Red cell folate and plasma homocysteine in preterm infants.

BACKGROUND: The supplementation of preterm infants with folic acid is routine practice in many neonatal units. However, the advent of preterm formula milks and breast milk fortifiers have increased folic acid intake. We measured red cell folate in preterm infants who received preterm formula milks and breast milk fortifiers to determine whether additional folic acid supplementation was still required. A potential benefit of folic acid supplementation is reduction of plasma total homocysteine (tHcy). tHcy appears to have a linear association with the risk of atherothrombotic vascular events in adults but its role in intraventricular haemorrhage and associated white matter damage in preterm infants is not known. As there is little information regarding tHcy in preterm infants, we also measured tHcy in this study. METHODS: Red cell folate and tHcy were measured at 1 and 4 weeks of age and before discharge in 28 consecutive infants <34 weeks' gestation. Factors which may have affected folate and homocysteine status were recorded. RESULTS: Red cell folate ranged between 266 and 1,513 ng/ml and deficiency (<140 ng/ml) was not observed in any sample. Red cell folate concentration tended to increase with increasing age. tHcy ranged from 0.8 to 12.2 micromol/l and fell within the 'normal' range for fasting adults. CONCLUSIONS: Preterm formula milks and breast milk fortifiers provide sufficient folic acid to prevent folate deficiency in preterm infants. Although tHcy fell within the 'normal' range for fasting adults, more research is needed to determine optimal concentration of tHcy for preterm infants.

Congenital ichthyosiform erythroderma: particulate staining pattern of TGK.

A case of late onset non-bullous congenital ichthyosiform erythroderma (CIE) was studied. This patient was not born as a collodion baby and did not have skin abnormalities until 9-10 years of age. She gradually developed erythroderma and fine scales, callosities of her feet, and a mild ectropion. Since recent work has revealed that in the majority of CIE patients, transglutaminase (TGK) is distributed in the cytoplasm of granular cells and horny cells (11), TGK was studied in our case. It was found that TGK was distributed along the cell periphery of horny cells and also in the cytoplasm of granular cells. In the control skins, TGK was stained along the cell periphery of horny cells and granular cells. The marginal band formation was normal. Involucrine and loricrin, the building materials of the marginal band whose-cross-linking is mediated by TGK, were normally stained in the upper epidermis. Cytoplasmic TGK of granular cells and normal development of the marginal band may serve as a helpful diagnostic marker of CIE, particularly because the often confusing collodion baby of lamellar ichthyosis may lack TGK staining and the marginal band altogether.

Expression and purification of human gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.

Coal workers' pneumoconiosis: a study of prevalence in coal mines of eastern Madhya Pradesh and Orissa states of India.

With objective to find out prevalence of Coal Worker's Pneumoconiosis and variation among readers in reading x-ray plates for pneumoconiosis, a retrospective epidemiological survey of Coal Worker's Pneumoconiosis was undertaken in 72 collieries of Madhya Pradesh and Orissa by re-reading of x-ray plates taken during the Periodical Medical Examination at the Occupational Health Units over a period of 5 years. Six readers, trained abroad in reading pneumoconiosis x-ray plates, were involved for the study. Each reader reported approximately one sixth of the available x-ray plates of all the collieries and classified on the 12 point scale of I.L.O. (International Labour Organisation) 1980 in special format. Total 43,504 chest x-rays were reviewed. The overall prevalence was found to be 3.03%, ranging from 1.52% to 4.76% between 10 areas (group of mines). Major category of profusion was category-I (81.09%), followed by category-II (17.84%). Only 3 cases of Progressive Massive Fibrosis (PMF) were detected. Round shaped opacities are predominant (89.59%) in Coal Worker's Pneumoconiosis. Among the opacities, 'p' type is more prevalent (48.29%) followed by 'q' type (40.62%). There was variation amongst the different readers and ranged from 1.14% to 6.76% for reporting the prevalence of Coal Worker's Pneumoconiosis. However, when analysis of six readers for inter reader variation was conducted, that shows no abnormal deviation in the reading of any of the readers.

Evidence for the interaction of avian 3-hydroxy-3-methylglutaryl-CoA synthase histidine 264 with acetoacetyl-CoA.

Previous work on HMG-CoA synthase has implied the presence of a reactive active site histidine, prompting our examination of the possible function of invariant histidine residues by site-directed mutagenesis. Mutations encoding H197N, H264N/A, and H436N HMG-CoA synthases were constructed, and the mutant enzymes were overexpressed in Escherichia coli BL21(DE3). Kinetic characterization of the isolated synthase variants indicates that, while H197N and H436N enzymes behave similarly to wild-type synthase, H264N and H264A synthases exhibit significant differences. Although the k(m) for acetyl-CoA is not substantially altered, H264N/A synthases catalyze production of HMG-CoA at a diminished (approximately 25-fold slower) rate. In contrast, H264N/A synthases can efficiently catalyze the acetyl-CoA hydrolysis partial reaction exhibiting a k(m) for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme. These mutants also retain the ability to form significant levels of the acetyl-S-enzyme reaction intermediate. The functional catalysis of partial reactions argues that the H264 mutant proteins retain substantial structural integrity. In this context, it appears significant that the H264N/A synthases exhibit a approximately 100-fold increase in the k(m) for acetoacetyl-CoA. In order to test whether the two orders of magnitude effect may be largely attributed to a decreased affinity of acetoacetyl-CoA for these enzymes and, more specifically, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA's thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of acetoacetyl-CoA, was investigated. This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild-type synthase with an apparent Vmax that is approximately 100-fold lower and an apparent k(m) that is 25-fold higher than the values obtained using the physiological substrate, acetoacetyl-CoA. H264A synthase also catalyzes the turnover of S-(3-oxobutyl)-CoA; the diminution in rate supported by the alternative substrate is comparable in magnitude to the effect observed for wild-type enzyme. In contrast, H264A exhibits comparable apparent k(m) values for S-(3-oxobutyl)-CoA and acetoacetyl-CoA. Thus, unlike wild-type synthase, there is no penalty in terms of efficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate. These data suggest that the imidazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen of the thioester functionality.

Avian cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase: evaluation of the role of cysteines in reaction chemistry.

The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6. The observation of optimal activity at alkaline pH and the insensitivity of pK to divalent cation concentration suggest that the pK reflects ionization of an amino-acid side chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization. Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by this mechanism-based inhibitor, stoichiometric modification occurs. Amino-acid analysis indicates that cysteine is the principal target in C129S enzyme, demonstrating the presence of a second reactive cysteine within this enzyme. To test whether another cysteine functions in reaction chemistry, conserved cysteines were identified by sequence homology analysis. Five cysteine residues (C59, C69, C224, C232, C268), invariant in the nine sequences available for various eukaryotic HMG-CoA synthase isozymes, were individually replaced by alanine in a series of mutant enzymes. Kinetic analyses of the isolated mutant HMG-CoA synthases indicate that none of these is crucial to the chemistry that results in production of HMG-CoA. These results further distinguish the HMG-CoA synthase reaction from the related condensation of acyl-CoA substrates catalyzed by beta-ketothiolase.

Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.

PURPOSE: To determine if lens epithelial lines can be established from cryopreserved whole rabbit lenses and from cryopreserved capsule-epithelial preparations (CEPs). METHODS: Lenses or freshly isolated CEPs were cryopreserved and subsequently thawed. Thawed whole lenses were cultured for 48 hours in growth medium and fixed, and whole mounts were examined for mitosis. In addition, CEPs were peeled from cryopreserved lenses and placed in tissue culture. Viability of cryopreserved cells was assessed measuring attachment efficiency and growth. RESULTS: Whole mounts from cryopreserved lenses that were thawed and placed in organ culture in a serum-containing medium exhibited numerous mitotic figures. Freshly isolated CEPs that were cryopreserved and CEPs from cryopreserved lenses generated cell lines. Attachment efficiency was 90% within 3 hours of plating. When 50,000 cells from cryopreserved CEPs were cultured in growth medium, 10(6) cells were noted after 7 days of culture. The cells completed 27 population doublings and showed no sign of senescence. CONCLUSIONS: Rabbit lens epithelial cell lines can be initiated from cryopreserved lenses or CEPs.

Avian 3-hydroxy-3-methylglutaryl-CoA synthase. Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate.

cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)

The superoxide dismutase mimic TEMPOL protects cultured rabbit lens epithelial cells from hydrogen peroxide insult.

The superoxide dismutase mimic, 4-hydroxy TEMPO (TEMPOL), was used to investigate the mechanism by which H2O2 damages cultured rabbit lens epithelial cells and to identify some of the targets of H2O2 insult. Most studies aimed at determining the mechanism by which H2O2 exerts its cytotoxic effect have used iron chelators to prevent the generation of the damaging hydroxyl radical. Since TEMPOL does not chelate transition metals, we were afforded an additional means of investigating the mechanism by which H2O2 exerts its cytotoxicity. Cells at low or high density were cultured in MEM containing 5 mM TEMPOL and exposed to a single sub-lethal dose of 0.05 or 0.5 mM H2O2, respectively. Analysis of EPR spectra indicated that TEMPOL was stable in MEM, did not destroy H2O2 and penetrated the intracellular fluid. TEMPOL prevented or curtailed the H2O2-induced inhibition of cell growth, blebbing of the cell membrane, the decrease in NAD+, the activation of poly ADP-ribose polymerase, an enzyme involved in DNA repair, and limited the induction of single strand breaks in DNA normally brought about by H2O2. TEMPOL did not prevent the H2O2-induced decrease in reduced glutathione, lactate production, and the activity of glyceraldehyde 3-phosphate dehydrogenase, or the H2O2-induced increases in oxidized glutathione and hexose monophosphate shunt activity. Addition of TEMPOL 1-15 min after exposure of cells to H2O2 offered partial protection from the inhibition of cell division. TEMPOL at 5 mM did not inhibit cell growth. These results, coupled with our other findings suggest that some of the H2O2-induced damage in cultured rabbit LECs is mediated by intracellular redox-active metals involved in the Haber-Weiss cycle. Cellular changes not protected by TEMPOL, including attack of H2O2 on the thiol groups of GSH (mediated through glutathione peroxidase) and G3PDH, are likely brought about by H2O2 itself and not by reactions of oxygen free-radicals generated from H2O2.

Management of missed abortion by intramuscular administration of 15(S) 15 methyl prostaglandin F2 alpha.

PIP: 22 subjects with a diagnosis of missed abortion were induced with 15(S) 15 methyl prostaglandin F2alpha (PGF2alpha) by the intramuscular route. The induction succeeded in all cases. The mean induction-abortion interval was 16 hours with a range of 2 hours 30 minutes to 32 hours 10 minutes. The average dose of 15 methyl PGF2alpha required was 1675 mcg. 9 subjects (42%) had complete abortions. 7 (31.8%) had no gastrointestinal side effects. The mean number of episodes of vomiting and diarrhea was 1.3 and 0.8 respectively. 15(S) 15 methyl PGF2alpha has a high degree of efficacy in the management of missed abortion.^ieng