Mammalian genes encoding a 35-kDa peroxisomal membrane protein (PMP35, peroxisome assembly factor-1) are compared using the polymerase chain reaction and DNA sequencing. DNA sequencing of the 915 bp of the PMP35 coding regions was in complete agreement with previously published rat data and showed 36 and 133 nucleotide substitutions, respectively, in mouse and man. The 12 and 35 respective amino acid changes encoded by these nucleotide substitutions are clustered and compatible with putative membrane-spanning regions. Rat/human and rat/mouse comparisons yield silent mutation rates of 0.33 and 0.21% per site per million years and replacement mutation rates of 0.082 and 0.076%; transitions account for 67% (human/mouse) and 83% (rat/mouse) of nucleotide replacements among PMP35 genes. PMP35 gene expression in mouse tissues as measured by reverse transcriptase-PCR was responsive to clofibrate and disproportionately high in neural tissue.
The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-β-D-maltoside. Complex I activity is measured in the presence of 2.5 mg · ml−1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.
The involvement of protein kinase C in differentiation of rat adipocyte precursor cells in serum-free culture was evaluated by using various protein kinase inhibitors. Induction of adipose conversion, which was maximal after 10 days of culture in the presence of 5 micrograms/ml insulin, 10 micrograms/ml transferrin, and 200 pM triiodothyronine, was inhibited by the addition of protein kinase C inhibitors, H-7 and staurosporine, in a dose-dependent fashion with the maximal effect at 10 microM and 10 nM, respectively. Inhibition of adipocyte differentiation by 12-O-tetradecanoylphorbol 13-acetate (10(-8) M), an activator of protein kinase C, was reversed by a concomitant addition of either 10 microM H-7 or 10 nM staurosporine. HA1004, a potent inhibitor of cAMP- and cGMP-dependent protein kinases, with minimal inhibitory activity on protein kinase C, did not affect adipose conversion. Furthermore, H-89, another isoquinoline derivative with a selective inhibitory action on cAMP-dependent protein kinase, was without effect on cellular differentiation. These results indicate that the potentiation of adipogenesis by H-7 and staurosporine is mediated by suppression of protein kinase C and that protein kinase C is involved in adipocyte differentiation in an inhibitory fashion.
We investigated glucose phosphorylation at various concentrations of glucose (1, 5, 10, 25, 50, 100 mmol/liter) in rabbit optic nerve. In the 3000 g supernatant of whole rabbit optic nerve homogenates from female albino rabbits (n = 10, 1.8-2.0 kg body weight, mean +/- SEM morning glycemia: 8.25 +/- 0.29 mmol/liter), the glucose phosphorylating activity (NADP reduction measured as change in optical density at 366 nm at pH 7.5) increased progressively with the increase in glucose concentration (r = 0.89; P < 0.05) and approached the maximum at a very high glucose level (100 mmol/liter), with values (mean +/- SEM) of 8.75 +/- 0.97 nanomol/min/mg protein and 11.57 +/- 1.15 at 1 and 100 mmol/liter glucose, respectively (+32.23%; P < 0.01). At a more alkaline pH (8.2; n = 5, mean +/- SEM morning glycemia: 8.83 +/- 0.07 mmol/liter) glucose phosphorylation was higher than at pH 7.5 and retained the glucose concentration dependence (r = 0.95, P < 0.01). These kinetic characteristics are reminiscent of those of the low-affinity enzyme glucokinase, which is typically present in the liver. By subtracting the activity at 1 mmol/liter glucose from that at higher glucose concentrations, we calculated the "glucokinase component," forms the "total" glucose phosphorylating activity. In five rabbits (of similar age and weight) with spontaneous hyperglycemia (mean +/- SEM: 11.71 +/- 0.60 mmol/liter), the optic nerve glucose phosphorylating activity was lower (value at 1 mmol/liter glucose: 5.42 +/- 1.31, -38.06%, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced FiO2 (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.
Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI; MPS VI) is a disorder which results from a deficiency in the lysosomal associated enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). A feline model of human MPS VI has previously been described and provides a system for the evaluation of enzyme replacement therapy protocols. As a preliminary study to human 4-sulfatase enzyme replacement therapy in feline we have compared the immunochemical properties of human and feline 4-sulfatase. By SDS-PAGE the molecular mass of purified feline and human 4-sulfatase were similar under both reducing and nonreducing conditions. There was, however, a detectable conformation difference between human and feline 4-sulfatase indicating some structural variation. Feline 4-sulfatase reacted weakly with a panel of monoclonal antibodies in an immunobinding assay (interacting with 4-sulfatase in free solution), but the same monoclonal antibodies reacted strongly with feline 4-sulfatase in an immunoquantification assay where the feline 4-sulfatase was bound to a polyclonal antibody (which presumably induces a conformation change in the feline 4-sulfatase to closer approximate the structure of human 4-sulfatase). A monoclonal antibody which selectively reacts with human 4-sulfatase has been used to develop an assay suitable for evaluating human 4-sulfatase enzyme replacement in cat tissues.
We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a Km of 275 ± 30 umol/liter (SD) and a Vmax of 4.0 ± 0.5 nmol/min · mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.
The linkage of the intragenic polymorphic (GATT)n repeat to a number of cystic fibrosis transmembrane conductance regulator gene mutations (Δ F-508, G542X, G551D, R553X, R1162X, W1282X, N1303K, R334W, and R347P) was studied. The linkage of Δ F-508, G542X, and N1303K to a six-copy allele and of R334W to a seven-copy allele of the repeat was found.
The two major species of arginine endopeptidase present in the soluble fraction of human submaxillary gland are glandular kallikrein and another enzyme tentatively named nonkallikrein arginine endopeptidase. In this study, we purified the latter enzyme to homogeneity and examined its catalytic properties. The newly found enzyme was clearly distinguishable from human tissue kallikrein in its molecular nature, action toward various synthetic substrates, and kinin-generated activity. The specificity of the action of the enzyme was further investigated using various basic amino acid-containing peptides as model substrates. HPLC analysis of peptide fragments produced, followed by their amino acid analysis, revealed that the enzyme preferentially hydrolyzed the Arg-Arg or Arg-Lys bonds in dynorphins A 1-10, 1-9, and 1-8, β-neoendorphin, adenorphin, and neurotensin.
We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC8 and OAG, and 1,3-DiC8 (a poor activator of PKC) on thromboxane A2 (TxA2)-independent phospholipase A2 (PLA2) activation in human platelets using collagen and A23187 as agonists. We measured PLA2 activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA2 synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13.85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC8, OAG, and 1,3-DiC8 increased TxA2-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC8, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC8) in priming TxA2-independent PLA2 activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA2-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligatory for priming PLA2 activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC8, OAG, and 1,3-DiC8 likely enhance PLA2 activation via intracellular Ca2+ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA1, or nonspecific PLA2. Since both 1,2-DiC8 and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA1 may not involve a PKC dependent mechanism. We, therefore, conclude that the mechanisms by which PMA and DAGs prime PLA2 activation are different and that the priming mechanism by DAGs may not involve PKC, but may require a rise in intracellular Ca2+.
Differences in Ahd-2 at the DNA sequence level were characterized in mouse strains with variable ethanol preferences. The 5′ region and the region surrounding the active site of Ahd-2 were compared to detect differences which could affect ethanol sensitivity. Only minor differences were found among the strains in the two regions. These differences cannot explain their variable ethanol preference and the implications of sequence identities among the divergent strains in these regions has yet to be determined.
Administration of 1,2-dimethylhydrazine (DMH) to rats produces colon cancer. The mechanism by which this agent induces colon cancer is unclear. This investigation was conducted to assess the effect of DMH on the hepatic RNA and the colonic RNA of rats. DMH (300 mg/kg body wt) was administered to rats by ip injections. After 24 h, the hepatic RNA and the colonic RNA were isolated and their component purine bases were analyzed by HPLC. DMH treatment resulted in the formation of 1-methyladenine, 1-methylguanine, N2-methylguanine, O6-methylguanine, and 7-methylguanine in RNA. These methylated products may play a role in cellular injury produced by DMH.
We show here that long-term streptozotocin diabetes affects differently the intracellular distribution of phosphofructokinase (PFK), the rate-limiting enzyme of glycolysis, in tibialis anterior and gastrocnemius muscles. Diabetes, which causes ultrastructural damage in both muscle fibers, induced a decrease in PFK binding to cytoskeleton in gastrocnemius muscle but not in the tibialis anterior muscle. However, the allosteric activity of cytoskeleton-bound and soluble PFK was reduced in both kinds of muscles, most probably due to the decrease in the level of glucose 1,6-bisphosphate, the potent allosteric activator of the enzyme. Levels of fructose 2,6-bisphosphate remained unchanged. A change in the allosteric properties of the cytoskeleton-bound PFK was found only in the diabetic tibialis anterior muscle; in contrast to normal muscle, where only the soluble but not the bound enzyme responded to allosteric effecters, in the diabetic tibialis anterior muscle, the bound enzyme exhibited allosteric properties similar to the soluble enzyme, The reduction in both cytosolic and cytoskeletal PFK, and, thereby, glycolysis in these two kinds of muscles, which results most probably from the reported high pathological intracellular Ca2+ concentration, may contribute to muscle damage in diabetes.
In the present study, we investigated the effects of different diacylglycerols in comparison with phorbol 12-myristate 13-acetate (PMA) on eicosanoid-independent phospholipase A2 (PLA2) activation in human platelets and neutrophils. Eicosanoid-independent PLA2 activation was measured under conditions where both cyclooxygenase and lipoxygenases were blocked by BW755C. In the presence of PMA (50 nM), the amount of mass arachidonic acid (AA) released represented 400 and 257% of control (without PMA) in A23187-stimulated platelets and neutrophils, respectively, while 1,2-dioctanoylglycerol (1,2-DiC8) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) had increased the eicosanoid-independent AA release by 150 and 117-134% of control, in platelets and neutrophils, respectively. Our results further demonstrate that 1,3-dioctanoylglycerol (1,3-DiC8), a poor activator of protein kinase C (PKC), is nearly as effective as diacylglycerols, such as OAG and 1,2-DiC8 (activators of PKC) in priming PLA2 activation, but is less effective than PMA as a priming agent. However, all three diacylglycerols were less effective than PMA as priming agents. Furthermore, diacylglycerols including 1,3-DiC8 exerted a much greater effect on PLA2 activation in platelets than in neutrophils. Neither 1,3-DiC8 nor 1,2-DiC8 and OAG had any significant priming effect on the accumulation of palmitic and stearic acids, while PMA caused a substantial accumulation of these fatty acids in platelets, but not in neutrophils. We also found that exogenously added OAG underwent significant hydrolysis even in unstimulated platelets, but not in neutrophils, suggesting that exogenously added OAG may be readily accessible for diacylglycerol (DAG) lipase/PLA1 in platelets. It is possible that the priming of PLA2 by diacylglycerols in both cell types may involve a PKC-independent mechanism, whereas that by PMA may involve both PKC-dependent and PKC-independent mechanisms. The differential effects of PMA and diacylglycerols on PLA2/DAG lipase activation observed between platelets and neutrophils may stem from the differences in the predominance of certain enzyme isoforms, requiring specific factors such as cytosolic/exogenous Ca2+, receptor-agonist interaction, enzyme-diacylglycerol interactions, and PKC and tyrosine kinase mediated phosphorylations.
The influence of enhancing the supply of hydrogen donors on respiratory rates, NAD(P)H fluorescence, and membrane potential was investigated. Addition of 5 mM malate to mitochondria during oxidation of 10 mM isocitrate, oxoglutarate, succinate, proline, or glycerol-3-phosphate under steady-state conditions resulted in an inhibition of respiration, coincident with a decrease in both transmembrane electrical potential and percentage reduction of NAD(P). Half-maximum inhibition of NAD(P) reduction in the resting state of 10 mM isocitrate respiration was reached at 10 mM malate. This inhibition was concluded to be due to oxaloacetate formed immediately from malate by succinate dehydrogenase. Addition of 5 mM isocitrate caused higher respiratory rates, accompanied by an increase in both Δψ and percentage of NAD(P) reduction, in mitochondria oxidizing 10 mM oxoglutatate, glutamate, proline, hydroxybutyrate, glycerol-3-phosphate, or 0.025 mM palmitoyl carnitine. The half maximum increase in percentage NAD(P) reduction with 10 mM 2-oxoglutarate as primary substrate was found at 0.24 mM isocitrate. Within the citric acid cycle, succinate dehydrogenase and NAD-isocitrate dehydrogenase play an important role in changes in the rate of NADH formation. Therefore, they participate in flux control. Furthermore, mitochondrial aspartate aminotransferase and oxidoreductases of the β-oxidation pathway of fatty acids are additionally involved in adjusting the rate of NADH formation.
The monomethyl ester of succinic acid (SME) was recently found to protect pancreatic islet B-cells against the impairment of glucose-stimulated insulin release caused by either glucopenia or starvation. The possible metabolic determinants of such a protective action are now scrutinized. After 180 min preincubation at 2.8 mM D-glucose in the presence of SME (10 mM), the oxidation of D-[U-14C]glucose, relative to either the utilization of D-[5-3H]glucose or the generation of 14C-labeled acidic metabolites, was higher than that after preincubation in the absence of SME, and became close to that otherwise found after preincubation at 16.7 mM D-glucose. Likewise, after 3 days of culture at a low concentration of D-glucose (2.8 nM), the presence of SME in the culture medium tended to increase the subsequent oxidation of D-[6-14C]glucose and utilization of D-[5-3H]glucose. These two variables increased as a function of the concentration of D-glucose in the culture medium, this coinciding with a modest increase in hexokinase activity and a more pronounced increase in glucokinase activity. The presence of SME in the culture medium failed, however, to exert any obvious effect upon the respiration of the islets, suggesting that the protective action of the ester against glucopenia may also involve variables distinct from the metabolism of either endogenous or exogenous nutrients. Likewise, the fact that SME infusion to starved rats prevents the impairment of glucose-induced insulin release otherwise attributable to starvation may involve enzymatic determinants, such as a less severe decrease in glucokinase activity, metabolic variables, such as a greater relative increase in D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization in response to a rise in extracellular D-glucose concentration, and other factors yet to be identified that-participate in the secretory sequence at a site distal to those metabolic events triggered by D-glucose in the islet cells.
The effects of growth hormone treatment and dietary alanine supplementation, individually and in combination, were studied in five patients with organic acidemias. Three patients had propionic acidemia, one had 3-hydroxyisobutyric acidemia, and one had a defect in isoleucine metabolism. Two patients with propionic acidemia had decreased growth hormone secretion in response to provocative stimuli (intravenous L-arginine and oral levodopa or clonidine); the remaining subjects had sufficient growth hormone secretion. Three of four subjects in whom IGF1 was measured showed subnormal concentrations at baseline (including two with normal growth hormone secretory responses). All patients showed an increase in linear growth with growth hormone. In the four patients studied, all had a significant increase in nitrogen retention over baseline with alanine or growth hormone alone, or with the combination of growth hormone and alanine, with a much greater effect of growth hormone. Lean body mass and body fat composition tended to become normal with treatment. Protein tolerance increased, and when the patients′ dietary protein intakes were increased between 20 and 60% they maintained positive nitrogen balance, without a significant increase in metabolite excretion. One patient with propionic acidemia expired during the time of the study, following a course of recurrent pancreatitis and an episode of acute basal ganglia infarction. All of the other subjects showed clinical improvement (decreased incidence of ketoacidotic episodes and decreased frequency of hospital admission and school absence) during treatment, and even the patient who expired remained metabolically stable up to and through the terminal event. We conclude that growth hormone may be of value in the management of patients with organic acidemia.
Previous studies of the molecular basis of 21-hydroxylase deficiency have shown four common gene conversion mutations in exons 7 and 8. Current molecular diagnostic protocols use allele-specific oligonucleotide hybridization (ASOH) to individually detect each of these mutations and the corresponding normal alleles. This method is costly, labor intensive, and may not provide quantitative results. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed and implemented single-strand conformational polymorphism (SSCP) analysis. We applied SSCP analysis to 12 families in whom mutations in exons 7 or 8 had been previously identified by ASOH. Using a single polymerase chain reaction (PCR) amplification, unique conformers can be assigned to three mutations: V281L, Q318X, and R356W. The fourth mutation, T insertion at nucleotide 1761, was detected by heteroduplex analysis of the same PCR product. Thus, we were able to identify all four mutations using a single PCR product on a single gel.
The incorporation of radioactivity from [1-14C]galactose into TCA-precipitable material was determined in skin fibroblasts derived from 11 galactosemic patients deficient in galactose 1-phosphate uridyl transferase (GALT-). "R" ratios (designated the R phenotype) were defined as the ratio between [14C]galactose incorporation and [3H]leucine incorporation. Results were expressed as a percentage of the controls. In the GALT-strains this ratio varied from strain to strain, presumably depending on the efficiency of the secondary route via the UDP-galactose pyrophosphorylase pathway. In 10 GALT-patients without late serious clinical manifestations, the R phenotype varied hom 37 to 57% of the control value. In the 11th patient, the R phenotype was only 20% of the control. Thus, we obtained a significantly lower R phenotype in one patient who was distinguished from the others by having very severe delayed neurological complications, although compliance to galactose-free diet was good. We suggest that, in this patient, the development of the UDP-galactose pyrophosphorylase pathway was not sufficient to ensure the availability of enough galactose for the necessary synthesis of glycoproteins and glycolipids. Thus the R phenotype may be an indicator of the risk of late neurological complications. The determination of the R phenotype of GALT-patients may therefore be valuable. However, further investigations of galactosemic patients with neurological complications are required to confirm this relationship.
To elucidate the effects of fatty acids on the uptake of glucose and insulin in the muscle, the effects of oleic and palmitic acids on the uptake of glucose and insulin were investigated in perfused hind-quarters of rats. In the absence of insulin, glucose uptake in the hindquarter was slightly, but not significantly, decreased by the addition of oleic and palmitic acids. In the presence of 100 or 500 microU/ml insulin, glucose uptake in the hindquarter (243 +/- 58 or 282 +/- 65 mumol/30 min) was significantly decreased by the addition of 1000 microM oleic acid and 500 microM palmitic acid (175 +/- 50 or 207 +/- 47 mumol/30 min). The decrease in insulin uptake rate, although not significant at 500 microU/ml insulin, is of similar magnitude to the decrease in glucose uptake. In the presence of 1000 microU/ml insulin, glucose and insulin uptake was not significantly altered by the addition of fatty acids. These results indicate that fatty acids directly inhibit the muscular glucose uptake via the decrease in muscular insulin uptake at a physiological concentration of insulin.
