.

Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12.6 kb consisting of the two contiguous HindIII fragments of 1.26 kb and 12.4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12.6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12.6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.     

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.     

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.     

Staphylococcal enterotoxin B: immunolabeling and visualization of target cells

Binding of staphylococcal enterotoxin B (SEB) to cultured cells and to tissue sections containing presumed target sites was detected by use of an immunofluorescence sandwich technique. A triple sandwich with successive incubations of SEB, rabbit anti-SEB, and fluorescein-conjugated goat anti-rabbit secondary antibody was applied to samples. Binding of SEB to rat basophilic leukemia (RBL) cells, mast cells of rat dorsal skin, and cells of leukocyte-enriched human plasma was observed. Our results point out and reinforce the reported involvement of SEB in various biological effects that appear to implicate leukocytes, either as mast cells residing in tissues or as white cells circulating in the bloodstream.     

Measurement of extracellular space in the rabbit AV node

We used quantitative histochemistry to measure the size of the extracellular space (ESC) in various regions of the rabbit heart. When inulin, sucrose, and sorbitol were used as ECS markers, the ECS of the AV-nodal tissue was found to be, respectively, 2.4, 2.2, and 2.5 times larger than that of left ventricular muscle. Glucose was also measured over a 50-fold serum concentration range as an extracellular marker for AV-nodal tissue, left ventricular muscle, and Purkinje fibers. Measurements with glucose also revealed that the ECS of the AV node was 2.5-2.8 times larger than that of ventricular muscle. In contrast, the ECS of the AV node was the same as that of Purkinje fibers when glucose was used as an extracellular marker. ATP content, measured as an intracellular marker, was similar in both AV-nodal and contractile tissue. Collectively, the data obtained with all extracellular markers indicate that the ECS of the AV-nodal region is approximately 2.5 times larger than that of adjacent contractile tissue. Differences in the size of the ECS in various regions of the heart probably have functional significance and should be considered appropriately during the interpretation of data obtained by biochemical and densitometric approaches.     

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.     

Antibiotic susceptibility, hemolysin production and haemagglutinating activity of uropathogenic Escherichia coli

The hemolysin production, haemagglutinating activity (HA) with human 0 group erythrocytes and antibiotic susceptibility of 130 uropathogenic Escherichia coli strains were studied. 43% of the strains produced hemolysins and 39% showed haemagglutinating activity. In 12% of the haemagglutinating strains HA was inhibited by D-mannose. 45% of the hemolytic strains showed haemagglutinating activity. There was a significant relationship between hemolysin production and haemagglutination activity (p less than 0.05). 85% of the 130 Escherichia coli strains were found to be multiple resistant to antibiotics.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.