Degradation of RNA in rat reticulocytes. II. Relationship between RNase and its corresponding inhibitor in rat reticulocyte

A latent RNase was partially purified by ammonium sulfate fractionation and Sephadex G-75 gel filtration from rat reticulocyte lysate. By mixing free RNase and its inhibitor in vitro, the latent RNase was shown to be a complex of each component. The latent RNase was activated by SH reagents, but no dissociation of the complex was observed. It was also activated by 6 m urea and 0.1 m H₂SO₄, and in these cases the enzyme was released as judged by gel filtration. From the results of this and precious paper, a possible mechanism of the regulation of RNase activity in rat reticulocyte is discussed.     

Erythrocyte sedimentation rate. I. Volume fraction dependence in saline solution

We have measured volume fraction dependence of the sedimentation curve of swine erythrocytes in a physiological saline solution at 10 degrees C, 20 degrees C, 30 degrees C and 40 degrees C. The sedimentation curves were found to consist of initial constant velocity region and final plateau region at the lower temperatures of 10 degrees C and 20 degrees C, while modified S-shaped curves were observed at the higher temperatures of 30 degrees C and 40 degrees C. The volume fraction dependence of the initial slope v of the sedimentation curve was fitted well to the following exponential type equation at all the temperatures: v = vs,exp (1 - H)exp[-(BH + CH2)] where vs,exp is the velocity in infinite dilution corresponding to the Stokes velocity and H is the volume fraction of erythrocytes. The volume fraction dependence of the relative velocity v/vs,exp was in close agreement with a semi-empirical equation derived for slurrys in the field of chemical engineering at the lower temperatures, while a small deviation between the observed and calculated curves was found at the higher temperatures. The volume fraction dependence of v at 20 degrees C was also analyzed on a theory recently developed by Oka. The explicit functional form of the medium up-flow factor phi (H) and the deformability factor f in the theory were determined using the experimental data.     

Induction of cellular DNA synthesis in G0-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses

tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.     

An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene.

A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions.     

Structural diversity of murine serum amyloid A genes. Evolutionary implications

Mouse serum amyloid A (SAA) gene family comprises four members that are closely linked in the chromosome 7. Two of these genes encoding major mouse SAA isotypes (SAA1 and SAA2) are highly homologous not only in exons but also in introns and flanking regions; this sequence homology extends 280 base pairs upstream of major cap sites and 430 base pairs downstream of polyadenylation sites, and the 5' boundary of this homology unit is marked by the CA/GT repeat. Sequence comparison also shows that one (SAA4) of the other two genes is related to the SAA1/2 gene, whereas the other gene (SAA3) evolved independently. Based on these results and the SAA gene arrangement, we discussed mouse SAA gene evolution.     

Accumulation of Galactosylsphingosine (Psychosine) in the Twitcher Mouse: Determination by HPLC

We developed a sensitive and simple procedure for determination of galactosylsphingosine (psychosine), using HPLC. The method involved extraction of lipids, separation by cation-exchange and C18 reverse-phase columns, and derivatization with o-phthalaldehyde. The fluorescent galactosylsphingosine was detected by HPLC. The amount of galactosylsphingosine was accurately assayed by simultaneous determination of glucosylsphingosine, as the internal standard. The detection limit was 0.5 ng/assay tube, and the quantitative range of the method was up to 750 ng. This procedure was applied to tissue from the twitcher mouse, an animal model of human globoid cell leukodystrophy, as well as tissue from normal and carrier mice. In the latter mice, a small amount of galactosylsphingosine was detected in the spinal cord (21.6-37.2 ng/100 mg wet weight) but not in the cerebrum and sciatic nerve. Marked accumulation of galactosylsphingosine was noted in the nervous tissues of the twitcher strain, even on postnatal day 4. The concentration of galactosylsphingosine was greater in the peripheral than in central nervous tissues. The spinal cord and brainstem contained more galactosylsphingosine than did the cerebrum and cerebellum. The concentration increased with age from 764 ng/100 mg in the sciatic nerve at 4 days to 5,910 ng/100 mg at 37 days. These data correlate well with the pathological changes; tissues containing higher concentrations of galactosylsphingosine show earlier and more severe pathological changes than those containing lower concentrations, thereby indicating the close link of galactosylsphingosine to the pathogenesis of the twitcher mouse.     

Distribution of Calcium and Boron in the Pectin Fraction of Tomato Leaf Cell Wall

Calcium was present in the pectin fraction of tomato leaf cellwall in association with pectin constituents and with a pectin-proteincomplex. Boron deficiency induced a decrease in the amount ofCa associated with pectin constituents. Most of the boron inthe pectin fraction obtained by pectinase treatment was in afree form. These results suggest that boron plays an importantrole in Ca metabolism in the cell wall. ¹Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu, Japan. (Received July 31, 1985; Accepted February 18, 1986)     

The minimal requirement of circulation for survival of undelayed and delayed flaps in rats

In an attempt to clarify the delay phenomenon, the regional blood flow was measured in 10 undelayed flaps and 65 delayed flaps in rats by the clearance method with the electrolytically generated hydrogen. The ultimate purpose of this study was to establish the minimal requirement of blood flow for flap survival. The dye distance and the survival length were also measured. Blood flow in the flap increased with the delay period. Elongation of the survival length and the dye distance corresponded well to the circulatory enhancement. The present findings support the theory that delay improves blood circulation. The distance from the base to the 0.04-ml flow point was found to be highly correlated with survival length in the delayed groups as well as in the undelayed group. Based on the results, the minimal requirement of circulation for flap survival was assumed to be approximately 0.04 ml/min per gram of tissue whether or not the flap was delayed.     

Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-

The Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), in Hanks' balanced salt solution, emitted a weak luminescence which was not affected by superoxide dismutase or catalase and was not augmented by resting human granulocytes. In contrast, activated granulocytes caused a dramatic increase in the luminescence of CLA. The light emission by CLA in the presence of activated granulocytes was inhibited by superoxide dismutase, but not by catalase or benzoate. Azide at 0.5 mM did not inhibit light emission significantly. These results indicate that O2-, rather than H2O2, HO., singlet oxygen, or HOCl, was the agent responsible for eliciting the chemiluminescence of CLA. Moreover, the intensity of light emission by CLA correlated with the rate of production of O2- either by activated neutrophils or by the xanthine oxidase reaction.