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Mitochondrial biogenesis: do liver mitochondria contain glycoproteins and glycosyltransferases?

1. Subcellular fractions isolated from livers of 19-day-old chicken embryos were analyzed in order to assess whether liver mitochondria contained glycosylated proteins or had mannosyl- or sialyl-transferases that could transfer sugars to mitochondrial macromolecules. 2. Proteins in liver mitochondrial membranes and matrix fractions were screened for their affinities for concanavalin A (Con A). 3. After separation by gel electrophoresis under denaturing conditions, a significant number of the proteins bound [125I]Con A, and the binding of the lectin was substantially inhibited by alpha-methyl-D-mannoside. 4. In addition, radio-iodinated matrix proteins were screened for lectin-binding properties by chromatography on Con A covalently linked to agarose. 5. A number of proteins, representing 14% of those loaded onto the column, became tightly bound to the agarose-linked lectin, and the molecular weights of several of those proteins are reported. 6. Mannosyltransferase activities were measured in fractions highly enriched for mitochondria. 7. In the reactions, mannose was transferred from guanosine diphosphomannose to materials insoluble in 0.3% trichloroacetic acid or in chloroform:methanol (2:1). 8. The fractions also catalyzed the transfer of mannose to materials extractable in chloroform:methanol and which migrated with the Rf of dolichol phosphate on Silica Gel H. 9. Dolichol phosphate stimulated the transfer of mannose to those materials extractable in the organic solvents. 10. Marker enzyme analyses indicated that the mannosyl transferase activity in the mitochondrial fraction could not be accounted for entirely by contaminating microsomal membranes. 11. Although sialyltransferase activity was detected also in the mitochondrial fractions, the levels of the activity and the kinetics of the reactions indicated that Golgi membranes were most likely the sources of the enzyme.

Animals↗

Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase.

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.

Aminolevulinic Acid↗

Production of delta-aminolevulinic acid: characterization of murine liver 4,5-dioxovaleric acid: L-alanine aminotransferase.

1. We report on the kinetic properties of murine liver 4,5-dioxovaleric acid:L-alanine aminotransferase (DOVA transaminase). 2. The transamination of 4,5-dioxovaleric acid (DOVA) led to the production of delta-aminolevulinic acid. 3. L-Alanine was the preferred amino group donor among the common 20 amino acids. 4. The optimum pH of the reaction was 7-8. 5. A Km of 220 microM for DOVA and a Km of 970 microM for L-alanine were obtained. 6. The reaction was inhibited by each of the following: glyoxylate, beta-chloroalanine, methylglyoxal, delta-aminolevulinate, pyruvate, heme, and gabaculine. 7. None of several xenobiotic inducers of microsomal mixed function oxidases tested had a significant effect on DOVA transaminase activity in studies performed with murine primary hepatocyte cultures.

Aminolevulinic Acid↗

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells.

1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2. 2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored. 3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels. 4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%). 5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway. 6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6-15% decrease), however a slight reduction (20%) in secretion rate was shown. 7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

Apolipoproteins E↗

Resting energy metabolism and cardiovascular disease risk in resistance-trained and aerobically trained males.

The objectives of this study were (1) to examine differences in resting metabolic rate (RMR) and cardiovascular risk factors among aerobically trained (n = 36), resistance-trained (n = 18), and untrained (n = 42) young males; and (2) to investigate the influence of body composition, dietary intake, and VO2max as possible modulators of differences in cardiovascular risk among groups. Results showed that RMR, adjusted for differences in fat-free weight (FFW), was 5% higher in aerobically trained males compared with resistance-trained males (P < .01), and 10% higher than that in untrained males (P < .01). Plasma levels of cholesterol and low-density lipoprotein cholesterol (LDL-C) were comparable between resistance-trained and aerobically trained males, but were lower (P < .05) than those in untrained males. (The percent intake of dietary fat was related to plasma cholesterol [r = .32, P < .01] and LDL-C [r = .30, P < .01].) When compared with untrained males, fasting triglyceride (TG) levels were 39% and 43% lower (P < .01) in resistance-trained and aerobically trained males, respectively. When compared with untrained males, the fasting insulin to glucose ratio (I/G) was 45% and 53% lower (P < .01) in resistance- and aerobically trained males, respectively. Mean arterial pressure (MAP) was 7% lower (P < .01) in aerobically trained compared with untrained males. Statistical control for differences in percent body fat or percent intake of dietary fat diminished the differences among the groups for plasma lipids, blood pressure, and the I/G ratio. We conclude that aerobically trained and resistance-trained males have higher resting energy requirements independent of FFW compared with untrained males. Aerobically trained and resistance-trained young males have comparable and favorable cardiovascular disease risk profiles compared with untrained males, and this appears to be related to their low level of adiposity and low intake of dietary fat.

Adolescent↗

A practical equation to predict resting metabolic rate in older men.

The accuracy of previous equations for predicting resting metabolic rate (RMR) in healthy older men is questionable because they are based on limited sample sizes and the absence of cross-validation procedures. The purposes of this study were to (1) examine biological predictors of RMR in healthy older men; (2) develop a practical equation to predict RMR from easily measured variables and examine its accuracy using cross-validation procedures; and (3) test the validity of existing equations in the literature to predict RMR in older men by comparison with measured RMR values. RMR, body composition, anthropometric measurements, leisure time activity (LTA), maximal aerobic power (VO2max), energy intake, and plasma thyroid hormone levels were determined in 89 healthy older men aged 50 to 78 years. Stepwise regression analysis showed that RMR was best predicted by fat-free weight ([FFW] R2 = 85%), free 3,5,3'-triiodothyronine (T3) level (R2 = 1%), and VO2max (R2 = 1%); these variables predicted RMR with a residual error of +/- 30 kcal/d. A practical equation was developed in a randomly selected subsample (N = 61) using easily measured variables as potential predictors, and was successfully cross-validated in a random subsample of older men (N = 28). The pooled equation to predict RMR is as follows: RMR (in kilocalories per day) = 9.7 (weight in kilograms) - 6.1 (chest skinfold thickness in millimeters) - 1.8 (age in years) + 0.1 (leisure time activity [LTA] in kilocalories per day) + 1,060. These variables accounted for 76% (R2) of the variation, and predicted RMR with a residual error of +/- 42 kcal/d.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

Hemostatic properties of the SV-40 transfected human microvascular endothelial cell line (HMEC-1). A representative in vitro model for microvascular endothelium.

HMEC-1 is a SV-40T transfected human microvascular endothelial cell line that constitutively expresses RNA transcripts for plasminogen activator inhibitor 1 (PAI-1), tissue-type plasminogen activator (t-PA), protein S (PS), von Willebrand factor (vWF), and thrombomodulin. Tissue factor (TF) can be induced in response to stimulation with tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1alpha) and phorbol 12-myristate 13-acetate (PMA). Proteins corresponding to PAI-1, t-PA, protein S and vWF genes were constitutively released in the culture supernatant. This cell line is a model that will be useful to investigate coagulation/fibrinolytic properties of microvascular endothelium.

Basal Metabolism↗

Potentiation of Leu 11 + natural cytotoxicity by human interleukin 2.

Human interleukin 2 was examined for its effect on potentiation of human natural cell-mediated cytotoxicity. Cells were isolated into Leu 11a + or Leu 11a- on a cell sorter. The data suggest that interleukin 2 potentiates cytotoxicity of the Leu 11a + population.

Cell Separation↗

Prospective study of natural cytotoxicity in peripheral blood of patients with nonlymphoid solid malignancies.

Natural killer (NK) cells have been implicated as an initial immunosurveillance mechanism for carcinogenesis in humans. Work in the murine system as well as the findings of depressed NK activity in patients with advanced malignancies and the discovery of increased incidences of cancer in humans congenitally deficient in NK ability have supported this. Few prospective studies have demonstrated a prognostic change in NK activity with respect to malignant disease course. In 32 healthy donors, NK activity against K562 was determined. No race or sex difference existed with respect to NK cell function. Esophageal (5), bronchogenic (3), breast (3), cervical (3), and endometrial (1) cancer patients who had received no prior chemotherapy were compared to controls. All patients subsequently received radiotherapy. Prior to such treatment NK activity could not be associated with stage of malignancy. Of the 15 patients studied, 11 were sequentially followed. Five of eight patients with stable or improving clinical courses as assessed by weight and Karnofsky scores were found to have increasing NK activity. Two of three patients with poor clinical courses presented with subnormal killing which never rose to normal while the third declined to subnormal before expiring. Esophageal, cervical, and endometrial carcinoma patients all presented with low or subnormal NK activity. Of these, only cervical and endometrial cancer patients exhibited an increase to normal levels.

Breast Neoplasms↗

Type I diabetes in an adolescent with common variable immunodeficiency.

A male adolescent with common variable immunodeficiency developed type I diabetes approximately 1 year after the initiation of immunoglobulin therapy. Immunologic evaluation revealed decreased numbers of T cells and an intrinsic B cell defect in immunoglobulin production. Lymphocytes from the patient failed to generate normal suppressor activity. There were no insulin or islet cell antibodies present in the patient's serum or in the commercial immunoglobulin preparations he received. The patient's HLA phenotype included HLA-DR3 and 4, placing him genetically at high risk for type I diabetes.

Adolescent↗

Prevalence of maternal HIV infection based on unlinked anonymous testing of newborn babies.

This pilot study established that unlinked anonymous testing of dried blood spots routinely collected on Guthrie cards for neonatal screening is a feasible method for monitoring HIV prevalence in women at the time of delivery. The method was sensitive, specific, and less expensive than more conventional ELISAs. 114,515 dried blood spots taken from cards collected in three Thames regions were tested for antibody to HIV-1. 28 samples were confirmed to be antibody positive by western blot (seroprevalence 0.24 per 1000). Unlinked anonymous screening of newborn babies should be extended to monitor the spread of HIV infection in the heterosexual population and to target preventive strategies and provision of health care.

Agglutination Tests↗

Prevalence of maternal HIV-1 infection in Thames regions: results from anonymous unlinked neonatal testing.

To monitor the spread of human immunodeficiency virus (HIV) in the heterosexual population, residues of blood samples collected routinely on absorbent paper for neonatal screening (Guthrie cards) in NE, NW, and SW Thames Regions in England have been tested for antibodies to HIV-1 since June, 1988. 323,369 dried blood spots were analysed to end March, 1991. Prevalence of anti-HIV-1 in newborn babies has remained stable in outer London and non-metropolitan districts whereas prevalence in inner London has increased from 1 in 2000 in the 12 months beginning June, 1988, to 1 in 500 in the first 3 months of 1991. Either exponential or linear growth in the numbers of new seropositives could account for the results. That obstetricians were aware of maternal HIV infection in only 20% of infected pregnancies, indicates the extent to which HIV infection goes unrecognised in the heterosexual community.

Confidentiality↗