Ontogeny of lactate dehydrogenase isozymes in chicken-quail hybrid embryos

The ontogeny of lactate dehydrogenase (LDH) isozymes was examined in avian hybrids and compared with the isozyme patterns of the parental species. Hybrids were obtained by crossing female Japanese quali (Coturnix coturnix japonica) with male domestic chickens (Gallus gallus domesticus). By use of starch gel electrophoresis and an enzyme-specific stain, traces of embryonic paternally derived LDH were detected in unincubated hybrid eggs. It was concluded that the embryonic genes coding for the B subunits of LDH are activated during the hours between fertilization and oviposition. In early blastoderms, a great excess of maternally stored LDH is present. In the hybrid, the predominantly maternal pattern of isozymes shifts during embryogenesis to a predominantly paternal pattern. This was considered evidence for differential allelic regulation of LDH inactivation. A progressive trend toward the establishment of the adult distribution of isozymes in various tissues was also observed in the hybrid and quail, and found to be similar to chicken LDH isozyme ontogeny.     

ER、bcl-2和p53在鸡与鹌鹑属间杂交种早期胚胎中的mRNA表达

通过人工授精获得鸡(♂10只)与鹌鹑(♀100只)属间杂交种蛋并同机入孵, 采用Wpkci引物和多重PCR鉴定66~120 h的鸡(♂)与鹌鹑(♀)属间杂交种活胚的性别后, 选取不同时间点雌、雄胚胎共300枚, 以b-actin为内标, 通过RT-PCR分别测定雌激素受体(ER)和细胞凋亡因子(bcl-2、p53)的mRNA相对丰度; 探讨ER、bcl-2和p53对杂交种早期胚胎发育及性别分化的影响。结果表明: (1) 杂交种胚胎ER mRNA 表达在66～84 h期间雌性极显著高于雄性(P＜0.01), 由此推测杂交种的性分化时间大致在胚胎发育的66~84 h范围内; (2) bcl-2和p53 的mRNA表达在杂交胚胎发育过程中具有明显的时序性, 说明bcl-2和p53基因对早期杂交胚的发育具有重要的影响。     

Expression of the paternal genes for lactate dehydrogenase, glutamate dehydrogenase and acetylcholinesterase in the development of hybrid fish between species from the families of Cobitidae and Cyprinidae]

The time of expression of the paternal genes of glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH) and acetylcholine esterase (AChE) was investigated in the development of fish hybrids. The species which differed by the thermostability of homologous enzymes were selected as parental pairs. The appearance of differences in the thermostability of homologous enzymes between the hybrids and the maternal species suggested the beginning of paternal enzyme synthesis in the hybrid embryos. Differences in the AChE thermostability appeared simultaneously with the enzyme activity at the stage of first muscle contractions (35 hrs of development), differences in the mitochondrial GDH thermostability appeared at the stage of hatching (50-60 hrs) and those in the LDH thermostability 12-17 days after hatching. The total activity of AChE and GDH sharply increased during the period of the paternal enzyme appearance whereas the activity of LDH suffered practically no changes. Differences in the AChE thermostability between the hybrids and the maternal species are the same for both the total AChE (in supernatant, 15,000 gX10 min.) and the solubilised AChE (in supernatant, 130,000 gX60 min.). AChE of the parental species and the hybrids have the same electrophoretic mobility. The differences in the thermostability of enzymes are preserved following the electrophoresis in polyacrilamide gel.     

Developmental genetics of teleosts: a biochemical analysis of lake chubsucker ontogeny

Developing embryos of the lake chubsucker, Erimyzon sucetta, were analyzed with regard to both gross morphological changes and specific enzymatic changes from the unfertilized egg stage until some 3 weeks posthatching. Total activities of three enzymes—lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase—were determined throughout the course of development. Each of these different enzymes exhibited a different pattern of change during ontogeny. Electrophoretic analysis of qualitative changes in isozyme patterns was accomplished for these three enzymes and for α-amylase, glucosephosphate isomerase, mannosephosphate isomerase, creatine kinase, esterase, glutamate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, malate dehydrogenase, hexose diphosphatase, phosphoglucomutase, and phosphogluconate dehydrogenase. Many of the enzyme systems investigated exhibited rich patterns of ontogenetic change, while a few remained relatively unchanged throughout the interval studied. Several of the enzymes in particular metabolic pathways exhibited coincident changes suggestive of coordinate control. The appearance of several rather “tissue-specific” isozymes was closely correlated with the morphological and functional differentiation of these particular tissues or organs.     

Chromosome localization of three syntenic gene pairs in the American mink (Mustela vison)

Twenty eight American mink X Chinese hamster somatic cell hybrids were analysed for the expression of mink enzymes and for mink chromosomes. The results of this analysis made it possible to assign the genes for phosphoglucomutase-1 and phosphogluconate dehydrogenase to chromosome 2, those for lactate dehydrogenase A and glucose phosphate isomerase to chromosome 7, and those for lactate dehydrogenase B and triosephosphate isomerase to chromosome 9.     

Glucose Utilization by Chick Embryo Heart Homogenates

Homogenates of early chick embryos and homogenates of early chick embryonic hearts utilized the phosphogluconate pathway of glucose catabolism to a greater extent, relative to the glycolytic-Krebs cycle pathway, than did homogenates of hearts from older chick embryos or adult chicks. An abrupt drop in the relative participation by the phosphogluconate pathway in embryo heart homogenates occurs at about 5 to 7 days of incubation. Heart homogenates from adult chicks catabolize glucose almost entirely by the glycolytic-Krebs cycle pathway, with negligible participation by the phosphogluconate pathway.     

Assignment of the rat genes coding for medium-chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and the beta subunit of propionyl-CoA carboxylase to chromosomes 2, 3, and 8, respectively

From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat genes coding for the enzymes medium-chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and the beta-subunit of propionyl-CoA carboxylase have been assigned to chromosomes 2, 3, and 8, respectively.     

Demonstrability of some oxidative enzymes in early rodent embryos with and without fixation

Several oxidative enzymes [NADH-TR (reduced nicotinamide-adenine dinucleotide-tetrazolium reductase), NADPH-TR (reduced nicotinamide-adenine dinucleotide phosphate-tetrazolium reductase), SDH (succinic dehydrogenase) and LDH (lactate dehydrogenase)] were studied by histochemical means during early development of rat and mouse. All investigated enzymes could be easily demonstrated in zygote and also to some extent in somitic stages without any pretreatment. However, in cleavage and early postimplantation stages enzyme activity could be revealed only after the embryos were pretreated in some way. This pretreatment can be fixation with formalin or acetone, freezing and thawing, slight mechanical damage or very prolonged incubation time. The formazan granules as a sign of enzymatic activity were present in all stages of embryonic development and were more abundant in reactions for NADH-TR and LDH than in reactions for NADPH-TR and SDH. Our results suggest that the investigated enzymes are present in all embryonic cells during early development. It seems that the permeability of embryonic cells for histochemical media must be increased otherwise the histochemical reactions cannot be accomplished.     

Initial expression of the genes for fructose 1,6-diphosphatase,malic enzyme,and aspartate aminotransferase in Japanese quail and chicken-quail hybrid embryos

The initial appearance of a number of enzymes involved in gluconeogenesis was investigated in the early embryogenesis of the Japanese quail (Coturnix coturnix japonica), the domestic chicken (Gallus gallus domesticus), and chicken-quail hybrids. Starch gel electrophoresis and enzyme-specific stains revealed genetic differences between muscle and liver fructose 1,6-diphosphatase (FDPase) as well as malic enzyme (ME) and mitochondrial aspartate aminotransferase (AAT) isozymes of the two species. ME and AAT were present in unfertilized unincubated eggs, indicating maternal storage of these enzymes. The initial expression of the paternally inherited genes in the hybrid occurred before oviposition in the case of ME, and between 12 and 18 hr incubation in the case of AAT. Initial expression of both parental sets of genes for FDPase occurred synchronously between 16 and 24 hr in the hybrid, corresponding to the time of initial appearance of this enzyme in the quail and chicken. Glucose 6-phosphate administration at 0 hr was found to cause no prevention or delay of initial enzyme activation. These results are interpreted in terms of early patterns of enzyme activation regulation and nutrition in the avian embryo.     

CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

The preparation of alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase from baker''s yeast

A procedure has been developed for the preparation of alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase from the same sample of baker's yeast. The two enzymes were obtained in good yield in a pure crystalline form. The method minimizes the work involved in preparing the two enzymes and would be of particular advantage for preparing the enzymes in radioactive form from yeast grown in a radioactive medium.     

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.

Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.     

Lactate dehydrogenase ontogeny,paternal gene activation,and tetramer assembly in embryos of brook trout,lake trout,and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.This study was supported by National Science Foundation Grant GB-7271.     

Testing homology of loci for two plumage colors, "lavender" and "recessive white," with chicken and Japanese quail hybrids

Homology for two plumage color loci was studied by hybridization between chickens and Japanese quail. First, chicken-quail hybrids were produced from homozygous "lavender" chicken cocks and "bleu" Japanese quail, and all 30 hybrids had the same parental slate blue plumage color. On the other hand, no hybrids with this plumage were obtained out of 18 progeny from the same cocks and wild-type quail. These results show that the slate blue plumage color is determined by homologous loci in Japanese quail and chickens. Second, all (n = 25) chicken-quail hybrids hatched from homozygous "recessive white" cocks and "recessive white" (n = 8) or "wild-type" (n = 17) quail had the same pattern of plumage color, with white feathers on the ventral face and colored feathers elsewhere. These results indicate that the recessive white mutations are not homologous in Japanese quail and chickens.     

Allelic expression at the sorbitol dehydrogenase and glucosephosphate isomerase loci in an interspecific hybrid ricefish

1. The expression of alleles encoding the enzymes sorbitol dehydrogenase (SDH; EC 1.1.1.14) and glucosephosphate isomerase (GPI; EC 5.3.1.9) was investigated in Oryzias melastigma, O. javanicus and in O. melastigma female x O. javanicus male hybrids by acrylamide gel electrophoresis. 2. It was not possible to investigate the expression of SDH or GPI in reciprocal hybrids as these fry failed to develop past the stage of embryonic body formation. 3. The delay in appearance of isozymes of paternal SDH subunit composition, and paternal and maternal GPI-B subunit composition is in keeping with observed effects of gene regulatory divergence where alleles of maternal origin interact more effectively with maternal cytoplasmic regulatory factors than do alleles of paternal origin.     

Asynchronous expression of the alcohol and supernatant malate dehydrogenase loci during Barbus hybrid development (Cypriniformes, Teleostei)

The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) and alcohol dehydrogenase (ADH) are reported for the tiger barb (Barbus tetrazona), the rosy barb (Barbus conchonius) and their reciprocal hybrids. The tissue distribution of s-MDH and ADH isozymes in both species is consistent with spatial profiles reported for other teleosts. The expression of alleles of paternal origin at the s-Mdh-B and Adh loci are delayed in reciprocal hybrids as compared to their expression intraspecifically; suggestive of a low degree of affinity between maternally derived regulatory factors and paternal regulative elements controlling structural gene activation.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.     

Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase,glucose 6-phosphate dehydrogenase,malate dehydrogenase,and lactate dehydrogenase

The activities of four enzymes were determined during the first four days of mouse embryogenesis. Two enzymes, fructose 1,6-diphosphate aldolase and malate dehydrogenase, increase about 30% in activity, and this increase is attributed to slow but continued enzyme synthesis. The other two enzymes, glucose 6-phosphate dehydrogenase (X-linked) and lactate dehydrogenase, remain constant for the first two days and then decline exponentially with half-times of 19 and 17 hr, respectively. These declines in activity cannot be explained by the appearance of soluble inactivators or by the disappearance of soluble activators. Likewise, although temporally related to the passage of the embryos from the oviducts into the uterine horns, the changes in enzyme activity do not result from this change in embryonic environment, and specific degradative processes beginning on day 2 of embryonic development are postulated.This study was supported by USPHS Grant HD 03132 and by a grant from the School of Medicine, University of California, San Francisco Medical Center. The senior author is the recipient of USPHS Research Career Development Award HD 35,565.