Study of the Huntington's disease IT-15 gene in different ethnic groups in Ecuador.

This study aims to establish the current state of the IT-15 (HTT) gene in different Ecuadorian ethnic groups and patients by determining CAG triplet repeats, compared with the ethnicity of individuals. A total of 412 individuals were studied using nested polymerase chain reaction and Sanger sequencing: 75 individuals were indigenous (Kichwas), 211 mestizos, and 65 Afro-Ecuadorians. We included 31 patients who were clinically diagnosed with Huntington's disease (HD) and relatives of the affected patients (n = 30). Moreover, we correlated the presence of HD in Ecuadorian patients with 46 genetic ancestry-informative insertion-deletion polymorphic markers. We found that 77.20% had <28 CAG repetitions, 18.80% had mutable alleles, 2.27% had incomplete penetrance, and 1.70% reflected >39 repetitions. The average of CAG repetitions was 24 ± 3 for indigenous people; 28 ± 2 for mestizos; and 24 ± 3.2 repetitions for the Afro-Ecuadorians. The ancestral component showed that the main ancestry corresponded to Native Americans (0.873) and European ascendants (0.145), Africans were less represented in the evaluated population (0.018). There was a significant difference between the number of CAG repeats in mestizos and indigenous people (P < .01), suggesting that the Ecuadorian mestizo population has a risk factor for the gene mutation.

Phylogeographic and genome-wide investigations of Vietnam ethnic groups reveal signatures of complex historical demographic movements.

The territory of present-day Vietnam was the cradle of one of the world's earliest civilizations, and one of the first world regions to develop agriculture. We analyzed the mitochondrial DNA (mtDNA) complete control region of six ethnic groups and the mitogenomes from Vietnamese in The 1000 Genomes Project (1000G). Genome-wide data from 1000G (~55k SNPs) were also investigated to explore different demographic scenarios. All Vietnamese carry South East Asian (SEA) haplotypes, which show a moderate geographic and ethnic stratification, with the Mong constituting the most distinctive group. Two new mtDNA clades (M7b1a1f1 and F1f1) point to historical gene flow between the Vietnamese and other neighboring countries. Bayesian-based inferences indicate a time-deep and continuous population growth of Vietnamese, although with some exceptions. The dramatic population decrease experienced by the Cham 700 years ago (ya) fits well with the Nam tien ("southern expansion") southwards from their original heartland in the Red River Delta. Autosomal SNPs consistently point to important historical gene flow within mainland SEA, and add support to a main admixture event occurring between Chinese and a southern Asian ancestral composite (mainly represented by the Malay). This admixture event occurred ~800 ya, again coinciding with the Nam tien.

Pyruvate kinase isozymes in cells isolated from fetal and regenerating rat liver.

There are at least three major mammalian isozymes of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40), designated K4, L4, and M4. Whereas parenchymal cells from adult rat liver contain only the type L isozyme, parenchymal cells isolated from fetal and regenerating liver were found to synthesize both the K4 and L4 isozymes. A small amount of K-M hybrid was seen in regenerating liver, but there were no detectable M-L or K-L hybrids. Thus, it appears that type L pyruvate kinase is not synthesized at the same time in the same liver cell with either of the other two isozymes. The intermediate electrophoretic bands seen with homogenates of whole fetal liver, and in some earlier work attributed to either hybrid isozymes or to the presence of M4, are contributed by nonparenchymal cells which, in the fetus, are largely hemopoietic. These additional bands of pyruvate kinase are electrophoretically and immunologically similar to the pyruvate kinase isozymes found in adult erythrocytes. The results reported here suggest a very rigorous control in the synthesis of K4 and L4 isozymes in parenchymal cells of both fetal and regenerating liver as opposed to developing neurons and glia, where the shift from synthesis of type K to type M subunits appears to occur gradually and results in the production of substantial amounts of hybrid isozymes.

Human interleukin 2. Quantitation by a sensitive radioimmunoassay.

Using polyclonal and monoclonal antibodies to human recombinant IL-2 (rIL-2), we developed a sensitive radioimmunoassay (RIA) for quantitation of human IL-2. In this assay, microtitration plates pre-coated with an anti-rIL-2 monoclonal antibody (35H10), recognizing residues 59-72 of human IL-2, are incubated with serial dilutions of test samples. Captured IL-2 is quantitated by adding an affinity-purified rabbit anti-rIL-2 antibody followed by an 125I-labeled goat anti-rabbit IgG. Antibodies to chemically synthesized IL-2 peptides could replace the polyspecific rabbit anti-rIL-2 antibody as the second specific reagent in the assay. This configuration was more sensitive than others tested, approaching the level of detection of the conventional IL-2 bioassay, thus allowing detection of as little as 100-200 pg IL-2. Serum or plasma fluids, however, inhibited the assay, reducing its sensitivity by approximately 5-fold. This RIA correlated well with the conventional bioassay in measuring IL-2 levels in sera from IL-2-treated patients. This, and similar, RIAs may serve as a useful adjunct or alternative to the conventional IL-2 bioassay in detecting and quantitating human IL-2 in culture supernatants and clinical samples.

Model building and quantitative analysis of a tandem immuno-capturing assay as a screening tool for breast cancer.

The onset of breast cancer appears to occur, on average, a decade earlier in Mexican women in comparison to American or European women. Early detection and prevention of breast cancer are of crucial importance to increase survival and improve quality of life. Based on the molecular elucidation of critical events leading to breast carcinogenesis, a tandem immuno-capturing blood test was developed as a quantitative population screening assay in view of providing a cost-effective and non-invasive alternative to population screening. Clinical analysis of 63 Mexican women within an age group of 35-70, revealed that Interstron activity increases from 800+/-65 IUJPA (Interstron Units) in the asymptomatic normal women to 994+/-100 IUJPA in the symptomatic/benign group, reaching 1289+/-81 IUJPA in the cancerous group. Accordingly, activity thresholds were established at 800 and 1200 IUJPA respectively, encompassing three risk groups: (i) Healthy Otherwise Normal (<800 IUJPA); (ii) Grey Risk Area (>800 and <1200 IUJPA), and (iii) At Risk group (>1200 IUJPA). Taking into account both baseline and clinical case reports, the Healthy Otherwise Normal group and the At Risk group were mostly homogeneous in nature, comprising a population of normal and cancer patients respectively. The Grey Risk group is heterogeneous, likely reflecting a transitional nature towards a potential early stage of breast disease development. Based on these results, a screening algorithm was developed as the underlining principle for population surveillance encompassing over 30,000 Mexican women. The current screening results have enabled us to objectively prioritize medical attention to approximately 1 in 8 women out of the general population mapped within the At Risk group. Overall, our findings suggest that monitoring Interstron activity units provides a valuable quantitative screening analysis as to selectively streamline the population of women in need of early medical counseling and/or mammography, thereby enhancing both the quality and cost-effectiveness of preventative population surveillance programs targeting breast cancer.

Immunoelectron microscopic study of the detection of the glycoproteins of influenza and Sendai viruses in infected cells by the immunoperoxidase method.

The envelope glycoproteins of influenza virus (HA and NA) and paramyxovirus (HN and F) were visualized on the surface of infected cells by immunoelectron microscopy using the indirect immunoperoxidase technique. In X7 influenza virus-infected fibroblasts, the hemagglutinin (HA) and the neuraminidase (NA) were observed on the cell membrane respectively 2 and 3--4 h after infection. The antigens were initially seen as discrete patches and later evenly distributed along the plasma membrane prior to budding. Antibody induction of HA and NA was observed as cytoplasmic inclusions, with peroxidase-positive activity, attributed to endocytosis. The redistribution of HA and NA supports the hypothesis of lateral mobility of the viral glycoproteins in cellular membranes as visualized by the immunoperoxidase method. The glycoproteins of Sendai virus, in infected Madin--Darby bovine kidney cells, were found to be evenly distributed along the plasma membrane and endoplasmic reticulum, the latter by the indirect microperoxidase method. The immunoperoxidase methods may be useful for investigating the polarized distribution of envelope glycoproteins.

Essential arginine residues occur in or near the catalytic site of L-amino acid oxidase.

Butanedione in borate buffer irreversibly inactivates L-amino acid oxidase. L-Phenylalanine and L-methionine, which are good substrates for the enzyme, protect against inactivation but glycine, which is a very poor substrate, and D-phenylalanine which is neither substrate nor inhibitor, do not provide significant protection. These results are consistent with the modification by butanedione of one or more arginine residues located in or near the catalytic site of L-amino acid oxidase.

Analysis of the renaturation kinetics of bovine muscle pyruvate kinase.

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine-HCl. Subsequent dilution of the enzyme into buffer containing 2-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with half-times that vary from 185 min at 0 degrees C to 4 min at 45 degrees C. In the temperature range 0-25 degrees C, 90% of the enzymatic activity is recovered. Above about 32 degrees C, the recovery drops off sharply, wih a yield of only 13% at 45 degrees C. Removal of inactive nonspecific aggregates and denatured monomer by gel filtration yields an enzyme with the same specific activity as the starting material. At enzyme concentrations below 3 microns/mL at 16 degrees C or below 25 micron/mL at 7.8 degrees C, the reactivation kinetics show a concentration dependence. At higher concentrations of protein and at temperatures of 16 degrees C or higher, no protein concentration dependence is seen, and the rate of reactivation is described by two first-order relaxations. The rate constants have apparent activation energies of 10.6 and 11.9 kcal/mol. Combinding the results presented here with earlier work from this laboratory [Cardenas, J. M., & Dyson, R. D. (1973) J. Biol. Chem. 248, 6938-6944; Cardenas, J. M., Hubbard, D. R., & Anderson, S. (1977) Biochemistry 16, 191-197] leads to the conclusion that a rapid, major folding produces two species which undergo transconformational steps. This is followed by subunit association which yields the native tetramer.

Pyruvate kinases of salmon: purification and comparison with the isozymes from birds and mammals.

Pyruvate kinase occurs as two major forms in coho salmon; the type M isozyme occurs primarily in muscle and heart, but type K has a more generalized tissue distribution, in parallel with the type K isozyme in other vertebrate systems. In order to assess the evolutionary relationships among the fish, avian, and mammalian isozymes of pyruvate kinase, we have purified the two isozymes from fish, have examined some of their physical properties, and have studied their immunological relationships to the avian and mammalian isozymes. Salmon type K is at least partially inactivated by antibody to bivine type L pyruvate kinase as well as by antibodies produced against chicken, bovine, and salmon type M isozymes. Salmon type M pyruvate kinase, on the other hand, is not significantly corss-reactive with the bovine type L isozyme, but is at least partially inactivated by antibodies produced against bovine or chicken type M isozymes. Mammalian type L pyruvate kinase is immunologically distinct from either mammalian type K or type M, but salmon type K has some structural features in common with all three mammalian isozymes. Thus, salmon fish type K pyruvate kinase could be similar to a primordial form that was antecedent to the three major differentiated isozymes of higher vertebrates.

Developmental changes in the pyruvate kinase isozymes of coho salmon.

Pyruvate kinase exists as two major isozymes in coho salmon. As in mammals and birds, one form is present in the early embryo and maintains a wide tissue distribution in adults. This salmonid type K shows anodal migration during electrophoresis at pH 7.5. The appearence of functional musculature in the developing embryos. In adult animals this second form is the only pyruvate kinase in muscle. Brain, kidney, liver and gill contain primarily the type K pyruvate kinase while heart contains both major forms along with three intermediate forms which presumably constitute a hybrid set. Since there is no additional isozyme restricted to gluconeogenic tissues, we conclude that a type L isozyme has not developed in these animals. The two major isozymes are immunologically distincy. Both forms are dubject to fructose 1,6-bisphosphate activation of phosphoenolpyruvate binding, but the magnitude of the effect is small. The affinities for phosphoenolpyruvate are similar, but salmon type K has hyperbolic saturation curves with this substrate and type M has sigmoidal saturation curves. While the immunological data indicates considerable divergence in structure, the kinetic parameters of the two forms have remained relatively similar.

Kinetic properties of pyruvate kinase hybrids formed with native type L and inactivated type M subunits.

Bovine type M pyruvate kinase, which normally has hyperbolic kinetics with its substrates, was inactivated by treatment with trinitrobenzenesulfonic acid. The inactivation probably occurs through trinitrophenylation of the epsilon-amino group of a lysine residue in or near the ADP binding site. Although 90 to 95% of the enzymatic activity is lost by this treatment, the molecular weight and sedimentation coefficient of the trinitrophenylated enzyme are quite similar to values obtained with the native enzyme. The inactivated, trinitrophenylated type M pyruvate kinase was hybridized in vitro with the native bovine type L enzyme, which has sigmoidal kinetics with phosphoenolpyruvate but can be activated by fructose 1,6-diphosphate to give hyperbolic kinetics. Four enzymatically active species were produced, designated L4, L3M, L2M2, and LM3, according to their subunit composition. L4 and L3M have sigmoidal kinetics with phosphoenolpyruvate and are activated by fructose diphosphate. Little or no sigmoidicity was seen for L2M2, although this species is activated to a moderate degree by fructose diphosphate. LM3 appears to have hyperbolic kinetics and is activated only slightly by fructose diphosphate. The kinetic results obtained with hybrids containing trinitrophenylated type M subunits are quite similar to the results previously reported by Dyson and Cardenas ((1973) J. Biol. Chem. 248, 8482-8488) using native type M and type L subunits, indicating that the properties of a type L subunit are profoundly affected by the nature of the other subunits present in the tetramer. In fact, type L and type M subunits in a given hybrid seem to have similar kinetic responses toward phosphoenolpyruvate and fructose diphosphate.

Single subunits of Sepharose-bound pyruvate kinase are inactive.

Bovine skeletal muscle pyruvate kinase was covalently coupled to Sepharose that had previously been activated by low concentrations of cyanogen bromide. Reaction conditions were chosen such that essentially all tetrameric enzyme molecules were covalently bound via a single subunit. Denaturation of the immobilized enzyme with guanidine hydrochloride followed by removal of noncovalently bound subunits amd denaturant resulted in essentially no enzymatic activity remaining bound to the resin. Thus, single immobilized subunits of bovine pyruvate kinase were inactive. Sepharose-bound enzymatic activity could be recovered by adding soluble renaturing enzyme subunits to the immobilized monomers. The former combine noncovalently with the latter, presumably resulting in re-formation of bound tetramers, and an average recovery of 61% of the original matrix-bound activity was observed. While interactions with other enzyme subunits appear to be necessary for catalytic activity of bovine muscle pyruvate kinase, these subunit interactions apparently can be provided by chemically modified subunits. Soluble, renaturing subunits from enzyme that had been inactivated by treatment with trinitrobenzenesulfonic acid were able to interact with matrix-bound single subunits, thereby restoring the enzymatic activity of the latter.

Mammalian pyruvate kinase hybrid isozymes: tissue distribution and physiological significance.

The purpose of this study was to examine the pyruvate kinase isozymic patterns of a wide variety of tissues from rats and mice, particularly regarding hybrid isozymes. For these studies, we employed longer electrophoresis times than used in most earlier studies in order to improve the resolution of closely spaced bands. The tissue distributions of types K, L, and M pyruvate kinases were found to be approximately the same as those reported earlier for rats and other mammals. In addition, K-M hybrids could be detected in most tissues examined in relative quantities which differed from one tissue to another in the same organism, in corresponding tissues from different species, and within a single tissue during development. Hybrid isozymes containing type L subunits occur in only a few tissues of either the fetus or the adult of either animal. In earlier studies utilizing L-M hybrid isozymes produced in vitro, we showed that the kinetic properties of a given subunit are profoundly affected by the nature of its neighbors within the tetramer (Dyson and Cardenas, ['73] J. Biol. Chem., 248: 8482-8488). Based on these altered kinetic properties, we suggest that there is little need for anorganism to suppress completely the gene activity for one subunit type of pyruvate kinase during the synthesis of larger quantities of a second subunit type.

Localization of pyruvate kinase isozymes in bovine kidney and comparison of these patterns with those of lactate dehydrogenases and aldolases.

Electrophoretic and immunofluorescence analysis were used to study the distribution of pyruvate kinase isozymes in the bovine kidney. Electrophoretic analysis demonstrated the presence of large amounts of K4 plus small amounts of K-M hybrids in cortical, medullary, and papillary sections cut from the kidney. Nearly all of the K-L hybrids seen in whole kidney extracts were found in cortical sections. Immunofluorescence of frozen sections revealed the presence of type L subunits in the tubules but the complete absence of this subunit type in flomeruli. Glomeruli do contain large quantities of pyruvate kinase isozymes, probably K4 and K-M hybrids, that cross-react with antibodies produced against type M pyruvate kinase. Type L-containing forms of pyruvate kinase and aldolase type B both appear to be found in cell types thought to be capable of catalyzing of gluconeogenesis, while type K pyruvate kinase and type A aldolase are found in predominantly glycolytic cell types of the kidney. Lactate dehydrogenase isozymic patterns appear to be less closely correlated with glycolytic versus gluconeogenic functions of the kidney but may be determined more directly by other metabolic functions.

Enolase isozymes in Coho salmon.

1. Analysis of the enolase isozymic distribution has been performed in tissues of the Coho salmon, using electrophoretic separation on cellulose acetate strips followed by localization of enzymatic activity. 2. A total of six electrophoretically distinct forms are seen in Coho salmon in patterns that differ both qualitatively and quantitatively from one tissue to another. 3. The isozymes in skeletal muscle and liver are sufficiently similar to one another that a purification procedure previously developed for trout muscle enolase by Cory & Wold (1966) can be used to partially purify enolase from either of the above-mentioned Coho tissues. The main form of enolase in Coho muscle has an isoelectric point of 7.57. 4. Both liver and skeletal muscle enolases can be reversibly denatured in guanidine HCl and subsequently renatured. Liver enolase appeared to renature somewhat faster than muscle enolase under the same conditions. 5. While polyploidy among salmonids may contribute to the complexity of enolase patterns in fish, the differences in isozymic patterns seen from one tissue to another indicate the presence of distinct, nonallelic genes, probably arising through gene duplication.

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.

Pyruvate kinase isozymes in adult and fetal tissues of chicken.

Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.