Formation of apolar ecdysteroid conjugates by ovaries of the house cricket Acheta domesticus in vitro.

The newly laid eggs of the house cricket Acheta domesticus contain apolar ecdysteroid conjugates, which we have hypothesized to be ecdysone long-chain fatty acyl esters [Whiting & Dinan (1988) J. Insect Physiol., in the press]. The ovaries of mature adult female A. domesticus in vitro convert [3H]ecdysone into apolar conjugates identical with those found in newly laid eggs. Comparison of the radioactive metabolites produced on incubation of [3H]ecdysone with various organs of adult female A. domesticus in vitro indicate that the fat-body is the major producer of polar ecdysteroid metabolites at this stage of development, whereas the ovaries are the major site of production of apolar metabolites. Apolar metabolites are also produced to a lesser extent by the crop, gut sections and the fat-body. Hydrolysis of radioactive metabolites produced by the ovaries with Helix enzymes releases only [3H]ecdysone, and thus ecdysone is not metabolized before conjugation by the ovaries. Formation of chemical derivatives (acetonide and acetates) of these 3H-labelled apolar conjugates strongly indicates that the position of conjugation is through the hydroxy group at C-22 of ecdysone. Extensive chromatographic analysis of the 3H-labelled apolar metabolites produced by the ovaries by t.l.c. and h.p.l.c. and comparison with authenticated reference compounds have conclusively demonstrated that the conjugates consist of ecdysone esterified at C-22 to a mixture of common long-chain fatty acids. The major fatty acyl esters have been identified and their percentage contribution to the mixture determined: laurate (0.5%), myristate (2.8%), palmitate (25.8%), stearate (8.4%), arachidate (1.0%), oleate (15.7%), linoleate (38.8%) and linolenate (2.1%). In addition there are three minor unidentified peaks, one of which has been tentatively identified as ecdysone 22-palmitoleate (2.6%). Comparison of this percentage composition with the previously published fatty acid composition of A. domesticus haemolymph [Wang & Patton (1969) J. Insect Physiol. 15, 851-860] reveals remarkable similarities, indicating that the acyl transferase(s) forming the conjugates have a broad specificity with regard to the fatty acyl substrate.     

Lipase production of Staphylococcus carnosus in a dialysis fermentor

Summary In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.     

Nyctohemeral Rhythms of Gonadal, Thyroid and Pineal Function in the Hyperprolactinemic Male Rat

In young adult male rats bearing a donor anterior pituitary gland grafted for 3 weeks under a kidney capsule, serum prolactin (PRL) concentrations were elevated and exhibited a rhythm with the highest values in the light phase. Serum PRL in control animals did not exhibit a significant rhythm. Eutopic pituitary PRL content, manifesting a biphasic (12-hr) rhythm with crests during the day and night in controls, exhibited a similar pattern in grafted rats though an overall reduction in pituitary PRL content was seen in the grafted animals. Neither the normal biphasic serum testosterone rhythm nor the normal 24-hr rhythm (nocturnal surge) of pineal N-acetyltransferase activity and melatonin content were altered in the hyperprolactinemic rats. Serum thyroxine (T₄) and triiodothyronine (T₃) and their free indices (FT₄ I, FT₃ I) and serum thyrotropin (TSH) were highest during the day in controls and grafted rats and a 12-hr rhythmic component was detected in data for these variables. In the grafted animals, the 12-hr component was reflected in an additional peak at night detectable by testing of means. The overall serum T₄ FT₄ I, and TSH levels were lower in grafted rats though overall T₃ and FT₃I levels did not differ between grafted and controls. T₃ uptake (T₃ U) values were similar between controls and grafted rats, in both cases exhibiting a fall during the night. Changes in serum thyronines could not be explained by changes in serum binding as assessed by the T₃U₃ and thus may represent changes in thyroidal secretion of T₄. The rhythm in serum PRL of grafted rats suggests the presence of rhythmic circulating factor(s) capable of influencing ectopic lactotrophs. The reduced eutopic pituitary PRL content suggests a role for PRL in influencing eutopic lactotrophs in the pituitary-grafted hyperprolactinemic male rat model. Though circulating testosterone and pineal melatonin synthesis were not altered in this model, thyroid function appeared to be so.     

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.     

The membrane dialysis bioreactor with integrated radial-flow fixed bed —a new approach for continuous cultivation of animal cells

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran^® carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l^?1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran^® carriers with 3–5 mm diameter showed an advantage compared to those with 1–2 mm diameter. For the 3–5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s^?1 and 0.75 mm s^?1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.     

Role of immune cells in protection against and control of reovirus infection in neonatal mice.

We studied the role of T cells in resistance to reovirus intestinal and central nervous system infection. Transfer of reovirus-immune adult spleen cells protected neonatal mice from (i) lethal infection with reovirus serotype 3 Dearing (T3D, footpad inoculation) and serotype 3 clone 9 (T3C9, oral inoculation) and (ii) hydrocephalus caused by serotype 1 Lang (T1L, intracranial [i.c.] inoculation). Cell-mediated protection was not serotype specific. While immune cells protected against T1L i.c., they failed to protect against 1/5,000 of the dose of T3D i.c. Two types of experiments showed that both CD4 and CD8 T cells are involved in reovirus resistance. First, immune cell-mediated protection against T3D was abrogated by in vivo treatment with anti-CD4 monoclonal antibody (MAb) and significantly inhibited by in vivo treatment with anti-CD8 MAb. Second, T3C9-infected neonatal mice treated with anti-CD4 and/or anti-CD8 developed a novel disease phenotype, an oily hair syndrome, associated with severe hepatobiliary pathology and increased viral titer in heart and liver. Immune cells and an MAb to the cell attachment protein sigma 1 (MAb G5) protected by different mechanisms. Immune cells were more effective than sigma 1 MAb G5 at controlling primary replication, while sigma 1 MAb G5 was more effective than immune cells at inhibiting neural spread of virus. We conclude that both CD4 and CD8 T cells are important for reovirus resistance, that cells and antibody act preferentially at different stages in pathogenesis in vivo, and that adoptively transferred immune cells can protect both the central nervous system and intestine.     

Effects of non-MHC loci on resistance to retrovirus-induced immunodeficiency in mice

Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1 ⁿand H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1 ^band other H-2 haplotypes including b, s, and q. The Fv-1 ^b, H-2 ^rstrain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2 ^r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII×RIIIS) F₁, F₂ and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V _\complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.Abbreviations MuLV murine leukemia virus - MCF mink cell focus-inducing MuLV - B6 C57BL/6 - BM5d the defective virus in LP-BM5 MuLV - MAIDS murine acquired immunodeficiency syndrome - RIIIS RIIIS/J - B10.RIII B10.RIII (71NS)/J - MLR mixed lymphocyte reaction - FACS fluorescence activated cell sorter     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora.

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

General recombination mechanisms in extracts of meiotic cells

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated s-rec and m-rec to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.