The promoter regulatory regions of the genes for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken and the rat have different species-specific roles in gluconeogenesis.

Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.

Hyperhomocysteinemia and low pyridoxal phosphate. Common and independent reversible risk factors for coronary artery disease.

BACKGROUND: High plasma homocysteine is associated with premature coronary artery disease in men, but the threshold concentration defining this risk and its importance in women and the elderly are unknown. Furthermore, although low B vitamin status increases homocysteine, the link between these vitamins and coronary disease is unclear. METHODS AND RESULTS: We compared 304 patients with coronary disease with 231 control subjects. Risk factors and concentrations of plasma homocysteine, folate, vitamin B12, and pyridoxal 5'-phosphate were documented. A homocysteine concentration of 14 mumol/L conferred an odds ratio of coronary disease of 4.8 (P < .001), and 5-mumol/L increments across the range of homocysteine conferred an odds ratio of 2.4 (P < .001). Odds ratios of 3.5 in women and of 2.9 in those 65 years or older were seen (P < .05). Homocysteine correlated negatively with all vitamins. Low pyridoxal 5'-phosphate (< 20 nmol/L) was seen in 10% of patients but in only 2% of control subjects (P < .01), yielding an odds ratio of coronary disease adjusted for all risk factors, including high homocysteine, of 4.3 (P < .05). CONCLUSIONS: Within the range currently considered to be normal, the risk for coronary disease rises with increasing plasma homocysteine regardless of age and sex, with no threshold effect. In addition to a link with homocysteine, low pyridoxal-5'-phosphate confers an independent risk for coronary artery disease.

Determination of total serum sulfite by HPLC with fluorescence detection.

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.

Metabolic regulation of gene transcription.

The impact of nutrients on gene expression has become an area of considerable interest as the number of genes coding for key regulatory proteins in metabolic pathways are studied in detail. This has been greatly aided by a number of new techniques developed to study gene transcription in animals. We will use as an example studies on the regulation of transcription of the gene coding for P-enolpyruvate carboxykinase, a key enzyme in hepatic and renal gluconeogenesis. The promoter for P-enolpyruvate carboxykinase contains a number of regulatory elements within 500 bp of the start-site of gene transcription that are required for the response of the gene to metabolic signals. These elements bind tissue-specific transcription factors in complex patterns of interactions, which result in the coordinate control of P-enolpyruvate carboxykinase gene expression. An analysis of the regulation of transcription of this gene involves the use of a number of techniques ranging from gene transfection into cells in culture to the introduction of chimeric genes containing the P-enolpyruvate carboxykinase promoter into transgenic mice. This review presents a progress report on the current status of research on the nutritional and hormonal regulation of transcription of the P-enolpyruvate carboxykinase gene.

Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects.

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, > 80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P < 0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Heteronuclear nuclear magnetic resonance studies of cobalt corrinoids. 15. The structure of glutathionylcobalamin: a 1H and 13C two-dimensional nuclear magnetic resonance study at 600 MHz.

Glutathionylcobalamin (GSCbl), the complex formed between glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl), has been implicated as an intermediate in the pathway for the formation of the cobalamin coenzymes. In chemical model studies, GSCbl has been shown to be a substrate for methylcobalamin formation in the presence of S-adenosylmethionine and a thiol reductant. Although GSCbl was first described in 1964, the structure of this compound, particularly the site of GSH coordination, has been unknown. GSCbl was prepared by reacting GSH (5-fold molar excess) with H2OCbl in 0.1 M sodium phosphate (pH 6.5) and was purified by gel-permeation chromatography on a Bio-Gel P2 polyacrylamide column. By use of a combination of homonuclear [homonuclear J-correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and absorption-mode nuclear Overhauser effect spectroscopy (NOESY)] and inverse detected heteronuclear [1H-detected heteronuclear multiple-quantum coherence (HMQC) and 1H-detected multiple-bond heteronuclear multiple-quantum coherence (HMBC) spectroscopies] two-dimensional NMR methods at 600 MHz, the complete 1H and 13C NMR spectra of GSCbl have now been assigned. Comparison of the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl to those of free GSH and GS- shows that by far the largest differences occur at the cysteine alpha and beta positions. This result strongly suggests that GSH is coordinated to the cobalt atom in GSCbl via the cysteine sulfur atom.

Serum transferrin receptor level is not altered in invasive adenocarcinoma of the breast.

The transferrin receptor is expressed on the surface of rapidly dividing cells that require iron as a co-factor for essential redox reactions and deoxyribonucleotide synthesis. Transferrin receptors are expressed on the surface of breast carcinoma cells but not on benign breast tumor cells. In this study, the authors investigated whether transferrin receptor concentrations in the serum were elevated in patients with invasive adenocarcinoma of the breast. The transferrin receptor was isolated and purified from human placenta by affinity chromatography. The serum transferrin receptor concentration was determined using an enzyme-linked immunosorbent assay in 19 patients with invasive breast adenocarcinoma, 12 of whom had involvement of axillary lymph nodes. These results were compared with those from 16 normal age-matched female controls. In the invasive breast cancer group, the range of transferrin receptor concentrations was 2.60-7.34 mg/L (mean, 4.44 mg/L) compared with 2.85-8.80 mg/L (mean, 5.49 mg/L) in the control group. Nine patients with in situ adenocarcinoma of the breast had transferrin receptor concentrations of 3.68-6.66 mg/L (mean, 4.94 mg/L). For both the invasive carcinoma group and the in situ group, the means were not significantly different from those of the control group (P = 0.06 and 0.32, respectively). It was concluded that the differential expression of transferrin receptor on the surface of malignant tumor cells in adenocarcinoma of the breast was not reflected by changes in circulating transferrin receptor concentrations.

Liver-specific expression of a phosphoenolpyruvate carboxykinase-neo gene in genetically modified chickens.

In order to investigate the potential of the avian liver for the expression of recombinant proteins in vivo, replication-competent retroviral vectors were used to introduce a recombinant rat phosphoenolpyruvate carboxykinase promoter-driven neomycin resistance gene (PEPCKneo) into early Line 11 Leghorn embryos. After hatching, these birds possessed apparently intact PEPCKneo sequences in most tissues examined, however, the neo protein was expressed preferentially in the liver (up to .45% of total cellular protein). Therefore, the tissue specificity of the PEPCK promoter from the rat was retained in the chicken, although hormone responsiveness was not observed. Retroviral vectors used to transmit the genes were more stable during passage in either fibroblast cells or in the animal if the inserted genes were oriented in the same (sense) direction as the viral genome. After Geneticin drug selection in cultured cells, PEPCKneo mRNA was the predominant recombinant species observed on Northern blots, whereas embryos expressed mostly the RNA species originating in the retroviral long terminal repeats. The results demonstrate the potential usefulness of liver-specific gene expression in chickens, as well as the transcriptional effects observed when a foreign promoter is introduced into the replication-competent vector.

Expression of the genes for the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase in avian liver during development.

Gluconeogenesis in the chicken has unique features due in part to the presence of two isozymes of PEPCK, a cytosolic form, PEPCK-C, and a mitochondrial form, PEPCK-M, which have novel patterns of expression. Here we show that, in contrast to mammals, in which PEPCK-C is not present in liver until after birth, avian PEPCK-C is expressed throughout embryonic life with mRNA levels gradually decreasing as development proceeds and becoming negligible at time of hatching. In addition two distinct mRNAs for PEPCK-M are expressed during development with specific patterns that vary among individual birds. These differences are likely to be genetic, as hormonal treatment of a chicken hepatoma cell line indicates that whereas the mRNA levels for PEPCK-C are hormonally regulated, the expression of PEPCK-M mRNA is unresponsive.

Immunomodulating effects of serum-material interactions.

The objective of this study is to evaluate an in vitro model to assess the effects of serum-material interactions on complement activation, macromolecular adsorption, and lymphocyte response. Minifilters of clinically available materials (PVA, EVAL-4A, and EVAL-D) used in extracorporeal therapies were evaluated. The test circuit consisted of a pump, sterile tubing, collection vessels, and the minifilter. A sham circuit similar to the test circuit was constructed, but without the filter. Serum flow rates and volumes processed were scaled down to those of clinical use. Post PVA serum showed the highest degree of complement activation, macromolecular solute adsorption, and lymphocyte suppressive response when incubated with Con-A, PHA, PWM, and Candida. Post EVAL-4A sera enhanced the response of lymphocytes to Con-A and PHA, while Post EVAL-D sera showed a slight suppression to these mitogens. Blood-material interactions have been shown to cause blood cellular changes. The in vitro model employed is simple to apply and does not require an animal or patient. The membrane modules used are a mini-type of clinically available extracorporeal filters, and there is a greater direct relevancy to clinical applications than there would be using specially formulated materials. This system would provide useful preclinical information in evaluating the effect of serum-material interactions.

Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme.

The amino acid sequence of the mitochondrial form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-M) from the chicken was deduced from the 3571 nucleotide sequence of three overlapping cDNA clones. The derived protein sequence, which includes 607 amino acids of the mature enzyme and a leader sequence, was aligned with nine tryptic peptides of PEPCK-M and the primary sequence of the cytosolic form of PEPCK from chicken. Secondary structure predictions for the two PEPCK isozymes indicated similar packing elements of conserved, hydrophobic beta strands in the central core of the primary sequence. This core protein, which contained three GTP-binding consensus elements, was 80% identical in the two chicken isozymes, although the overall level of identity was only 63% for amino acids and 60% for nucleotides. The untranslated regions of the two cDNAs were dissimilar, although both mRNAs have potential for significant secondary structure. The PEPCK-M mRNA contained several G-C-rich regions which demonstrated free energies of formation in dyad symmetry programs up to -70 kcal/mol. The 1.6-kilobase (kb) 3'-untranslated region contained several repeat elements including one of 11 base pairs, which was present 30 times; but, a signal sequence for polyadenylation was not present. Each of the three PEPCK-M cDNA clones recognized two mRNAs of 4.2 and 3.4 kb in the livers and kidneys of starved or normally fed chickens. However, the level of these two related PEPCK-M mRNAs changed in response to cAMP treatment, with the larger mRNA predominant at 20 and 160 min and the 3.4-kb mRNA present at intermediate times. In contrast, the level of the 2.8-kb PEPCK-C mRNA increased dramatically upon addition of the cyclic nucleotide, particularly in the liver where it was not detected without cAMP induction. Thus, PEPCK-M and PEPCK-C, clearly represented the products of two distinct genes, which were distinguished by altered protein sequences and non-cross-hybridizing, differentially regulated mRNAs.