Prostaglandin induction of spawning behavior in Cichlasoma bimaculatum (Pisces cichlidae)

Prostaglandin (PG) stimulates female spawning behavior in goldfish and in some other teleosts in which female reproductive behaviors consist of postovulatory oviposition acts. This study examined the effects of PG on female sexual behavior in a teleost fish, Cichlasoma bimaculatum, in which female reproductive behaviors involve both preovulatory courtship and substrate cleaning behaviors, and post-ovulatory oviposition behavior. In females of established pairs, PGF2 alpha injection (5 micrograms, im) at any stage of the spawning cycle, or in the parental phase, rapidly induced substrate cleaning which soon merged into oviposition behavior (without egg release). These results support a role for PG in oviposition behavior of Cichlasoma. However, indomethacin (1 mg, ip), a PG synthesis inhibitor, did not block oviposition in ovulated females which had begun to spawn. Indomethacin may not have lowered PG levels sufficiently. Alternatively, as shown by J.J. Polder (1971, Neth. J. Zool. 21, 265), oviposition behavior may be induced or maintained by other factors associated with the spawning situation.     

Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

A comparison of the changes in the non‐neuronal cell populations of the superior cervical ganglia following decentralization and axotomy

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non‐neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double‐labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 68–79, 2002     

Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypeptides (61 kDa and 28 kDa) were observed in urease-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypeptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for enzyme activity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacteria and ultrathin cryosectioned bacteria the enzyme was located on the cell surface and in material apparently shed from that surface.