Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.     

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Changes in soil microflora associated with control of Sclerotium Rolfsii by Furfuraldehyde

The efficacy of 2‐furfuraldehyde for control of Sclerotium rolfsii was studied in laboratory and greenhouse experiments. Mycelial growth of the fungus was reduced proportionally with concentrations of 0.1–0.5 ml furfuraldehyde l^‐1 agar medium, and viability of sclerotia diminished on exposure to 2‐furfuraldehyde vapours. Detectable populations of bacteria and fungi, including Trichoderma spp., were reduced significantly (9=0.05) when furfuraldehyde was added to the agar used for soil dilution plates of untreated soil. Repeated treatments of natural soil with the fumigant significantly increased populations of Trichoderma spp. and bacteria, but diminished numbers of actinomycetes. Increasing dosages applied to soil artificially infested with S. rolfsii caused a reduction of disease on lentil, Lens culinaris. Results indicate that the compound, when applied to field soil, changes the composition of soil microflora and has potential for integrated control of S. rolfsii.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Fluctuations in follicular structures of rat thyroid glands during 24 hours: fine structural and morphometric studies

The thyroid follicles of adult male Wistar rats were examined at six evenly spaced times over 24 hr with a morphometric technique. Follicular structures were subjected to distinct variations during 24 hr, with respect to volume and numerical densities of follicles in the thyroid gland, and diameters, volumes, cell numbers, and luminal surface areas of individual follicles. Variations in follicular structures were divided into two phases: a large follicular phase at 1200, 1600, and 2000 hr and a small follicular phase at the other times. Although volume densities of follicles in the gland varied with a small amplitude, diameters, volumes, and cell numbers of individual follicles exhibited distinct fluctuations during 24 hr. Numerical densities of follicles in the gland changed distinctly during the small follicular phase as well. Degenerating follicular cells appeared in the follicular lumen especially at 1600 hr. No mitotic follicular cells were found throughout the experiment. Furthermore, one to three follicular cells of two adjacent follicles were often in contact with each other at 0400, 0800, and 1200 hr, and these follicles were lined by the common basement membranes. These results suggest that the variations in follicular structures during the small follicular phase occur in the form of follicle separation and fusion. Moreover, the morphological and morphometric variations in follicles reflect those in subcellular structures of follicular cells previously reported by us.     

Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(NP)nN, where n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5'-(dimethoxytrityl)deoxyribonucleoside 3'-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5'-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazolide) and can be stores in the reaction solution for up to 2 weeks at 4 degrees C with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95% complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78%, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5'-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5'-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ cGMP phosphodiesterase and photoreceptor potential in gecko retina

The possible involvement of phosphodiesterase (PDE) activation in phototransduction was investigated in gecko photoreceptors by comparing the in situ PDE activity with the photoreceptor potential. In the dark, intracellular injection of cGMP into a gecko photoreceptor caused a long-lasting depolarization. An intense light flash given during the depolarization phase repolarized the cell with a short latency comparable to that of the light-evoked hyperpolarizing response, which indicates that the activation of PDE in situ is rapid enough to generate the photoreceptor potential. PDE activity in situ was estimated quantitatively from the duration of the cGMP-induced depolarization, since it was expected that the higher the PDE activity, the shorter the duration. Under steady illumination, the enzyme exhibited a constant activity. On exposure to a light flash, PDE became activated, but recovered in the dark with a time course that was dependent on the intensity of the preceding stimulus. When PDE activity and photoreceptor sensitivity to light were measured in the same cell after a light flash, both recovery processes showed similar kinetics. Theoretical analysis showed that the parallelism in the recovery time courses could be explained if cGMP is the transduction messenger. These results suggest that PDE activation is involved not only in the generation but also in the adaptation mechanisms of the photoreceptor potential.     

A histochemical study on postnatal growth of the soleus muscles of male and female mice

Postnatal changes in histochemical properties of the soleus muscles were examined in male and female mice aged 5 to 40 weeks. The fiber type composition did not significantly differ between sexes 5 weeks after birth. In males the percentage of type I fibers increased from 5 to 22 weeks of age but did not change thereafter. In females the percentage of type I fibers increased from 5 to 40 weeks of age. As a result, females had significantly higher percentage of type I fibers than males 30 and 40 weeks after birth. The smaller increase in the percentage of type I fibers during postnatal growth in males is suggested to be attributable to the higher testosterone level. In males the cross-sectional area of both type I and type II fibers increased with age. In females, however, the cross-sectional area of type II fibers increased with age whereas that of type I fibers increased from 5 to 10 weeks of age but thereafter decreased gradually. The ratio of mean fiber cross-sectional area of type I fibers to that of type II fibers decreased slightly from 5 to 10 weeks of age but did not change thereafter in males. In females the ratio increased from 5 to 10 weeks but thereafter decreased gradually. The ratio was significantly higher in females than in males in all age groups. The percentage area occupied by type I fibers increased with age in both sexes. The increase was greater in females than in males, however. Furthermore, females had significantly higher value than males in all age groups. No significant age difference and sex difference were observed in the total number of muscle fibers.