Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Accumulation of closed-circular polyoma DNA molecules containing host DNA during serial passage of the virus

The high-multiplicity serial passage of plaque-purified polyoma virus in baby mouse kidney cultures results in the accumulation of closed-circular virus DNA molecules containing covalently linked sequences homologous to reiterated mouse cell DNA. Such molecules can be encapsidated within virions and are probably defective in plaque formation.     

The occurrence of the tropical bedbug (Cimex hemipterus, fabricius) in poultry barns in Israel

The occurrence of the tropical bedbug (Cimex hemipterus Fabricius) in poultry houses in Israel is described. Despite the heavy infestation serious losses have not been registered and no clinical signs observed. Treatment of the barns and accessories with 2% malathion emulsion gave good results. The parasite invaded human habitations as well.     

Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes.

Human granulosa cells were immortalized by transfection of the primary cells with a mutated p53 gene in combination with the Harvey-ras oncogene, yielding established cell lines designated HGP53. Here we report that forskolin, 8-Br-cAMP and FSH modulate cell growth and steroidogenesis in HGP53 cells. Low concentrations of 8-Br-cAMP or FSH stimulated cell proliferation, while higher doses attenuated cell proliferation. Progesterone production was already evident at an FSH concentration of 0.3 mIU/ml and was maximally stimulated (50-135-fold) at 50 mIU/ml of FSH. Expression levels of steroidogenic acute regulatory protein (StAR), adrenodoxin and cytochrome P450scc were enhanced 64-, 48- and 3.1-fold respectively by FSH stimulation. Dexamethasone enhanced FSH/cAMP-induced steroidogenesis and this effect involved a marked elevation in the intracellular level of adrenodoxin and P450scc, concomitantly with a marked decrease in StAR. Conversely, basic fibroblast growth factor attenuated FSH-stimulated progesterone production, and this effect involved reductions in adrenodoxin, P450scc and StAR levels. These data suggest that the rate of steroidogenesis may be determined by the ratio of StAR and P450scc, rather than by the level of each protein alone. Whereas FSH at a low dose slightly reduced apoptosis induced by serum withdrawal from HGP53 cells, higher doses enhanced it. Dexamethasone dramatically attenuated FSH- or forskolin-enhanced apoptosis. In conclusion, FSH-dependent mechanisms of differentiation, luteinization and apoptosis can be preserved in human granulosa cells immortalized by mutated p53. Moreover, this system lends itself to studies on cross-talk between the endocrine and paracrine factors that control these processes.     

Further characterization of the slow muscarinic responses inXenopus oocytes

In immature follicular ocytes of the frogXenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29–0.39 M), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32–56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 M 4-phorbol 12,13-dibutyrate (PDBu). Ca-dependence of the ACh-evoked K current resembles that of ACh-evoked Cl currents, described earlier, and suggests mediation by a similar mechanism, i.e. mobilization of Ca from intracellular stores. On the other hand, most of the features described here are in a sharp contrast to those reported for adenosine-evoked, cAMP-mediated slow K current. Thus, we suggest that purinergic and muscarinic receptors inXenopus follicular oocytes are coupled to potassium channels through different molecular mechanisms.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate - PDE phosphodiesterase     

The association between melatonin and sleep stages in normal adults and hypogonadal men

The role of melatonin in normal sleep-wake regulation has been inferred from the temporal relationships between its cycle and the 24 h cycle in sleep propensity. Pharmacological doses of melatonin were reported to have sleep-inducing effects in insomniacs. The current study investigated the relationship between melatonin and sleep stages in groups of hypogonadal men with abnormal melatonin levels. We were also interested in examining what would happen to these relationships during testosterone replacement therapy. Male patients with hypogonadotropic hypogonadism (IGD, n = 6), constitutional delayed puberty (DP, n = 6), and Klinefelter's syndrome (KS, n = 5) before and during testosterone replacement therapy were studied. Six patients with KS and normal testosterone levels were also studied. Results were compared with those obtained in normal controls (n = 6). Serum samples were obtained at 15 min intervals from 1900-0700h in a controlled light-dark environment with simultaneous polysomnographic sleep recordings. Serum melatonin levels were the highest in IGD and DP and lowest in KS patients. A lower percentage of sleep stage 2 and higher percentage of stage 3/4 were observed in IGD and DP groups while KS patients had higher percentage of stage 2 and lower percentage of stage 3/4 as compared to controls. Slow wave sleep was the highest in IGD and the lowest in KS groups. Serum melatonin levels were lowest in KS groups. Serum melatonin levels were lowest in sleep stage 3/4, higher in stage 2 and highest in REM sleep when all groups were combined and averaged together. However, in the IGD group, melatonin levels were actually lowest in REM sleep. Also in the KS group, melatonin levels were lower in REM than during sleep stage 2. Serum melatonin levels were lowest in sleep stage 3/4 in all groups, higher in stage 2, and highest in REM sleep. During waking periods, melatonin levels were the highest in untreated IGD, DP and KS patients. Testosterone treatment given to these patients, although normalized, their melatonin levels did not statistically significantly change these correlations. These data demonstrate that relative melatonin concentrations are associated with sleep stages in hypogonadal and normal men. The results also indicate that the association between melatonin and the reproductive hormones are independent of the synchronizing effects of melatonin on sleep homeostasis.     

Nidovirus infections: experimental model systems of human neurologic diseases

The presence of terminally differentiated slow- and non-dividing cells in the central nervous system (CNS) provides a safe harbor for viral persistence and latency and constitutes a unique immunologic environment for viral infections. Studies of experimental model systems of viral infections of the CNS provide insight into mechanisms of viral persistence and immune-mediated pathology. Nidoviruses are comprised of 2 families of viruses, coronaviruses and arteriviruses, and are common pathogens of humans and a variety of animal species. Both families of viruses contain neurotropic strains that produce experimental neurologic diseases in rodents. These include acute meningitis and encephalitis; acute poliomyelitis; and chronic inflammatory, immune-mediated, demyelination. Coronavirus-induced demyelinating disease mimics many of the pathologic features of Multiple Sclerosis (MS).