Molecular cloning and nucleotide sequence of complementary DNAs encoding human short chain acyl-coenzyme A dehydrogenase and the study of the molecular basis of human short chain acyl-coenzyme A dehydrogenase deficiency.

Complementary DNAs encoding the precursor of human placental short chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) (EC 1.3.99.2) were cloned and sequenced. The cDNA inserts in these clones were 1,852 bases in length combined, and encoded the entire 412-amino acid precursor SCAD (mol wt 44,303). This sequence included the 24-amino acid leader peptide moiety (mol wt 2,576) and 388 amino acids corresponding to the mature protein (mol wt 41,727). The comparison of SCAD and medium chain acyl-CoA dehydrogenase sequences revealed a high degree of homology, suggesting that these enzymes evolved from a common ancestral gene and belong to a gene family. We also studied mutant human SCAD in cultured skin fibroblasts from three patients with hereditary SCAD deficiency. Labeling fibroblast cultures with [35S]-methionine followed by immunoprecipitation with anti-SCAD antibody revealed that a normal size variant SCAD protein was synthesized. In all of the three SCAD-deficient cell lines, the size of variant SCAD mRNA as determined by Northern blotting using one of the normal SCAD cDNA as a probe was also normal, and no difference was observed on Southern blots in the restriction patterns of mutant genomic DNA using EcoRI, TaqI, HincII, and BamHI. These results suggest that the defects in SCAD in these cell lines are caused by a point mutation.

A reappraisal of the subunit molecular mass of chicken liver cytosol 5'-nucleotidase.

The subunit molecular mass of chicken liver cytosol 5'-nucleotidase was earlier reported to be 51 kDa upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Naito, Y. and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159-168). By immunoblot analyses after SDS-gel electrophoresis, however, a fraction from the liver homogenized in the presence of leupeptin showed multiple bands with molecular masses around 57 kDa, and SDS-extracted proteins directly from the liver exhibited a single 70 kDa component. These results indicate that the 51 kDa peptide observed in the cytosol 5'-nucleotidase preparation might, in fact, be a proteolytic artifact.

Demonstration and characterization of estrogen receptor in chimpanzee sex skin: correlation between nuclear receptor levels and degree of swelling.

The presence of cytosol and nuclear estrogen receptors has been demonstrated in chimpanzee sex skin. Both receptors sedimented at approximately 4S in sucrose gradients containing 0.6 M KCl. Both had a steroid specificity and high affinity for estrogen that conform to an estrogen receptor. The absence of such receptors in the pigmented skin surrounding the sex skin and in the abdominal skin indicates their discrete localization in the sex skin. While the cytosol receptor remained low (less than 10 fmol/mg cytosol protein), the nuclear estrogen receptor showed a fluctuation during the menstrual cycle, attaining the highest level during the late follicular phase (90.4 +/- 8.4 fmol/mg nuclear extract protein; nearly 3-4 times the level during the early follicular and luteal phases). When ovariectomized animals were treated with mestranol, the concentration of nuclear estrogen receptor increased from below detection to 74 fmol/mg nuclear extract protein. An increase in serum progesterone during the luteal phase, despite concurrently elevated serum estradiol levels, was associated with a reduction of the nuclear estrogen receptor as was the administration of progesterone and mestranol to estrogen-primed ovariectomized animals. Changes in the concentration of the nuclear estrogen receptor were positively correlated with changes in the degree of the sexual swelling. These results strongly suggest that estrogen controls the sexual swelling by acting through specific receptors in the sex skin, and that counteraction by progesterone of estrogen stimulation of the sexual swelling is effected through a reduction in nuclear estrogen receptors.

Lysyl oxidase activity in the mouse uterine cervix is physiologically regulated by estrogen.

Lysyl oxidase activity in the mouse cervix was examined during the estrous cycle, pregnancy, and the postpartum period. It was highest in estrus and lowest in diestrus. On day 1 of pregnancy, the activity was comparable to that seen in estrus. It then declined continuously until day 17 of pregnancy, when it was approximately equal to that seen during diestrus. The activity showed a slight increase toward term, then a precipitous increase occurred on the first postpartum day. The activity fell by the seventh postpartum day to a value comparable to that seen in diestrus. Enzyme activity fell to one fourth of its former value 3 days after ovariectomy. The injection of 1 microgram 17 beta-estradiol or more in 0.9% NaCl 10 days after ovariectomy raised the activity, in 18 h, to the level seen in estrus. One milligram of progesterone had no effect on the enzyme activity, but a combination of 1 microgram 17 beta-estradiol and 1 mg progesterone raised the activity to that seen with 1 microgram 17 beta-estradiol alone. These results suggest that lysyl oxidase activity in the mouse cervix is under the control of estrogen.

Immunohistochemical analysis of rat S-adenosylmethionine synthetase isozymes in developmental liver.

Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver-type and kidney(non-hepatic)-type enzymes. The developmental expression of these two isozymes proteins has been investigated in rat liver using immunohistochemical techniques. The liver-type AdoMet synthetase is expressed only in adult liver, but not in fetal liver. On the other hand, the kidney-type AdoMet synthetase is predominantly expressed in fetal liver and faintly detected in adult liver. It was also found that both isozymes were localized to the hepatocytes of rat liver. These results clearly show that AdoMet synthetase isozymes are developmentally regulated within hepatocytes. In addition, in rat kidney we have shown that the kidney-type AdoMet synthetase is predominantly localized to the distal tubule.

Immunocytochemical localization of cytosol 5'-nucleotidase in chicken liver.

We investigated the localization of cytosol 5'-nucleotidase in chicken liver by use of a pre-embedding immunoenzyme technique. Cytosol 5'-nucleotidase was purified from chicken liver and a monospecific antibody to this enzyme was raised in a rabbit. Fab fragments of the antibody were conjugated with horseradish peroxidase. Tissue sections of the fixed chicken liver were incubated with the peroxidase-Fab fragments, followed by DAB reaction for peroxidase. By light microscopy, dark-brown staining was present in the cytoplasm of parenchymal cells, Kupffer cells, and endothelial cells. The latter two types of cells were stained more strongly than the former. By electron microscopy, reaction deposits were present in the cytoplasmic matrix but not in cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes, or in nuclei. In control sections incubated with peroxidase-conjugated Fab fragments from non-immunized rabbit, no specific reaction was noted. The results indicate that cytosol 5'-nucleotidase is contained more in the sinus-lining cells and less in the parenchymal cells, and that the enzyme is present in the cytoplasmic matrix of these cells.

Effects of vitamin E deficiency and glutathione depletion on stress protein heme oxygenase 1 mRNA expression in rat liver and kidney.

Heme oxygenase 1 (HO-1) is a stress protein and has been suggested to provide defense mechanisms against agents that may induce oxidative injury. Vitamin E (VE) is considered to function as an important cellular antioxidant. Rats were fed a VE-deficient (0E) or a VE-sufficient (10E) diet for 6 weeks and then were intraperitoneally administered buthionine sulfoximine (BSO), a glutathione (GSH)-depleting reagent. Whereas HO-1 mRNA levels were undetectable in untreated 0E and 10E rat livers, BSO administration induced HO-1 mRNA expression in both 0E and 10E rat livers. High levels of HO-1 mRNA expression were observed in particular in BSO-treated 0E rat livers. The time-course of changes in HO-1 mRNA expression in 0E rat liver after BSO administration showed that HO-1 mRNA expression was transiently induced at 2.5 hr after BSO treatment, the earliest time examined. In addition, to determine whether VE deficiency and GSH depletion affect the expression of HO-1 mRNA in other tissues, we also examined the time-course of HO-1 mRNA expression in BSO-treated 0E rat kidney. The expression pattern of HO-1 mRNA in the kidney was very similar to that in the liver, and the peak was also observed at about 2.5 hr after BSO administration. Interestingly, histologic assessment of liver and kidney showed that VE deficiency and GSH depletion induced injury in the kidney, but not in the liver.

Pharmacological preconditioning with doxorubicin. Implications of heme oxygenase-1 induction in doxorubicin-induced hepatic injury in rats.

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide, and iron. HO-1, an inducible form, is thought to contribute to resistance to various types of oxidative stress. Doxorubicin (DOX) produces clinically useful responses in a variety of human cancers. We reported previously that prior administration of DOX ameliorated subsequent hepatic ischemia and reperfusion injury. The aim of this study was to examine whether this pharmacological preconditioning was useful for another type of hepatic injury induced by a non-surgical method. When a high dose of DOX (10 mg/kg body weight) was administered directly to rat liver via the portal vein, serum aspartate transaminase (AST) and alanine transaminase (ALT) levels increased markedly 24 hr after the injection. Under this condition, zinc-protoporphyrin IX, a specific inhibitor of HO-1, caused both serum AST and ALT levels to be elevated further. When a low dose of DOX (5 mg/kg body weight) was administered to rats via the tail vein as pharmacological preconditioning 3 days before the injection of a high dose of DOX via the portal vein, the levels of serum AST and ALT in rats clearly were improved as compared with rats without the preconditioning. Expression of HO-1 in the liver was confirmed 3 days after the administration of a low dose of DOX. In addition, prior administration of zinc-protoporphyrin IX abolished the effect of DOX preconditioning. Immunohistochemical analysis showed that the positive staining of HO-1 protein induced by a low dose of DOX was localized to histiocytes infiltrating periportal areas. These results strongly suggest that pharmacological preconditioning with DOX may generally help to attenuate subsequent oxidant-induced hepatic injury.

Regenerative response in acute renal failure due to vitamin E deficiency and glutathione depletion in rats.

In this study, we investigated some factors contributing to renal regeneration after acute renal failure (ARF) induced by vitamin E (VE) deficiency and glutathione (GSH) depletion. Acute renal failure was induced by feeding rats a vitamin E-deficient diet for 6 weeks and then injecting buthionine sulfoximine (BSO), a glutathione-depleting agent. The level of hepatocyte growth factor (HGF), a renotropic factor for regeneration in the kidney, showed a transient increase at 5 hr after the BSO treatment. Subsequently, renal ornithine decarboxylase (ODC) activity, a marker of G1 phase, and labeling index (LI) of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis (S phase), reached peaks at 10 and 53 hr after the injection, respectively. Thus, it appears that the increase in ornithine decarboxylase activity and subsequent elevation in proliferating cell nuclear antigen labeling index following the increase in the hepatocyte growth factor level in the kidneys are closely related to the renal regenerative response after acute renal failure.

Human cardiac myosin heavy chain gene mapped within chromosome region 14q11.2----q13.

Our previous report demonstrated that two human cardiac alpha- and beta-myosin heavy-chains (MHCs) which correspond to MYH6 and MYH7 respectively, according to Human Gene Mapping nomenclature, were mapped to human chromosome 14 and that human cardiac and skeletal MHC genes do not cosegregate. For further analysis, the regional mapping method was used. DNA from 4 human deletion and 3 human duplication cell lines were prepared for southern blotting, hybridized with human cardiac alpha- and beta-MHC DNA probes, and the hybridization intensity relative to 46,XX or 46,XY DNA was estimated. The results showed that two human cardiac MHC genes segregated with the 14cen----q13 region of the long arm of human chromosome 14. In situ hybridization of 3H-labeled human cardiac alpha-MHC probe to normal human metaphase chromosome independently confirmed this result.

Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase.

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver.

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.

Purification and properties of carnitine acetyltransferase from rat liver.

Carnitine acetyltransferase was purified from rat liver after induction of the enzyme by feeding with di(2-ethylhexyl)phthalate. Two enzyme sources were used: the mitochondrial fraction and the homogenate of the liver. The purification procedure was essentially the same for the two enzyme sources. The enzyme purified from the mitochondrial fraction consisted of two different polypeptides with molecular weights of 36,500 and 27,000, whereas that from the homogenate consisted of one polypeptide with a molecular weight of 67,500. Amino acid compositions and peptide maps of the limited proteolytic products of the two enzyme preparations were nearly the same. Their antibodies were cross-reactive. Catalytic properties of the two preparations were nearly the same: the specific enzyme activities, double reciprocal plots of initial velocity study, substrate specificities for acylcarnitines having various carbon chain lengths, apparent Michaelis constants for substrates. On electrophoresis of the immunoprecipitate obtained after incubation of the mitochondrial extract, the two immunoreactive polypeptides with molecular weights of 36,500 and 27,000 were found. But only one polypeptide, with molecular weight of 67,500, was detected when the protease inhibitors were added to the mitochondrial extract. It was concluded that the enzyme in the mitochondrial fraction was a monomeric form but was converted into a dimeric form by proteolytic modification after the disruption of mitochondria. The preparation from the post-mitochondrial fraction, which had a lower specific activity, contained two polypeptides whose molecular weights were 69,000 and 67,500. They could not be separated from each other throughout the purification. The peptide maps of the products of the limited proteolysis were very similar.

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid.

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.

Biosynthesis and turnover of carnitine acetyltransferase in rat liver.

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.

Progesterone increases serum CA-125 in endometriosis.

Serum level of CA-125 was monitored up to 48 hours after injection of 125 mg P-in-oil into patients with or without endometriosis in the follicular phase of their menstrual cycle. Circulating CA-125 levels remained unchanged at around 5 U/ml during the observation period in patients without endometriosis; however, a significant elevation was observed at 30 hours after injection in those patients with endometriosis (from 19.6 U/ml before injection to 30.0 U/ml at 30 hours after injection, P less than 0.05).

Soluble transferrin receptor-transferrin complex in serum: measurement by latex agglutination nephelometric immunoassay.

The transmembrane protein, transferrin receptor (TfR), exists in serum as a soluble form that lacks cytoplasmic and transmembrane domains (residues 1-100). The level of soluble TfR in serum is a sensitive indicator of total erythropoiesis and iron deficiency. This study revealed that the major part of soluble TfR was saturated by transferrin (Tf) in serum, forming a stable complex which was more immunoreactive than intact TfR. Thus, we proposed that serum soluble TfR should be measured as the TfR-Tf complex (TRC), using prepared TRC for assay standardization. We developed a new assay for TRC, employing antibody-coated latex agglutination nephelometry (LA). Rapid and reproducible measurements were achieved using an automated analyzer. The values obtained by this LA assay were closely correlated with those obtained by conventional enzyme immunoassay (r = 0.967). The mean level of TRC in 179 adult healthy subjects was 1.62 mg/l. Patients with iron-deficient anemia showed significantly higher TRC levels than the healthy subjects.

The repetitive activation of extracellular signal-regulated kinase is required for renal regeneration in rat.

In this study, we investigated the activation of p42 extracellular signal-regulated kinase (ERK2) during renal regeneration after HgCl2-induced acute renal failure (ARF) in rat. ERK2 activation was observed at 5 and 29 hr after HgCl2 injection, respectively. The tyrosine phosphorylation of hepatocyte growth factor receptor (c-MET) occurred between 2.5 and 5 hr after the treatment. On the other hand, the phosphorylation of epidermal growth factor receptor (EGFR) was transiently observed at 29 hr after the injection. The peak of ornithine decarboxylase activity as a marker of G1 phase was at 10 hr, and subsequently the labeling index of proliferating cell nuclear antigen as a marker of S phase increased at 53 hr. These results indicate that the repetitive activation of ERK2 related to the phosphorylation of c-MET and EGFR is required for the renal regeneration in HgCl2-induced ARF of rat.

Changes in serum lactate dehydrogenase isozyme-X activity observed after cadmium administration.

After subcutaneous (s.c.) administration of cadmium (Cd), activity of lactate dehydrogenase isozyme-X (LDH-X) appeared in the serum of treated mice within 24 h. The determination of the activity of this enzyme in serum serves as a reliable and convenient method for early detection of testicular damage induced by Cd.