Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Effect of captopril on renal vascular resistance, renin, prostaglandins and kinin in the isolated perfused kidney

Vasodilatory and natriuretic effects of captopril were studied in the isolated hog kidney perfused with modified Krebs-Ringer solution. Renal arterial infusion of captopril caused increases in releases of renin, prostaglandins (PGE2, 6-keto-PGF1 alpha and PGF2 alpha) and kinin, and was accompanied by a decrease in the renal vascular resistance and an increase in urinary sodium excretion. Indomethacin administered with captopril diminished the saluretic effect of captopril and evoked an increase in kinin, but was associated with a marked decrease in prostaglandin and renin releases, while renal vascular resistance remained decreased. Indomethacin alone did not alter vascular resistance and kinin; however, renin and prostaglandin releases were decreased. Aprotinin administered with captopril showed a decrease in releases of prostaglandins, renin and kinin without any change in vascular resistance. These results suggest that increased release of kinin induced by captopril contributes to a reduction in renal vascular resistance. Increased prostaglandin release after captopril administration may be caused by an increase in kinin without direct involvement of captopril in prostaglandin synthesis. Renal prostaglandins may enhance sodium excretion and mediate renin secretion in captopril perfusion.     

Effect of Phleomycin on Polyoma Virus Synthesis in Mouse Embryo Cells

The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.     

Separation of the Polypeptides of Chlamydia and Its Cell Walls by Polyacrylamide Gel Electrophoresis

The polypeptide composition of Chlamydia was examined by acrylamide gel electrophoresis. When the polypeptide patterns of purified infectious elementary bodies (EB) of C. psittaci meningopneumonitis strain, 6BC strain, and C. trachomatis T'ang strain were compared, no significant differences were observed. The polypeptide patterns of whole EB and reticulate bodies (RB) appeared to overlap, but differences were found. In EB cell walls, nine main and several minor bands of polypeptides were observed in gels containing sodium lauryl sulfate, and the eighth main band from the top of the gel stained positive with periodic acid-Schiff reagent. On the other hand, the polypeptides in bands 3, 6, and 8 in EB cell walls were missing or minor in RB cell walls, and the ninth band was clearly stained by PAS. Band 8 was also stained slightly. Purified subunits, which occur as a lattice structure on the inside layer of EB cell walls but are largely missing in RB cell walls, contained bands 4, 6, and 8, and band 8 was PAS positive. These results indicate that significant polypeptide synthesis or reorganization in the cell walls occurs during the growth cycle.     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

Endothelin activates the vascular renin-angiotensin system in rat mesenteric arteries

The effects of endothelin on the vascular renin-angiotensin system were examined in isolated perfused rat mesenteric arteries by measuring vascular renin activity and angiotensin II released into the perfusate. Infusion of endothelin (10(-9)M and 10(-11)M) increased the vascular renin activity and angiotensin II release. Pretreatment with nicardipine (10(-6)M), a calcium channel blocker, significantly suppressed these effects of endothelin. These results suggest that endothelin activates the vascular renin-angiotensin system via intracellular calcium metabolism. Vascular angiotensin II produced by endothelin may modulate the local effect of endothelin on the resistance vessels.     

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.     

Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic Compounds

The effects of allyl, sulfur and cyanogenic compounds on thegermination of upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds were examined. Mercaptoethanol and methylmercaptan aswell as KCN, substrates for rßcyanoalanine synthase(CAS), and H₂S and thiocyanate, the products of the CAS catalyzingreaction, were effective in promoting germination, suggestingthe involvement of CAS in germination. Most of allyl compounds, especially allylthiourea, as well asethylene which activated CAS [Hasegawa et al. (1994) Physiol.Plant. 91: 141], promoted the germination in an abnormal typewhich occurred by the predominant growth of cotyledons as didC₂H₄ [Katoh and Esashi (1975) Plant Cell Physiol. 16: 687].However, they failed to activate CAS unlike ethylene, and toliberate free ethylene during an incubation period. It was thuspossible that an C₂H₄-like double bond within allyl compoundscan act to promote seed germination. (Received June 10, 1996; Accepted August 21, 1996)