Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

短串联重复序列D7S2201基因座的群体遗传学研究

用扩增片段长度多态性技术分析短串联重复序列D7S2201基因座的遗传多态性,在262个中国成都地区汉族无关个体及119个泰国曼谷地区泰人无关个体中分别发现7个和5个等位基因,首次获得该基因座在两群体中的频率分布,其等位基因片段大小范围为100～124bp。两群体的基因型频率分布均符合Hardy Weinberg平衡。该基因座在两群体中的个人识别能力（PD）、杂合度（H）、多态性信息含量（CPI）及非父排除率（PE）分别为0.7038、0.5992、0.4789、0.2900和0.7351、0.5882、0.5012、0.2770。家系调查证实了等位基因的传递遵循孟德尔遗传规律。χ2检验表明两群体间等位基因频率分布无显著性差异。 Abstract：The polymorphism of a new short tandem repeat (STR) locus D7S2201 was analyzed by using AmpFLP. Seven alleles were observed in 262 unrelated Chinese individuals living in Chengdu and five alleles in 119 unrelated Thai individuals living in Bangkok, the ranges of fragment size were 100～124bp. The genotypes distributions of D7S2201 locus in the two populations were in accordance with Hardy Weinberg equilibrium. The discriminating power (PD), observed heterozygosity (H), polymorphism information content (CPI) and power of exclusion (PE) were 0.7038, 05992, 04789, 02900 in Chinese population and 0.7351, 0.5882, 0.5012, 0.2770 in Thai population respectively. Family studies confirmed Mendelian inheritance of alleles. No significant difference was observed between the two populations.     

Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Regulatory flexibility of methylotrophic yeasts in chemostat cultures: Simultaneous assimilation of glucose and methanol at a fixed dilution rate

The influence of the composition of methanol/glucose-mixtures as only sources of carbon and energy on growth and regulation of the synthesis of enzymes involved in methanol-dissimilation was studied under chemostat conditions at a fixed dilution rate with the methylotrophic yeasts Hansenula polymorpha and Kloeckera sp. 2201. Both carbon sources were found to be utilized completely independently of the composition of the C₁/C₆ mixture. Using mixtures of ¹⁴C-labelled methanol and glucose the growth yield for glucose was found to be constant for all C₁/C₆-mixtures tested and both yeasts. The growth yield for methanol, however, was reduced by up to 25% when the proportion of methanol in the inflowing medium was lower than 20% (w/w with respect to glucose) for H. polymorpha and 50% (w/w with respect to glucose) for Kloeckera sp. 2201 respectively. During growth with C₁/C₆-mixtures containing higher C₁-proportions of methanol regular growth yields for methanol were recorded which corresponded to the growth yields found with methanol as the only carbon source.The regulation of the synthesis of the enzymes of the dissimilatory pathway for methanol was found to be under multiple control. Although glucose was present in the medium methanol had a positive effect on the synthesis of these enzymes. Thus, in addition to derepression induction by methanol was also observed. This inductive effect was found to increase with increasing proportions of methanol in the mixture. Depending on the enzyme, 10–40% methanol in the mixture resulted in a maximal induction with enzyme specific activities equal to those found in cells grown with methanol as the only carbon source. No further enhancements in enzyme specific activities were observed during growth on mixtures containing more than 40% methanol.Abbreviations and terms C₁ Methanol - C₆ glucose - C₁/C₆ mixture compositions are given in % (w/w) - C₀ concentration of ¹⁴C in the inflowing medium (DPM ml^-1) - C(t) concentration of ¹⁴C incorporated in cells as a function of time t (DPM ml^-1) - d dilution rate (h^-1) - DPM disintegrations per minute - q _s q _C1 and q _C6 are specific rates of consumption of substrate, methanol and glucose respectively [g (g cell dry weight)^-1 h^-1] - q _O2 and q _CO2 are the specific rates of oxygen consumption and carbon dioxide release [mmol (g cell dry weight)^-1 h^-1] - RQ respiration quotient (q _CO2 q _O2 ^-1) - s _C1 and s _C6 are the residual concentrations of methanol and glucose in the culture liquid (g l^-1) - s _O/C1 and s _O/C6 are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - Sp.A. enzyme specific activity - x cell dry weight concentration (g l^-1) - Y _X/C1 and Y _X/C6 are growth yields on methanol and glucose respectively (g cell dry weight (g substrate)^-1 - Y _C/C1 growth yield with methanol with respect to carbon (g carbon assimilated (g carbon supplied)^-1 - _m maximum specific growth rate (h^-1)     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.