Developmental changes of the glycolytic enzymes in the human fetal heart

The ontogeny of hexokinase, phosphofructokinase, phosphoglucoisornerase, aldolase, pyruvate kinase and lactate dehydrogenase activities which are associated with glycolysis, an important energy yielding process, has been studied in human fetal heart for periods ranging from 13 weeks to above 33 weeks of gestation. Hexokinase, phosphoglucoisomerase and pyruvate kinase activities show similar developmental profiles exhibiting maximum activity at 25–28 weeks ofgestation. Phosphofructokinase activity, on the other hand, shows a minimum at this period and exhibits a peak value at early stages (13–16 weeks of gestation). Though considerable activity for aldolase is observed at an early period, it declines thereafter, but again increases in the later period. The probable role and correlations of these glycolytic enzymes with energy demand and general functional development in human fetal heart in ontogeny are evaluated.     

Developmental changes in malonate-related enzymes of rat brain.

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.     

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.     

Developmental changes in the activities of enzymes of free multienzyme complexes involved in the Benson-Calvin cycle

The electrophoretically homogenous preparations of free multienzyme complexes involved in the Benson-Calvin cycle with mol wt of 520 ± 20 and 240 ± 10 kD were isolated from 15–25-day-old cotton (Gossypium hirsutum L.) leaves of the same story (the third and the fourth upper leaves). Enzyme preparations were obtained at three stages of plant development: at the stage of 6–8 true leaves, during flower-bud formation, and during flowering. Comparison studies of developmental changes in activities of ribose phosphate isomerase (RPI), phosphoribulokinase (PRK), and Rubisco were carried out. RPI and PRK activities of multienzyme complexes differed insignificantly (by 2.3% on the average). Significant changes were observed in Rubisco activity (by 30% on the average). The optimum enzymatic activities of these complexes as well as the highest photosynthetic rate were revealed at the stage of reproductive organ formation. This correlation indicates the major role of multienzyme complexes involved in the Benson-Calvin cycle in the developmental control of photosynthesis and epigenesis.     

Modulation of TCA cycle enzymes and electron transport chain systems in experimental lung cancer

The modulatory effect of Withania somnifera along with paclitaxel on tricarboxylic acid (TCA) cycle key enzymes and electron transport chain complexes were investigated against lung cancer induced by benzo(a)pyrene in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in lung cancer bearing animals were observed. Upon W. somnifera along with paclitaxel administration the above biochemical changes were inclined towards normal control animal values. Activities of mitochondrial enzymes and electron transport complexes were analyzed in the experimental groups to determine the efficiency of energy production. This study further confirms the chemotherapeutic effect of W. somnifera along with paclitaxel which is found to be more effective in the treatment of lung cancer. Thus these results are consistent with our hypothesis that the combination chemotherapy of W. somnifera along with paclitaxel as a promising chemotherapeutic agent.     

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.     

Pathway classification of TCA cycle

The structural analysis of large metabolic networks exhibits a combinatorial explosion of elementary modes. A new method of classification has been developed [called aggregation around common motif (ACoM)], which groups elementary modes into classes with similar substructures. This method is applied to the tricarboxylic acid cycle and metabolite carriers. The analysis of this network evidences a great number of elementary flux modes (204) despite the low number of reactions (23). The ACoM is used to class these elementary modes in a low number of sets (8) with biological meanings.     

Developmental changes of amino acids in ovine fetal fluids

We recently reported an unusual abundance of arginine (4-6 mM) in porcine allantoic fluid during early gestation. However, it is not known whether such high concentrations of arginine are unique for porcine allantoic fluid or whether they represent an important physiological phenomenon for mammals. The present study was conducted to test the hypothesis that arginine is also the most abundant amino acid in ovine allantoic fluid. Allantoic and amniotic fluids, as well as fetal and maternal plasma samples, were obtained from ewes between Days 30 and 140 of gestation. Glycine was the most abundant amino acid in maternal uterine arterial plasma, representing approximately 25% of total alpha-amino acids. Alanine, glutamine, glycine, plus serine contributed approximately 50% of total alpha-amino acids in fetal plasma. Fetal:maternal plasma ratios for amino acids varied greatly, being less than 1 for glutamate during late gestation, 1.5-3 for most amino acids throughout gestation, and greater than 10 for serine during late gestation. Marked changes were observed in amino acid concentrations in amniotic and allantoic fluids associated with conceptus development. Concentrations of alanine, citrulline, and glutamine in allantoic fluid increased by 20-, 34-, and 18-fold, respectively, between Days 30 and 60 of gestation and were 24.7, 9.7, and 23.5 mM, respectively, on Day 60 of gestation (compared with 0.8 mM arginine). Remarkably, alanine, citrulline, plus glutamine accounted for approximately 80% of total alpha-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed approximately 60% of total alpha-amino acids in allantoic fluid on Day 140 of gestation. These novel findings of the unusual abundance of traditionally classified nonessential amino acids in allantoic fluid raise important questions regarding their roles in ovine conceptus development.     

Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK _m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

Developmental changes in interstitial collagens of fetal rat genital ducts

The distribution of interstitial collagen types I and III was studied by immunocytochemistry in the mesenchyme of progressing and regressing mesonephric and paramesonephric ducts of male and female rat fetuses from the age of 15 days until birth. Immunocytochemistry revealed a collagen-poor mesenchymal area around the genital ducts and in continuation with the coelomic epithelium on the lateral edge of the mesonephric ridge of 15-day-old fetuses. Ultrastructurally, collagen fibrils were accumulated along the continuous lamina densa of the mesonephric ducts, whereas they were absent on the medial side of the male and female paramesonephric ducts. In males, the amount of collagen fibrils increased with the histological maturation of the mesenchyme around the mesonephric duct, whereas around the regressing paramesonephric duct collagens disappeared from the basement membrane region and the surrounding mesenchyme of the 16-day-old male duct. After the completion of the paramesonephric regression, the mesenchyme acquired a uniformly collagen containing interstitial matrix. In females, the collagens increased in the mesenchyme around the progressing paramesonephric duct, and the original site of the regressing mesonephric duct became occupied with a collagen-containing mesenchyme by the age of 19 days. The results suggest a close structural linkage between the mesonephric duct and the established early paramesonephric duct. The differences in the developmental maturation of the periductal mesenchyme and the observed changes in the composition of the interstitial matrix probably reflect the functional differences in the regulatory factors acting on the progression and regression of the male and female genital ducts.     

Molecular changes in fetal Down syndrome brain

Trisomy of human chromosome 21 is a major cause of mental retardation and other phenotypic abnormalities collectively known as Down syndrome. Down syndrome is associated with developmental failure followed by processes of neurodegeneration that are known to supervene later in life. Despite a widespread interest in Down syndrome, the cause of developmental failure is unclear. The brain of a child with Down syndrome develops differently from that of a normal one, although characteristic morphological differences have not been noted in prenatal life. On the other hand, a review of the existing literature indicates that there are a series of biochemical alterations occurring in fetal Down syndrome brain that could serve as substrate for morphological changes. We propose that these biochemical alterations represent and/or precede morphological changes. This review attempts to dissect these molecular changes and to explain how they may lead to mental retardation.     

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.