Effect of Oxygen on Heme and Porphyrin Accumulation from δ-Aminolevulinic Acid by Suspensions of Anaerobically Grown Staphylococcus epidermidis

The effect of various conditions on the accumulation of porphyrins and heme by resting suspensions of anaerobically grown cells of Staphylococcus epidermidis was examined. Anaerobically grown cells contain 10 to 15% of the amount of protoheme found in cells grown aerobically. Resting suspensions of anaerobically grown cells, when incubated aerobically in buffer with delta-aminolevulinic acid and glucose for 60 min, exhibited a fourfold increase in protoheme content. At high levels of delta-aminolevulinic acid, there was also a significant accumulation of porphyrins with the solubility and chromatographic properties of coproporphyrin and uroporphyrin. Protoporphyrin was not accumulated. When oxygen was excluded from the incubation mixture, accumulation of protoheme was prevented, but accumulation of coproporphyrin and total porphyrin was enhanced. Nitrate served as an electon acceptor as indicated by its reduction to nitrite; however, nitrate did not substitute for oxygen in causing the accumulation of protoheme. These results suggested that oxygen is required for one of the late steps of heme synthesis in S. epidermidis, possibly for the conversion of coproporphyrinogen to protoporphyrin. The inability of nitrate to substitute for oxygen suggests a role for molecular oxygen as a substrate rather than as an electron acceptor for heme synthesis.     

Biosynthesis and Thermal Properties of PHBV Produced from Levulinic Acid by Ralstonia eutropha

Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0–40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2–6 g L⁻¹ LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L⁻¹ ammonium chloride and 2 g L⁻¹ casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L⁻¹ with a PHBV concentration of 12.61 g L⁻¹ (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.     

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

Generation of O₂^– and H₂O₂ as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid – a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O₂^– production. The H₂O₂ level was not significantly affected. In toxin-treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid-induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro- and anti-oxidant systems are involved in response to fusaric acid treatment although differential response of H₂O₂ and its metabolism-related enzymes between the whole leaf and cell culture assays was found.     

Separation of β-Factor Synthesis from Stimulated β-Carotene Synthesis in Mated Cultures of Blakeslea trispora

Mated cultures of Blakeslea trispora, grown in a potato extract-glucose-thiamine medium, produced 10 to 15 times more beta-carotene than either unmated culture. Mated, but not unmated, cultures produced a family of compounds (beta factor) which stimulated carotenogenesis in unmated cultures. In fact, carotenogenesis was stimulated sixfold more in minus cultures than in plus cultures. By altering the relative amounts of plus and minus inocula used in fermentations of mated cultures, it was possible to separate the synthesis of beta factor from the synthesis of extra beta-carotene. The plus strain appeared to produce the beta factor; the minus strain appeared to produce most of the extra beta-carotene. Kinetic studies of beta-factor formation suggested that physical contact between the two strains may be required to initiate beta-factor synthesis.     

Transport of Gabapentin, a γ-Amino Acid Drug, by System L α-Amino Acid Transporters: A Comparative Study in Astrocytes, Synaptosomes, and CHO Cells

Abstract: The system L transporter is generally considered to be one of the major Na⁺-independent carriers for large neutral α-amino acids in mammalian cells. However, we found that cultured astrocytes from rat brain cortex accumulate gabapentin, a γ-amino acid, predominantly by this α-amino acid transport system. Uptake of gabapentin by system L transporter was also examined in synaptosomes and Chinese hamster ovary (CHO) cells. The inhibition pattern displayed by various amino acids on gabapentin uptake in astrocytes and synaptosomes corresponds closely to that observed for the system L transport activity in CHO cells. Gabapentin and leucine have K _m values that equal their K _i values for inhibition of each other, suggesting that leucine and gabapentin compete for the same system L transporter. By contrast, gabapentin exhibited no effect on uptake of GABA, glutamate, and arginine, indicating that these latter three types of brain transporters do not serve for uptake of gabapentin. A comparison of computer modeling analysis of gabapentin and l -leucine structures shows that although the former is a γ-amino acid, it can assume a conformation that can resemble the L-form of a large neutral α-amino acid such as l -leucine. The steady-state kinetic study in astrocytes and CHO cells indicates that the intracellular concentrations of gabapentin are about two to four times higher than that of leucine. The uptake levels of these two substrates are inversely related to their relative exodus rates. The concentrating ability by system L observed in astrocytes is consistent with the substantially high accumulation gradient of gabapentin in the brain tissue as determined by microdialysis.     

Effect of PACAP on Bacterial Adherence and Cytokine Expression in Intestinal Cell Cultures

International Journal of Peptide Research and Therapeutics - Bacterial adhesion is a crucial event of intestinal pathological conditions evoked by bacterial infections. Pituitary adenylate cyclase...     

Effect of Nitrate, Ammonium and Some Amino Acids on Growth and Nitrate Reductase Activity in Suspension Cultures of Atropa belladonna

Growth and nitrate reductase activity (NRA) of Atropa belladonnacells were studied in medium supplemented with NaNO₃, NH₄NO₃,and amino acid precursors to tropane alkaloids. Growth and NRAwere stimulated by NH₄⁺ and by proline, by proline plus ornithine,but not by glutamate, in NO₃^–-containing medium. Testedamino acids inhibited neither utilization of inorganic nitrogennor growth. (Received September 30, 1988; Accepted August 28, 1989)     

钴- 原卟啉对大鼠脑损伤区域血红素氧合酶-1 及损伤周边区脑含水量的影响

目的:研究探讨钴-原卟啉对大鼠脑损伤的保护作用。方法:用钴-原卟啉灌(50mg/kg)胃处理后,建立液压脑损伤模型,采用免疫组织化学SP三步法检测大鼠脑损伤区域血红素氧合酶-1(HO-1)的表达情况,用干湿重法测定损伤周边区脑含水量的变化。结果:正常组、假手术组大鼠HO-1均无表达;模型组脑损伤区域的HO-1阳性细胞数为(3.45±047);实验组12h、24h、3d和7dHO-1的阳性细胞数分别为:(10.9±1.35)、(10.62±1.88)、(12.4±1.57)和(10.99±2.42)。正常组、假手术组脑损伤周边区脑含水量分别为(78.7±0.4)%和(78.6±0.7)%;模型组脑含水量(89.3±0.3)%;实验组12h、24h、3d和7d脑含水量分别为:(83.1±0.3)%、(83.6±0.6)%、(83.9±0.4)%和(83.2±0.3)%。结论:钴-原卟啉(CoPP)能够诱导大鼠脑损伤区域HO-1的表达,减轻脑损伤周边区域的水肿程度,故钴-原卟啉对大鼠脑损伤具有保护作用。     

Effects of Abscisic Acid on Nucleic Acid Synthesis and the Induction of Nitrate Reductase in Lemna polyrhiza

Abscisic acid (ABA) at low concentrations brings about the formationof turions (dormant fronds) in Lemna polyrhiza within 3 to 5days after application. The incorporation of ³H-thymidine intoDNA, separated by polyacrylamide-gel electrophoresis, is inhibitedby 80 to 90 per cent within 1 to 3 h of ABA application. Theincorporation of ¹⁴C-orotate and ³²P into RNA is not inhibiteduntil 3 to 9 h after ABA application, but 70 per cent inhibitionis reached after 24 h on 10^–5 M ABA. There is little inhibitionof ¹⁴C-leucine incorporation into protein until 2 to 3 daysafter application of ABA. The capacity of nitrate to inducenitrate reductase in cultures previously grown on nitrate-freemedium is not affected by ABA even up to 3 days after application.The results are discussed in relation to the mode of actionof ABA.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

Effect of Emulsan on Biodegradation of Crude Oil by Pure and Mixed Bacterial Cultures

Crude oil was treated with purified emulsan, the heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population as well as nine different pure cultures isolated from various sources was tested for biodegradation of emulsan-treated and untreated crude oil. Biodegradation was measured both quantitatively and qualitatively. Recovery of ¹⁴CO₂ from mineralized ¹⁴C-labeled substrates yielded quantitative data on degradation of specific compounds, and capillary gas chromatography of residual unlabeled oil yielded qualitative data on a broad spectrum of crude oil components. Biodegradation of linear alkanes and other saturated hydrocarbons, both by pure cultures and by the mixed population, was reduced some 50 to 90% after emulsan pretreatment. In addition, degradation of aromatic compounds by the mixed population was reduced some 90% in emulsan-treated oil. In sharp contrast, aromatic biodegradation by pure cultures was either unaffected or slightly stimulated by emulsification of the oil.     

Optically Active Compounds Derived from Salicylaldehyde and Pyridoxal: Syntheses and Their Chelate Formation Properties in the Interaction with α-Amino Acid

Synthesis of chiral compounds derived from salicylaldehyde and pyridoxal was carried out. In aqueous media containing copper ion, these compounds were analyzed to form Schiff base copper chelate spontaneously with α-amino acid. This complex formation process was reversed by the addition of EDTA. Applicabilities of the compounds to the resolution of α-amino acids are discussed.     

Effects of Oxygen Tension and Glucose Repression on Mitochondrial Protein Synthesis in Continuous Cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae grown over a range of oxygen and glucose concentrations were used to determine the effects of these two physiological regulators on mitochondrial protein synthesis in vivo. Quantitative estimates were obtained of the contribution of the mitochondrial protein-synthesizing system to the formation of mitochondrial membranes in cells grown over a wide range of dissolved oxygen concentrations, under conditions of glucose limitation or glucose excess in the cultures. The nature of the products of the mitochondrial system formed under these conditions was examined by selectively labeling membrane proteins in vivo in the presence of cycloheximide, and fractionating the products by gel electrophoresis under dissociating conditions. These results have been correlated with changes in the lipid composition of the cells and with the synthesis and assembly of components of the mitochondrial adenosine 5'-triphosphatase complex.     

Bacterial Growth in Mixed Cultures on Dissolved Organic Carbon from Humic and Clear Waters

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 μm of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.     

Genetic Dissection of Behavioural and Autonomic Effects of Δ9-Tetrahydrocannabinol in Mice

Marijuana and its main psychotropic ingredient Δ⁹-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release γ aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions.     

Transformation of Bile Acids by Mixed Microbial Cultures from Human Feces and Bile Acid Transforming Activities of Isolated Bacterial Strains

Microbial transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7α-hydroxy group in both primary bile acids and epimerization of the 3α-hydroxy group in all metabolic bile acids. Extensive epimerization of the 7α-hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7α-Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7α-dehydroxylase activity was revealed in one of the isolates, an unidentified gram-positive nonsporeforming anaerobic bacterium. 3α-Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3α- and 7α-dehydrogenations. No microorganism accounting for 7α-epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.     

Effect of Gabaculine on the Synthesis of Heme and Cytochrome f in Etiolated Wheat Seedlings

The effect of gabaculine (3-amino 2,3-dihydrobenzoic acid), an inhibitor of tetrapyrrole synthesis, on the accumulation of heme and cytochrome f in etiolated wheat (Triticum aestivum var Mardler) seedlings has been examined. Gabaculine treatment resulted in decreased amounts of heme and of holocytochrome f detected spectroscopically and by peroxidase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of the cytochrome f polypeptide detected immunochemically on Western blots was much less affected by gabaculine treatment, indicating that apocytochrome f synthesis was not tightly coupled to heme availability. Gabaculine treatment did not affect the size of the cytochrome f polypeptide, indicating that heme addition is not required for proteolytic removal of the presequence.