Peroxisomal oxidation of thiazolidine carboxylates in firefly fat body, frog retina, and rat liver and kidney.

D-amino acid oxidase is a widely distributed peroxisomal enzyme whose principal natural substrates are still unknown. Thiazolidine carboxylates, their derivatives and relatives, and the intermediates in their metabolism are among the more plausible substrate candidates. Using a cytochemical procedure, we have explored the distribution of peroxide-generating enzymatic activity against two thiazolidine carboxylates. We find that these compounds are effective substrates for peroxisomal oxidation in a variety of tissues that contain peroxisomal D-amino acid oxidase. Reaction was seen in the "classical" peroxisomes of rat liver and kidney, the peroxisomes of the fat body of firefly and of Drosophila and the peroxisomes of frog retina. Interestingly, both with the thiazolidine compounds and with more traditional D-amino acid oxidase substrates, the fireflies' photocyte granules, which are peroxisomes, lack activity.     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

Arthrite rhumatoide traitée par noréthynodrel associée à mestranol: Aspects cliniques et tests de laboratoire

A cause de l''effet favorable de la grossesse sur l''activité de l''arthrite rhumatoïde, on s''est demandé si une pseudo-grossesse, produite par un progestatif de synthèse n''entraînerait pas une rémission du moins partielle de cette maladie.Noréthynodrel associée à mestranol (Enovid), 30 mg./jour, a été administrée à 44 femmes pendant quatre à 24 mois. A cause d''effets secondaires indésirables, 11 patientes furent soustraites de l''investigation. Les résultats s''appuient sur 33 cas. Une rémission apparente complète s''est manifestée chez sept patientes aux stades précoces de la maladie; chez 15, une amélioration objective des signes inflammatoires a été observée; chez quatre, une amélioration subjective seule a été notée; chez sept, il n''y a eu aucune amélioration. Dix-sept femmes sur 36 ont présenté une altération d''un ou plusieurs tests hépatiques. Trois présentèrent un ictère cholestatique. Les 17-OH plasmatiques se sont élevés à trois ou quatre fois la normale.De cette étude il ressort que noréthynodrel associée à mestranol peut produire une atténuation des signes inflammatoires de l''arthrite rhumatoïde. L''effet est palliatif, mitigé et non curatif et ne résulte pas nécessairement de l''état de pseudo-grossesse en soi.     

Characterization of primar cell cultures derived from rat renal proximal tubules

Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E₁. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D₃-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na⁺-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron. This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC.     

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 μM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.     

Vitamin D metabolism and expression in rats fed on low-calcium and low-phosphorus diets

1. Cholecalciferol, radioactively labelled with both (14)C and (3)H, was administered weekly for 7 weeks to rats that had been depleted of vitamin D for 4 weeks before repletion with the radioactive vitamin. This permitted measurement of the steady-state effect on vitamin D metabolism of low-calcium and low-phosphorus regimens, as compared with a normal mineral intake. These dietary manoeuvres were carried out during the last 3 weeks of repletion. Cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol were determined in plasma, intestine, kidney and bone. Ca(2+)-binding-protein content was measured in intestine and kidneys of comparable animals. 2. In rats on the low-calcium diets, 1,25-dihydroxycholecalciferol concentration was elevated in plasma, bone, kidney and intestine, and intestinal Ca(2+)-binding protein was increased to over twice the concentration found in the control animals. 3. The low-phosphorus regimens led to a decrease in plasma phosphate and 1,25-dihydroxycholecalciferol in all tissues studied, for the latter to the point where it was undetectable in plasma and bone. Intestinal and renal concentrations of Ca(2+)-binding protein were unchanged in the low-phosphate-intake group and decreased in the very-low-phosphate-intake group. 4. It is concluded that in the rat, unlike in the chick, hypophosphataemia is not associated with a stimulation of the production of 1,25-dihydroxycholecalciferol or its expression in the synthesis of Ca(2+)-binding protein. Therefore the plasma phosphate concentration does not appear to be directly involved in the regulation of the functional metabolism of vitamin D.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier