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.     

The Impact of a Line Probe Assay Based Diagnostic Algorithm on Time to Treatment Initiation and Treatment Outcomes for Multidrug Resistant TB Patients in Arkhangelsk Region,Russia

BackgroundIn the Arkhangelsk region of Northern Russia, multidrug-resistant (MDR) tuberculosis (TB) rates in new cases are amongst the highest in the world. In 2014, MDR-TB rates reached 31.7% among new cases and 56.9% among retreatment cases. The development of new diagnostic tools allows for faster detection of both TB and MDR-TB and should lead to reduced transmission by earlier initiation of anti-TB therapy.MethodsA culture-based diagnostic algorithm used prior to LPA implementation was compared to an LPA-based algorithm that replaced BacTAlert and Löwenstein Jensen (LJ) for drug sensitivity testing. A total of 295 MDR-TB patients were included in the study, 163 diagnosed with the culture-based algorithm, 132 with the LPA-based algorithm.ResultsAmong smear positive patients, the implementation of the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 50 and 66 days compared to the culture-based algorithm (BacTAlert and LJ respectively, p<0.001). In smear negative patients, the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 78 days when compared to the culture-based algorithm (LJ, p<0.001). However, several weeks were still needed for treatment initiation in LPA-based algorithm, 24 days in smear positive, and 62 days in smear negative patients. Overall treatment outcomes were better in LPA-based algorithm compared to culture-based algorithm (p = 0.003). Treatment success rates at 20 months of treatment were higher in patients diagnosed with the LPA-based algorithm (65.2%) as compared to those diagnosed with the culture-based algorithm (44.8%). Mortality was also lower in the LPA-based algorithm group (7.6%) compared to the culture-based algorithm group (15.9%). There was no statistically significant difference in smear and culture conversion rates between the two algorithms.ConclusionThe results of the study suggest that the introduction of LPA leads to faster time to MDR diagnosis and earlier treatment initiation as well as better treatment outcomes for patients with MDR-TB. These findings also highlight the need for further improvements within the health system to reduce both patient and diagnostic delays to truly optimize the impact of new, rapid diagnostics.     

Influence of growth hormone and thyroxine on thermotropic effects of respiration and 1-anilino-8-naphthalene sulfonate fluorescence and on lipid composition of cardiac membranes

The effects of hypophysectomy and subsequent administration of growth hormone and/or L-thyroxine on thermotropic properties of State 3 respiration (ADP-induced), cholesterol, phospholipid and fatty acid composition of phospholipid fraction were examined in myocardial mitochondria of rats. Temperature-dependence of 1-anilino-8-naphthalene sulfonate fluorescence was determined in vesicles prepared from lipids of heart mitochondria. Transition temperature obtained from the Arrhenius plots of respiration occurred at 21 and 24°C for heart mitochondria of normal and hypophysectomized rats, respectively. Most notably, after hypophysectomy the rate of respiration was lower below 24°C, but was progressively higher above that temperature when compared to normal rats. The energy of activation was 148 and 36% larger below and above the transition temperature, respectively. Growth hormone restored almost completely the energy of activation and respiratory rates to normal levels. Administration of L-thyroxine, with or without growth hormone, did not significantly change the rate of respiration but decreased the transition temperature to 17.7–17.0°C. Lipid and phospholipid content, as well as percent distribution of phospholipids and their fatty acid composition were not statistically different among the different groups of rats. Only cholesterol content was increased after hypophysectomy. Administration of growth hormone and thyroxine did not significantly change the total unsaturation index of fatty acids, but growth hormone increased the content of arachidonic acid (20 : 4) by 70% but decreased the docosahexaenoic acid (22 : 6) three times which may have a beneficial effect on mitochondrial membranes. These and other results suggest that hormones exert different effects on subcellular organelles in different tissues, like heart and liver.     

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.     

Identification of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates

Hereditary sensory and autonomic neuropathy (HSAN) type II is an autosomal recessive disorder characterized by impairment of pain, temperature, and touch sensation owing to reduction or absence of peripheral sensory neurons. We identified two large pedigrees segregating the disorder in an isolated population living in Newfoundland and performed a 5-cM genome scan. Linkage analysis identified a locus mapping to 12p13.33 with a maximum LOD score of 8.4. Haplotype sharing defined a candidate interval of 1.06 Mb containing all or part of seven annotated genes, sequencing of which failed to detect causative mutations. Comparative genomics revealed a conserved ORF corresponding to a novel gene in which we found three different truncating mutations among five families including patients from rural Quebec and Nova Scotia. This gene, termed "HSN2," consists of a single exon located within intron 8 of the PRKWNK1 gene and is transcribed from the same strand. The HSN2 protein may play a role in the development and/or maintenance of peripheral sensory neurons or their supporting Schwann cells.     

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

Anoxic and metabolic stresses in large‐scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro‐apoptotic proteins and over‐expression of anti‐apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro‐apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc‐finger nuclease‐mediated gene disruption. Zinc‐finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale‐down systems under conditions that mimic growth in large‐scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX‐ and BAK‐deleted cells produce two‐ to fivefold more IgG than wild‐type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double‐knockout cultures achieve equal or higher cell densities than unmodified wild‐type cultures and reach viable cell densities relevant for large‐scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330–340. © 2009 Wiley Periodicals, Inc.     

Selectivity signatures of three isoforms of recombinant T-type Ca2+ channels

Voltage-gated Ca(2+) channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca(2+) over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca(2+) permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca(2+)-channel alpha1 subunits, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca(v)3 channels select Ca(2+) over Na(+) by affinity. Ca(v)3.1 and Ca(v)3.2 discriminate Ca(2+), Sr(2+) and Ba(2+) based on the ion's effects on the open channel probability, whilst Ca(v)3.3 discriminates based on the ion's intrapore binding affinity. All Ca(v)3s were characterized by much smaller difference in the K(D) values for Na(+) current blockade by Ca(2+) (K(D1) approximately 6 microM) and for Ca(2+) current saturation (K(D2) approximately 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na(+)/Ca(2+) current at close to physiological Ca(2+) concentrations, which was the strongest for Ca(v)3.3, smaller for Ca(v)3.2 and the smallest for Ca(v)3.1. In addition to intrapore Ca(2+) binding site(s) Ca(v)3.2, but not Ca(v)3.1 and Ca(v)3.3, is likely to possess an extracellular Ca(2+) binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca(2+) current in native cells.