Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus

Bacteroides melaninogenicus requires vitamin K for normal growth. Cells incubated in a vitamin K-free medium form defective cell envelopes. Studies with vitamin K-grown "K(+)" and vitamin K-depleted "K(-)" cells showed that [(14)C]choline and [(14)C]glycerol were not taken up, but several amino acids and acetate were incorporated to the same degree by both types of cultures. However, K(-) cells incorporated succinate to a greater degree than did K(+) cultures. The relative incorporation of succinate into ceramide phosphorylethanolamine and ceramide phosphorylglycerol was depressed compared with incorporation into phosphatidylethanolamine in K(-) cultures. B. melaninogenicus can be grown in serial subculture in the absence of vitamin K in the presence of 2.5 mg/ml of succinate. Under these conditions the relative incorporation of [2,3-(14)C]succinate and (32)P into ceramide phosphorylethanolamine and ceramide phosphorylglycerol is markedly depressed. Stimulation of phosphosphingolipid synthesis by vitamin K was shown by comparing the uptake of (32)P and lipid phosphorus levels of a succinate-grown, vitamin K-depleted culture supplemented with (32)P plus 0.1 micro g/ml vitamin K(1) with a similar culture supplemented with (32)P only. The phosphosphingolipids from the vitamin K-supplemented cells incorporated greater amounts of (32)P and had higher levels of phosphorus than did the ceramide phosphorylethanolamine and ceramide phosphorylglycerol of the culture without added vitamin K. It was further shown that vitamin K added to a vitamin K-depleted culture stimulated synthesis of ceramide phosphorylethanolamine and ceramide phosphorylglycerol 38 min and 60 min, respectively, following the addition of the vitamin; incorporation of (32)P into other phospholipids was unaffected.     

The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement

In seminiferous tubules of vitamin A-deficient rats, the remaining spermatogonia were A spermatogonia. These cells were topographically arranged as single and paired cells and clones of 4, 8, or more cells. The bromodeoxy-uridine-labeling index and the mitotic index of these cells were found to be 9% and 1%, respectively, indicating that these cells were slowly proliferating. Administration of vitamin A (retinol-acetate) resulted in a reinitiation of spermatogenesis in such a way that the epithelium became stage-synchronized. The rate of development of the spermatogenic cells between 7 and 21 days after vitamin A replacement was found to be similar to that in normal rats. At 24-30 h after administration of vitamin A, a 4- to 6-fold increase in the labeling index was found. In contrast, after 2 days, the labeling index was low, while the mitotic index was elevated (10%). A high labeling index was found again after 3 days. Assuming that during the first 7 days after vitamin A replacement the rate of development of the spermatogenic cells also was normal, it could be deduced that the spermatogonia labeled 24-30 h after vitamin A administration were A1 spermatogonia. These cells would then divide into A2 spermatogonia after about 2 days, which in turn would traverse their S phase after about 3 days. Hence, spermatogenesis in vitamin A-deficient rats would be arrested shortly before the S phase of the A1 spermatogonia. After administration of vitamin A, the spermatogonia synchronously start the series of six divisions leading to the formation of spermatocytes and, ultimately, they develop into mature spermatids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permanent automatic synchronization of micro algae achieved by photoelectrically controlled dilution

Summary A photoelectrically controlled automatic dilution device and the experimental set-up for its application in maintaining cultures of micro algae permanently synchronized during repeated life cycles are described. The accuracy of dilution, synchrony, growth parameters and photosynthetic response were determined for cultures of Scenedesmus obliquus and Chlorella pyrenoidosa.

---

Zusammenfassung Es wird eine automatische photoelektrisch gesteuerte Verdünnungsanlage beschrieben, die so mit einer Schaltuhr gekoppelt ist, daß sie Synchronkulturen von Mikroalgen zu Beginn des Entwicklungscyclus auf eine vorgegebene Dichte verdünnt. Diese Anlage gewährleistet die permanente Synchronisation von Mikroalgen während beliebig vieler aufeinander folgender Entwicklungscyclen. Die Genauigkeit der Verdünnung sowie die Reproduzierbarkeit der Synchronkulturen wurden an mehreren Wachstumsparametern und am photosynthetischen Verhalten der Zellen überprüft. Die Abweichungen lagen während der Meßperiode von acht Entwicklungscyclen innerhalb der meßmethodisch zu erwartenden Grenzen.

     

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.     

Induction of apoptosis in neoplastic cells by depletion of vitamin B12

Methionine synthase, a critical enzyme in deoxyribonucleotide biosynthesis for DNA replication, requires vitamin B12 as a cofactor. We have tested the hypothesis that depletion of cells of vitamin B12 would block growth of neoplastic cells and divert them into apoptosis and could form the basis of a new therapeutic strategy for cancer treatment. Using nitrous oxide to inactivate vitamin B12 we show that, in a variety of cell lines in vitro, methionine synthase is rapidly inhibited, the cells cease proliferation and undergo apoptosis. The kinetics of cell death, once started, are similar to those observed following methotrexate treatment or serum withdrawal. This is the first observation of apoptosis being induced following depletion of an essential metabolite as opposed to the more conventional strategy of adding a toxic drug to damage cells thereby triggering apoptosis. Moreover, vitamin B12 depletion has no effect on the nonproliferating cell population.     

Rapid depletion and restoration of striatal met-enkephalin after ketamine

The acute administration of ketamine depletes methionine-enkephaline levels in rat striatum, as measured by radioimmunoassay. The maximum decrease of about 25% was observed 30 minutes after 100 mg.kg^?1, i.p. injection of the drug, enkephalin levels returning to normal within 90 minutes. Neither higher doses nor a double administration of ketamine produced any further depletion. These results indicate that ketamine interacts with the enkephalinergic system and suggests the presence of a limited and rapidly releasable pool of methionine-enkephaline in the brain.     

DNP complex of the spermatogenic cells of rats with hypovitaminosis A

Retinol deficiency in rats was shown to cause atypical changes of DNP-complex in spermatogenic cells and a decrease in their fertility. The final stages of spermiogenesis were found to be the most sensitive: ca. 25% of testicular sperms were characterized by abnormal chromatin structure and deficiency in spermiospecific argininerich histones what might be related to a sharp decrease in arginine content in blood plasma. Similar results were obtained with rats kept on retinoic acid. Thus, retinoic acid fails to restore retinol-dependent processes involved in the formation of sperms with normal DNP-complex.     

Effect of vitamin A depletion on nonvisual phototransduction pathways in cryptochromeless mice

Mice exhibit multiple nonvisual responses to light, including 1) photoentrainment of circadian rhythm; 2) "masking," which refers to the acute effect of light on behavior, either negative (activity suppressing) or positive (activity inducing); and 3) pupillary constriction. In mammals, the eye is the sole photosensory organ for these responses, and it contains only 2 known classes of pigments: opsins and cryptochromes. No individual opsin or cryptochrome gene is essential for circadian photoreception, gene photoinduction, or masking. Previously, the authors found that mice lacking retinol-binding protein, in which dietary depletion of ocular retinaldehyde can be achieved, had normal light signaling to the SCN, as determined by per gene photoinduction. In the present study, the authors analyzed phototransduction to the SCN in vitamin A-replete and vitamin A-depleted rbp-/- and rbp-/-cry1-/-cry2-/- mice using molecular and behavioral end points. They found that vitamin A-depleted rbp-/- mice exhibit either normal photoentrainment or become diurnal. In contrast, while vitamin A-replete rbp-/-cry1-/-cry2-/- mice are light responsive (with reduced sensitivity), vitamin A-depleted rbp-/-cry1-/-cry2-/- mice, which presumably lack functional opsins and cryptochromes, lose most behavioral and molecular responses to light. These data demonstrate that both cryptochromes and opsins regulate nonvisual photoresponses.     

Proteome alterations of cortex and hippocampus tissues in mice subjected to vitamin A depletion

Vitamin A regulates the development and maintenance of the central nervous system. Studies of vitamin A depletion (VAD) and mutations of retinoid receptors in rodents have revealed a dysfunction of motor and cognitive abilities. However, the molecular mechanisms underlying these behavioral changes are not well understood. In this study, VAD mice were examined and abnormal motor behavior related to psychosis symptoms was found. With the use of two-dimensional gel electrophoresis (2-DE), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometric (MS) technologies, 44 and 23 altered protein spots were identified in the cortex and hippocampus, respectively, in VAD mice. By Western blot, the up-regulation of mitogen-activated protein kinase 1 (MAPK1) and proteasome subunit beta type 2 (PSMB2) in the cortex and that of dihydropyrimidinase-related protein 2 (DPYSL2) and PSMB2 in the hippocampus were observed in VAD mice. Bioinformatic analysis using DAVID revealed that altered proteins induced by VAD showed significant enrichment of (i) glycolysis, cytoskeleton, mitochondrion and glutamate metabolism in the cortex; and (ii) actin binding, dopamine receptor signaling and transmission of nerve impulse in the hippocampus. The up-regulations of DPYSL2, MAPK1 and PSMB2 may indicate the activated neuronal defensive mechanism in VAD brain regions, which may underlie the VAD-related psychosis behavior.     

A multicompartmental model of vitamin A kinetics in rats with marginal liver vitamin A stores

A linear, first-order, constant-coefficient multicompartmental model is presented which describes the dynamics of [3H]retinol turnover in adult rats with normal plasma retinol concentrations but low liver stores (less than 100 micrograms of retinol equivalents). To fit plasma and tissue (liver, kidney, and rest of carcass) tracer and tracee data, eight physiological compartments were required in the model: two in plasma (proposed to correspond to the retinol transport complex, and retinyl esters in plasma lipoproteins) and two each in liver, kidneys, and other extrahepatic tissues. Extensive recycling of retinol among plasma, liver, and the rest of carcass was also required. The model predicted that 44% of whole body vitamin A (143 micrograms) was in extrahepatic tissues. The vitamin A utilization rate (system disposal rate) was 6.9 micrograms of retinol equivalents/day. The system residence time (mean sojourn time) for vitamin A was 21 days, and the fractional catabolic rate for the system was 5%/day. The mean transit time (turnover time) for vitamin A in its plasma retinol transport complex was 0.078 days (1.9 hr); the residence time was 0.98 day, versus 11 days in the liver, 9 days in carcass, and 0.54 days in kidneys. The model predicted that, of the plasma turnover, 48% recycled to the liver and 52% to extrahepatic tissues. The liver retinol secretion rate was 48 micrograms/day, more than half of which was from recycled plasma retinol. Since the plasma retinol turnover rate (87 micrograms/day) was 13 times the system disposal rate, the data suggest that this is a high response system in which changes in the dynamics of recycling of retinol allow for rapid adjustment in vitamin A distribution in response to changes in nutritional, metabolic, or physiological conditions; and in which plasma retinol levels are controlled homeokinetically by changes in hepatic and extrahepatic recycling of holo retinol-binding protein.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Utilization of vitamin A in rats with inflammation

Reductions in the concentration of retinol (vitamin A) in serum, lung and kidney were observed in rats subjected to inflammation-inducing treatments (turpentine oil injection of thermal injury). At the same time, the liver showed an almost normal vitamin A content. Feeding of retinol to vitamin A-depleted rats with inflammation revealed that intestinal absorption of retinol was still active in the inflamed state, and the livers of these rats showed good incorporation of retinol. The livers of normal and vitamin A-depleted rats subjected to the inflammatory treatments showed a normal RBP content (retinol-binding protein) and hepatic release of holo-RBP into the serum was not impaired functionally. These results suggest the possibility that the decreases of vitamin A in the lung, serum and kidney may be due primarily to enhanced local consumption of vitamin A related to the inflammation, rather than to a reduced supply of vitamin A from the liver or to decreased intestinal absorption. In bovine serum albumin (BSA)-sensitized rats produced by direct intubation of BSA into the lungs, the level of vitamin A in the lung decreased prior to that in the liver or serum, supporting the hypothesis that the decrease in vitamin A in the inflamed lungs of these rats may be due mainly to the consumption of vitamin A in the lung in response to inflammation.     

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.