Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

The effect of starvation on RNA: DNA ratios and growth of larval striped bass, Morone saxatalis

Hatchery reared larval striped bass, Morone saxatilis , 8-days-post-hatching were subjected to various feeding/starvation regimes over a period of 14 days.
Batches of larvae from each treatment were sampled over the 14-day period and subdivided for determination of notochord length and RNA:DNA ratio. The best growth was found in fully fed F1000 larvae (exposed to 1000 Artermia nauplii l⁻¹), which reached 8.2 mm after 11 days and 9.6 mm after 14 days. Starved animals after 11 days had notochord lengths of 4.9 mm. Growth curves from feeding-delayed larvae indicated that animals fed after up to 5 days starvation were capable of complete recovery. F100 larvae (exposed to 100 Anemia nauplii 1^−l) had a slower growth rate than F1000 larvae, reaching a notochord length of 7.3 mm after 14 days. RNA:DNA ratios over time closely followed notochord growth curves, with clear differences between starved, F100 and F1000 larvae being established after only 2 days. Equilibrium RNA:DNA ratios of 3.0 and 2.25 were established in F1000 and F100 larvae, respectively, 6.8 days after the beginning of the experiment. The average lag time between a change from the starved to the fed condition and a change in RNA:DNA ratio as determined by the divergence of the nucleic acid curve from the starved condition was 0.66 days.
In treatments where starvation followed various periods of feeding, larvae regressed in notochord length such that the final length at 14 days reflected the degree of feeding. RNA:DNA ratios in these animals again closely followed growth curves with a lag time of 0.81 days.
It was concluded that RNA:DNA ratios provided very accurate indices of growth in striped bass larvae which were highly sensitive to feeding status.     

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.     

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.