Toxicity of 4-nitroquinoline 1-oxide for Crithidia fasciculata.

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cystein, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

Properties of Two Homologous Alkaline Proteases from Streptomyces rectus

Some physicochemical properties of two thermostable proteases from Streptomyces rectus are described. The enzymes were judged to be identical with respect to molecular weight, inactivation with serine protease inhibitors, and in primary structure by peptide analysis. Amino acid analysis indicated the enzymes had identical compositions except for their amide content. The molecular weights of the enzymes were judged to be 28,000 by sedimentation equilibrium, 26,200 by sedimentation diffusion, and 29,100 from amino acid analysis. Titration of the proteases with diisopropylfluorophosphate and phenylmethane sulfonylfuoride indicate equivalent weights of 28,500 and 32,800 g, respectively, for the proteins. The pentapeptide around the serine residue reacting with diisopropylfluorophosphate was isolated and had the composition: Asx(1), Gly(1), Thr(1), Ser(1), Met(1).     

Structural Rearrangement and Subunit Composition of RNA from Released Soehner-Dmochowski Murine Sarcoma Virions

Two types of genomic, high-molecular-weight RNA species were found in Soehner-Dmochowski murine sarcoma virions released from virus-induced rat tumor cells grown in tissue culture. The type of RNA species observed depended on the length of exposure of the tumor cells to radioactive precursor. Early RNA of virions labeled up to 4 h with radioactive uridine had a sedimentation coefficient of 50S, and late RNA of virions labeled for 24 h had a sedimentation coefficient of 58S. Thermal transitions of early and late RNA indicated a difference in the configuration or structure of these two types of RNA. The late RNA may represent either a different configurational state of the early RNA or an aggregate molecule of two early RNA components joined together. Heat dissociation revealed that the major subunit of both RNA types was a 28S species, which was not susceptible to degradation by the addition of micrococcal nuclease to virions. A transitional, intermediate RNA species with a sedimentation coefficient of 37 to 40S was detected when early RNA was dissociated by dimethyl sulfoxide or heat at temperatures suboptimal for complete conversion. No free RNA subunit components were detected in virions harvested at intervals as short as 30 s or 5 min. A model for the assembly of genomic RNA from 28S RNA subunits is proposed.     

Electrotonic coupling between cercal afferents and giant interneurons in the American cockroach

Stimulation of the cercal nerve of the female American cockroach evokes a short-latency action potential in one giant axon in the ipsilateral connective of the ventral nerve cord. Neither procion yellow nor cobalt passes from the nerve cord into the cercal nerve, and the short-latency response disappears several weeks after removal of the cercus. Therefore, the short-latency spike is not due to a branch of the giant interneuron extending into the cercal nerve, but is presumably due to electrotonic coupling of cercal afferents to the giant. Responses of the presumed electrotonic junction to drugs, varied ionic concentrations and tonicity, and to cold are described. These responses and the impermeability of the junction to procion yellow suggest that the coupling is not by means of a gap junction. There is evidence for electrotonic coupling to another giant axon in the female, but this junction does not ordinarily transmit a spike. Electrotonic coupling is rare in males. In some females action potentials in giant interneurons excite cercal afferents electrically, and the afferents then re-excite the giants chemically. Electrotonic coupling may reduce fatigue and habituation of chemical synapses by depolarizing presynaptic terminals whenever the giants are active.     

The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of 8 prostaglandins (PG) on growth and sulfate incorporation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA₁, PGB₁, PGE₁ and PGE₂ reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA₁ was the most potent, being effective at a concentration of 2.5 μg/ml [6.8 μM] while the others required 25 μg/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 μg/ml. At the higher concentration, PGA₁ was highly cytotoxic, and PGB₁ as well as PGE₂ reduced cell growth. The cytotoxicity of PGA₁ was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA₁ and PGB₁ was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.     

A radioimmunoassay for 13,14-dihydro-15-keto prostaglandin F2α with chromatography and internal recovery standard

Antibodies to the 13,14-dihydro-15-keto metabolite of prostaglandin F_2α(PGF_2αM) were raised in sheep using a bovine thyroglobulin conjugate of PGF_2αM. Labeled 13,14-dihydro-15-keto prostaglandin A₂ (PGA₂M), 13,14-dihydro-15-keto prostaglandin E₂ (PGE₂M) and PGF_2αM were prepared from their corresponding high specific activity parent prostaglandins with swine kidney homogenate and purified using reverse phase liquid-liquid partition chromatography. A rapid method of column chromatography for use prior to radioimmunnoassay was developed.Mathematical corrections for the effect of recovery tracer on the logit/log transformation are presented with a new approach to expression of radioimmunoassay cross reactions allowing continuous expression of the variation of these cross reactions with displacement. These mathematical approaches are widely applicable to other radioimmunoassay systems and transformations.The assay was used for measurement in groups of human volunteers: males, females, women at delivery and paired venous umbilical cord bloods. A correlation between venous cord and maternal peripheral PGF_2αM levels is demonstrated with a gradient from the cord plasma to maternal plasma.     

Separation of the Infectious Ribonucleic Acid of Potato Spindle Tuber Virus from Double-Stranded Ribonucleic Acid of Plant Tissue Extracts

The infectious ribonucleic acid (RNA) of potato spindle tuber virus (PSTV) can be separated by hydroxyapatite chromatography from double-stranded RNA detectable in low amounts in both infected and uninfected plant tissue extracts. The chromatographic behavior of ribonuclease-sensitive PSTV RNA resembles that of transfer RNA.     

Rapid prenatal and postnatal detection of inborn errors of propionate,methylmalonate, and cobalamin metabolism: A sensitive assay using cultured cells

Summary A sensitive, reliable, and easily performed procedure is described for the prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism using cultured amniotic cells and skin fibroblasts. With this assay, control fibroblast lines incorporated a mean of 6.89 nanoatoms ¹⁴C/mg protein from [1-¹⁴C]propionate into trichloroacetic acid (TCA)-precipitable cell material in 10h. Twenty-five mutant fibroblast lines from patients with propionicacidemia or one of the methylmalonicacidemias fixed 0.04 to 0.93 nanoatoms ¹⁴C/mg. Considerable variation was observed, both among and within discrete mutant classes, with respect to the residual amount of propionate pathway activity, possibly reflecting further molecular heterogeneity in these disorders.We applied this procedure to cultured amniotic cells from controls and 4 midtrimester pregnancies at risk for methylmalonicacidemia and diagnosed one fetus with a methylmalonyl CoA apomutase defect and 3 fetuses which were unaffected.Presented in part at the annual meeting of the Society for Pediatric Research, St. Louis, Missouri, April 1976.