DNA linkage based diagnosis of Wilson disease in asymptomatic siblings.

BACKGROUND & OBJECTIVES: Wilson disease (WD) is an autosomal recessive disorder caused by defects in ATP7B gene located in chromosome 13q14, and manifested as hepatolenticular degeneration as a result of accumulation of copper. No information on the mutation in the ATP7B gene and haplotypes using linked markers is available for WD patients in India. Hence, the present study was undertaken to identify, by a PCR-based molecular diagnostic test, presymptomatic siblings of WD affected individuals in families with multiple offspring. METHODS: Genomic DNA was prepared from the peripheral blood of the patients, siblings and his/her first degree relatives. The repeat-markers flanking WD locus were amplified by PCR using fluorescent labeled primers. Amplified DNA fragments were analyzed by polyacrylamide gel electrophoresis in ABI 377 DNA sequencing system. Genotypes of the samples were determined using Genescan software. Haplotypes were determined based on segregation of the alleles in the families under study. RESULTS: Among 15 WD affected families with multiple children, 4 cases were identified where younger siblings shared same genotype as the patient at all three markers analyzed. Further, eight different haplotypes were detected in the four patients. INTERPRETATION & CONCLUSION: The siblings of the WD patients carrying the same genotype at the markers linked to WD locus were presymptomatically diagnosed individuals. Presence of eight different haplotypes in the four patients suggested mutational heterogeneity at the WD locus. The test helps clinicians for therapeutic intervention in suspect WD cases by copper chelating agents prior to manifestation of overt clinical symptoms.

Role of fatty acid binding protein in the modulation of inhibitory effect of fatty acids on fatty acid synthase and ATP-citrate lyase in developing human brain.

Inhibition of the activities of fatty acid synthase and ATP-citrate lyase (ATP-CL) by fatty acids and their CoA esters has been studied. Purified fatty acid binding protein from human fetal brain reverses this inhibition. This protein also activates the enzyme when added alone. ATP-citrate lyase and fatty acid synthase activity gradually increased with the advancement of gestation showing a relationship between high demand of fatty acid synthesis in developing brain and supply of its precursors.

Modulation of bilayer fluidity by lipid peroxidation of human placental syncytiotrophoblast membranes during embryogenesis.

Placental syncytiotrophoblast while regulating the passage of nutrients from maternal blood to the fetal circulation exposes itself to the risk of oxidative attack by the oxygen free radicals. Extent of lipid peroxidation (LPO) investigated in placental brush border membrane (BBM) and basal membrane (BM) revealed a decreasing trend with gestational progress. Steady-state fluorescence anisotropy measurement of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled malondialdehyde treated placental membrane vesicles suggested modulation of BBM and BM fluidity by lipid peroxidation in all gestational ages. alpha-tocopherol content in both the placental membranes which increased as gestation progressed has been proposed to play a significant role in decreasing LPO of placental membranes during intrauterine development.

Inhibition of two HMP shunt pathway enzymes by fatty acids and their CoA esters in developing human brain: role of fatty acid binding protein.

Inhibitory effects of fatty acids and their CoA esters on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities of human fetal brain cytosol have been studied. Purified human fetal brain fatty acid binding protein reverses the inhibitory effects of palmitoyl-CoA and oleic acid on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in human fetal brain cytosol. This protein, when added alone, activates the enzymes. Levels of fatty acid binding proteins as well as the activities of these two HMP shunt pathway enzymes, which provide cofactors like NADPH for reductive biosynthesis, increase with gestation. These results indicate that a relationship exists between the high demand for fatty acids and synthesis of cofactors for lipid biosynthesis in developing brain.

Effect of fatty acid binding proteins on developing human placental malate dehydrogenase activity.

Role of fatty acid binding proteins (FABPs) in modulating inhibition of human placental malate dehydrogenase by palmitoyl-CoA and oleate has been studied. Activity of human placental cytosolic malate dehydrogenase is detected throughout the gestation, showing a peak at midgestation (20-25 weeks). Inhibition (50%) of the enzyme activity is obtained by 20 microM palmitoyl-CoA or 35 microM oleate. FABPs enhance the activity of malate dehydrogenase in absence of palmitoyl-CoA or oleate and also protect against palmitoyl-CoA or oleate inhibition. Such a modulatory effect of FABP may be due to the binding of long chain fatty acyl-CoA or fatty acid rather than a direct effect of FABPs on the enzyme.

Role of rusticyanin in the electron transport process in Thiobacillus ferrooxidans.

Effect of diethyl dithiocarbamate (DEDC), an antimicrobial agent, on growth of Thiobacillus ferrooxidans, possibly by inhibiting rusticyanin present in the periplasmic space of the microorganism, has been studied to gain more insight into the electron transport chain in the bioleaching process. DEDC is found to form a stable complex with rusticyanin in solution and also in polyacrylamide gel. The spectrum of the complex is identical to that of Cu-DEDC complex, suggesting binding of DEDC with copper moiety of rusticyanin and resulting in inhibition of growth. In vitro reduction of purified rusticyanin by Fe(II) in absence of acid-stable cytochrome c is very slow, indicating the importance of cytochrome c in electron transport. Thus, in the iron oxidation process, acid-stable cytochrome c is the primary acceptor of electron, transferring the electron to rusticyanin at pH 2.0, which, in turn, affects electron transfer to iron-cytochrome c reductase around pH 5.5.

Relationship between fatty acid synthesis, transport and total lipid content during human fetal lung development.

Levels of fatty acid binding proteins (FABPs), lipids as well as activities of fatty acid synthesizing enzymes such as fatty acid synthase and ATP-citrate lyase increase with gestation showing maximum at term in human fetal lung. However, the activity of ATP-citrate lyase showed the same trend up to 30 weeks of gestation before declining slightly at term. These results indicate the importance of supply and/or synthesis of fatty acids when lung surfactant synthesis begins; thereby showing a correlation between the FABPs, lipid pattern and the activities of fatty acid synthesizing enzymes during prenatal lung development.

A comparative study of 5'nucleotidase and alkaline phosphatase in human placenta during development.

Activities and a few properties of alkaline phosphatase and 5′-nucleotidase were compared in the developing human placenta. Both the enzymes were mostly membrane-bound and displayed similar developmental patterns with the highest activities at 24/26 weeks of the placenta. L-Phenylalanine, L-tryptophan and L-leucine were inhibitors of alkaline phosphatase, whereas they had no effect on the 5′-nucleotidase. Alkaline phosphatase from a late stage of gestation appeared to be almost heat-stable. An appreciable part of 5′-nucleotidase was also resistant to heat inactivation and this fraction varied with gestational age of the tissue. For both the enzymes, Vmax changed without altering Km values with periods of gestation. Ca2+ , Mg2+ and Mn2+ ions stimulated the alkaline phosphatase activity and Hg2+ , Zn2+ , Cu2+ ,Ni2+ were inhibitory. 5′-Nucleotidase was not activated by any of these cations. EDTA and Concanavalin A inhibited both the enzymes, although the extent of inhibition was different and also varied with gestation.