Two major regulatory steps in cholesterol synthesis by human renal cancer cells.

Two major mechanisms regulating cholesterol biosynthesis exist in a human renal cancer cell line, Caki-1. Caki-1 is a newly established cell line whose characteristics of rapid growth and active cholesterol synthesis qualify it as a potentially valuable tool for elucidation of regulatory mechanism of cholesterol synthesis and transport. In the absence of exogenous cholesterol, cholesterol is the dominant sterol arising from labeled acetate and mevalonate. As expected, in the presence of exogenous cholesterol, the conversion of acetate to cholesterol and the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) is markedly reduced and this inhibition is released when cholesterol is removed from the medium. An unexpected and possibly unique finding is the inhibition of the conversion of mevalonate to cholesterol in the presence of exogenous cholesterol. This second major control process results in the accumulation of squalene and may involve additional late steps in cholesterol biosynthesis or metabolism. The occurrence of two major mechanisms regulating cholesterol synthesis may be a unique property of renal cancer cells or a previously unrecognized characteristic of a variety of cultured cells.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

The increase in activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in skeletal muscles of rats after subcutaneous administration of N,N′-dimethyl-para-phenylenediamine

Summary After subcutaneous administration of N,N-dimethyl-para-phenylenediamine (DPPD) in rats, a myogenic myopathy was produced in the skeletal muscles. In this communication, the results of the application of various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases and biochemical techniques for the estimation of activities of oxidoreductases in the experimental skeletal muscles are presented. The most striking result was the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase which increased dramatically during the early phase of the muscle disease. The increase in activity of the pentose phosphate shunt enzymes was the first pathological alteration and was present as early as 8 h after a single injection of DPPD. Histochemical techniques for demonstration of activity of both enzymes are therefore highly suited for the detection of minor diseases and the early onset of major diseases of the neuromuscular system. Some glycolytic enzymes as well as some enzymes of the aerobic part of the metabolism showed an early decrease or increase in activity indicating a metabolic imbalance in the muscle fibres. There were more fibres with an intermediate pattern of the energy yielding enzymes in the experimental muscle specimens then in specimens from the control groups. The activity of the catabolic hydrolytic enzymes was strongly increased in pathological muscles. The aerobic muscles were more vulnerable to DPPD than the anaerobic muscles.     

Rhodotorula matritense sp. nov., isolated from powdered red pepper (Capsicum frutescens L.)

A new species of Rhodotorula Harrison was recovered in May 1978 from Spanish powdered red pepper (Capsicum frutescens L.) in Madrid, Spain. It could not be identified with any hitherto described species of yeast and it was assigned to the genus Rhodotorula Harrison as representative of a new species on the bases of both its morphological and physiological characteristics, for which the name of Rhodotorula matritense is proposed.     

Alpha-ketoglutarate supply for amino Acid synthesis in higher plant chloroplasts: intrachloroplastic localization of NADP-specific isocitrate dehydrogenase

Isocitrate dehydrogenase was found in Pisum sativum chloroplasts purified on sucrose density gradients. A chloroplast-enriched pellet obtained by differential centrifugation formed two chlorophyll-containing bands. The lower one containing intact chloroplasts had NADP-specific isocitrate dehydrogenase and triose-phosphate isomerase activities. Mitochondria and peroxisomes were observed to band well away from the intact chloroplast region, as indicated by peak activities of fumarase and catalase, respectively. The presence of isocitrate dehydrogenase in chloroplasts suggests that chloroplasts may generate at least some of the α-ketoglutarate required for glutamate synthesis.     

Respiratory responses to intravenous and intrapulmonary CO2 in awake dogs

Ventilatory responses to CO2 inhalation and CO2 infusion were compared in the awake dog. The CO2 was introduced directly into the systemic venous blood via a membrane gas exchanger in a femoral arteriovenous shunt circuit, and the extracorporeal blood flow, QX, was maintained constant at one of two rates: low, 0.5 l/min; or high, 2.0 l/min. A total of 13 experiments was performed in four dogs comprising 50 control and 25 inhalation and infusion observations at each of the two flow rates. Comparison of CO2-response curve slopes, S = delta V E/delta PaCO2, between CO2 inhalation and infusion showed no significant difference either within or between flow rates. The mean value of S for all conditions was 1.88 l/min per Torr with a 95% confidence interval of 1.66 -2.14. An independent additive ventilatory drive amounting to 28% of low-flow control VE was found at the highflow rate. We conclude that at constant blood flow the responses to both CO2 inhalation and infusion are hypercapnic and not significantly different.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.