In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

Energy-dependent transport of nickel by Clostridium pasteurianum.

The mechanism of nickel transport by Clostridium pasteurianum was investigated by using 63NiCl2 and a microfiltration transport assay. Nickel transport was energy dependent, requiring either glucose or sucrose; xylose and o-methyl glucose did not support growth, butyrogenesis, or transport. Transport was optimum at pH 7 and 37 degrees C, and early-stationary-phase cells had the highest propensity for nickel transport. The apparent Km and Vmax for nickel transport approximated 85 microM Ni and 1,400 pmol of Ni transported per min per mg (dry weight) of cells, respectively. On the basis of metal specificity, nickel appears to be transported primarily by a magnesium transporter, although an alternative nickel transporter may also be involved. ATPase inhibitors (N,N'-dicyclohexylcarbodiimide, tributyltin chloride, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and quercetin), protonophores (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and gramicidin D), metal ionophores (valinomycin, monensin, and nigericin), benzyl viologen, carbon monoxide, and oxygen inhibited nickel transport. Nickel transport was coupled indirectly to butyrogenesis and was dependent on the energy state of the cell.     

Impaired production of gamma-interferon by newborn cells in vitro is due to a functionally immature macrophage

The decreased production of gamma-(PHA-induced) interferon (IFN) by leukocytes of normal newborns could be due to functionally immature T cells, macrophages, or both. We studied gamma-IFN production by macrophages and T cells, alone and in combination, obtained from 50 cord blood samples and 14 adult blood samples in a series of experiments. Adherent macrophages were cultivated for 7 days before the addition of T cells. After 48 hr, PHA-stimulated macrophage-T cell supernatants were harvested and assayed for IFN by a microassay. Macrophage-T cell cultures of autologous and nonautologous cells in 14 adults showed enhanced IFN production (GMT 121 +/- 5 IU) as compared with Ficoll-Hypaque mononuclear cells (GMT 42 +/- 5 IU). No IFN was detected in supernatants from PHA-stimulated Ficoll-Hypaque cord cells alone or macrophage-T cord combined cultures. Combined cord macrophages and adult T cells produced minimal IFN (GMT 13 +/- 3 IU); however, cord T cells combined with adult macrophages showed enhanced IFN production (GMT 195 +/- 47 IU). This cord macrophage dysfunction was not due to an inhibitor and improved with the time of in vitro cultivation. These results indicate that the neonatal macrophage is primarily responsible for the impaired gamma-IFN response by the newborn cells.     

Mutator Gene of Escherichia coli B

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.     

A ‘Problematic fossil’ revealed:Pycnoporidium ? eomesozoicum Flügel, 1972 (Late Triassic,Tethys)—not an enigmatic alga but a strophomenid brachiopod (Gosaukammerella n.g.)

Summary The microproblematicumPycnoporidium ? eomesozoicum Flügel, 1972, from Upper Triassic reefs of the Alpine-Mediterranean region, Turkey Oman and Iran (originally interpreted as possible alga) represents the type species of a new strophomenid brachiopod genus (Gosaukammerella n.g.). The genus is characterized by a very small, millimeter-sized plano-convex shell, whose ventral valve is attached to the substratum (mainly sponges) by symmetrically arranged outgrowths developing from a pseudopunctate, lamellose foliated shell wall and composed of densely spaced subparallel ‘tubes’ comparable with productide spines secreted by papillose extensions of the mantle.Gosaukammerella seems to be the only reliable candidate for the existence of post-Paleozoic strophomenid (productid ?) brachiopods. Gosaukammerella eomesozoica is restricted to possibly cryptic, shaded reef environments inhabited predominantly by sponges serving as substrates for micromorphic brachiopods.     

Hepatic Gluconeogenesis and the Activity of PDH in Individual Tissues of GTG-Obese Mice Following Adrenalectomy

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 ± 0.7g; GTG: 44.1 ± 0.4g; p<0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 ± 1.6 mmol/L; GTG: 29.4 ± 1.9 mmol/L; p<0.05) and serum insulin levels (GTG ADX: 76 ± 10μ.U/mL; GTG: 470 ± 63μU/mL; p<0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 ± 10 nmol glucose/10⁶ cells; GTG: 403 ± 65 nmol glucose/10⁶ cells; p<0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 ± 27 μmol/liver; GTG: 614 ± 60 pmol/Iiver; p<0.05) and fatty acid content (GTG ADX: 101 ± 9 mg fatty acid/g liver; GTG: 404 ± 40 mg fatty acid/g liver; p<0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.     

Intercellular junctions in the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela)

Summary Intercellular junctions in the uterine epithelium of the ovoviviparous urodele Salamandra salamandra were studied in pregnant and non-pregnant females by freeze-fracture technique. Junctional complexes consist of zonulae occludentes (tight junctions) and numerous maculae adhaerentes (desmosomes); z. adhaerentes and nexuses (gap junctions) could not be identified. Tight junctions are of the flexible type exhibiting loosely interconnected fibrils. The fibrillary network appears stretched more often in pregnant females possibly due to the mechanical stress of pregnancy. The structure and occurrence of the junctions identified, especially that of the tight junctions, is discussed with regard to the functions of the uterus during pregnancy.Zusammenfassung Mit Hilfe der Gefrierbruchtechnik wurden im Uterus-epithel trächtiger und nichtträchtiger Feuersalamanderweibchen (Salamandra salamandra) Zonulae occludentes und Maculae adhaerentes, jedoch keine Z. adhaerentes sowie Nexus identifiziert. Die Z. occludentes sind flexibel. Ihr fibrilläres Netzwerk ist bei trächtigen Weibchen öfter gestreckt; das ist möglicherweise auf die stärkere Ausdehnung des Uterusgewebes während der Trächtigkeit zurückzuführen. Das Vorkommen der verschiedenen Kontakt-strukturen, namentlich das der Z. occludentes, wird im Hinblick auf die Funktionen des Uterus während der Trächtigkeit diskutiert.We are indebted to Mrs. K. Ott for excellent technical assistance and to Miss Dr. U. Beigel for linguistic help