Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed by tsA mutant of simian virus 40

Primary rat embryo cells were transformed by a tsA mutant (tsA640) of simian virus 40 (SV40). Proliferation of all four independent diploid transformants was suppressed at a nonpermissive temperature (40.3 degrees C), being accompanied by a marked increase in the fraction of cells with a 4N DNA content (a 4N peak in the flow cytofluorogram). However, in this case, the fraction of cells with a 2N DNA content (a 2N peak in the flow cytofluorogram) was preserved. Both effects (suppression of proliferation and increase in the 4N peak) diminished when transformed cells were superinfected with wild-type SV40. The increased 4N peak was preserved, albeit not completely, for at least 24 hours, when cells were further incubated in the presence of hydroxyurea at the nonpermissive temperature. On the other hand, the preserved 2N peak all but disappeared within 24 hours, when cells were further incubated in the presence of colcemid at the nonpermissive temperature. These results suggest that the thermolabile large T antigen of SV40 directly or indirectly induces an accumulation of cells with a 4N DNA content, at the nonpermissive temperature, by prolonging the G2 (and/or late S) period.     

On the enzyme system obtained from some mutants of Bacillus brevis deficient in gramicidin S formation

Twenty mutants of Bacillus brevis which were deficient in gramicidin S formation were isolated by N-methyl-N′-nitrosoguanidine treatment. In addition to three groups which have been previously classified, further two groups were established according to their characteristics of amino acid activating enzymes concerned with gramicidin S formation. The fourth group mutants had a phenylalanine activating enzyme, but they had an enzyme complex from which one specific enzyme among proline, valine and leucine activating enzymes was deleted. Some of them also the ability to form d-phenylalanyl-l-prolyl diketpiperazine (DKP) even though they had phenylalanine and proline activating enzymes. The fifth group mutants contained both a phenylalanine activating enzyme and a complex of prodine, valine, ornithine and leucine activating enzymes like as a wild strain, but did not synthesize gramicidin S, and also one of them could not form even DKP.Combination of enzymes from DKP (+) mutants of the fourth or fifth groups with the first group mutant which had an intact proline, valine, ornithine and leucine activating enzyme complex showed gramicidin S formation, but the combination of enzymes from DKP (−) mutants except a proline activating enzyme minus mutant with the first group mutant could not synthesize gramicidin S.     

Detection of mucosal and serum antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay (ELISA) with rough-surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB-CP). A successful method for indirect coating of glass bead surfaces with HITB-CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent-antibody assay. The assay was applied to the measurement of antibodies to HITB-CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.     

Construction of plasmid vectors for cloning of promoters using the dihydrofolate reductase gene of Escherichia coli K-12

Vectors for cloning promoter-DNA fragments were derived from pTP 30-5 which was constructed in our previous work (M. Iwakura et al. (1982) J. Biochem. 91, 1205). The selection was based on the expression of trimethoprim resistance of transformed bacteria in which the enhancement of dihydrofolate reductase production was directed by the cloned promoter. A linear relationship between the content of dihydrofolate reductase and the strength of trimethoprim resistance was observed. It is suggested that the promoter activities can be estimated by trimethoprim resistance without measuring the enzyme activities.     

Bioinformatics tools for the study of microbial diversity

Although computers are capable of storing a huge amount of data, there is a need for more sophisticated software to assemble and organize raw data into useful information for dissemination. Therefore we developed tools that assist in gathering and categorizing data for the study of microbial diversity and systematics. The first tool is for data retrieval from heterogeneous data sources on the INTERNET. The second tool provides researchers with a polyphasic view of microbes based on phenotypic characteristics and molecular sequence data.     

Brain transfer of a new neuromodulating ACTH analog, ebiratide, in rats.

The transfer of ebiratide into the brain was examined in rats. Its brain levels after intravenous administration (2-20 mg/kg), determined by radioimmunoassay, peaked at 5 min and declined almost in parallel with the plasma levels. Brain/plasma ratios (B/P) were constantly about 0.05 ml/g at all doses. Brain distribution study at 5 min after 125I-ebiratide at an effective dose (0.4 microgram/kg) revealed that unchanged ebiratide had larger B/P than the metabolites and region selectivity. The combined use with unlabeled ebiratide resulted in marked decreases in B/P, particularly in the hippocampus, suggesting a specific uptake of this peptide.     

Neural control of contraction in isolated submucosal gland from feline trachea

To determine the autonomic innervation to myoepithelial cells of submucosal gland, we applied electrical field stimulation (FS) to the intrinsic nerves in isolated submucosal glands from feline tracheae. FS induced contraction that was voltage or frequency dependent and abolished by pretreatment with tetrodotoxin. DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) did not produce any significant contraction, and pretreatment with hexamethonium did not alter the response to FS. Atropine inhibited the contractile response to FS and neostigmine augmented the response to FS. Serotonin also augmented the response to FS, whereas the response to methacholine remained unchanged in the presence of serotonin. Phentolamine reduced the response to FS by 15% of control, whereas propranolol induced no significant changes in the response to FS. No significant inhibitory responses were observed by FS. Our findings indicate that the contraction of tracheal submucosal glands is mediated mainly by cholinergic nerves via muscarinic receptors and in small part by adrenergic nerves via alpha-receptors, and serotonin potentiates the contractile response to FS at the postganglionic nerve.     

A lambda-shaped retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus

We identified a morphologically uncommon piscine retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus. This lentis muscle has a shape similar to the Greek small letter lambda (λ). The two legs of the muscle are attached to the retinal periphery at the ventral eyecup, while the tip is connected to the lens surface by a ligament. Scanning electron microscopy showed that the fibers of the lentis muscle run along the length of both the anterior and posterior legs. Immunolabeling with antiacetylated tubulin antibody and neuronal tracing with DiI of the whole lentis muscle revealed that the anterior leg is innervated by one or more nerves. The topographic distribution of ganglion cells in the retina was investigated to identify the visual axis. Three high cell density areas were observed in the dorso-temporal, ventro-nasals and ventro-temporal retina. These findings suggest that the λ-shaped lentis muscle may enable accommodatory movement of the lens toward the temporal as well as the nasal and/or ventral retina.