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V R Bommineni

Publications and source records attributed to V R Bommineni.

6 recordsLinked to original sources
PubMed1996

Microtubular disruption prolongs the expression of human bilirubin-uridinediphosphoglucuronate-glucuronosyltransferase-1 gene transferred into Gunn rat livers.

DNA delivered to the liver by asialoglycoprotein receptor-mediated endocytosis is degraded in lysosomes within 48 h. To test the hypothesis that microtubular disruption should promote transgene persistence by interrupting endosomal translocation to lysosomes, plasmids containing bacterial chloramphenicol acetyltransferase (pSV2-CAT) or human bilirubin-UDP-glucuronosyltransferase-1 (pSVK3-hBUGT1) genes were complexed with asialoglycoprotein-polylysine conjugates, and 1 mg of the complexed DNA was injected intravenously into bilirubin-UDP-glucuronosyltransferase-deficient Gunn rats. 30 min before DNA injection, one group received 0.75 mg of colchicine/kg of body weight intraperitoneally, which was shown by immunofluorescent confocal microscopy to disrupt the microtubular network. Control rats received normal saline. In colchicine-pretreated rats receiving pSV2-CAT, hepatic chloramphenicol acetyltransferase activity persisted for 9-14 weeks, whereas in the saline-pretreated group the activity was detectable for 48 h only. In colchicine-pretreated Gunn rats receiving pSVK3-hBUGT1, the DNA persisted in liver for 10 weeks, bilirubin glucuronides were excreted in bile, and serum bilirubin levels declined by 25-35% in 2-4 weeks and remained reduced for 8 weeks. Without colchicine pretreatment, the DNA was detectable in liver for 2 days only, and serum bilirubin levels were not reduced. Thus, microtubular disruption provides a noninvasive method for prolonging the effect of liver-targeted gene therapy.

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PubMed1995

Fate of DNA targeted to hepatocytes by asialoglycoprotein polylysine conjugates.

The data indicate that partial hepatectomy results in a decrease in degradation of targeted DNA. This appears to be due to an inhibition of the endocytotic pathway shortly after hepatectomy and is associated with the accumulation of targeted DNA within a population of light endosomal vesicles. It is likely that these vesicles serve as a reservoir from which targeted DNA gradually escapes and can be found in the nucleus. The DNA targeted to liver is capable of expressing the marker gene, and the DNA in the vesicles is transfection competent, suggesting that a substantial portion is intact. Overall, the receptor-mediated delivery system is highly efficient in transporting foreign DNA to liver cells. Partial hepatectomy and the ensuing cellular events provide a means of inhibiting the degradative portion of the endocytotic pathway. Pharmacological agents that can mimic the cellular processes that occur after partial hepatectomy may be useful in increasing the duration of foreign gene expression without the trauma of surgery.

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PubMed1994

Depolymerization of hepatocellular microtubules after partial hepatectomy.

Asialoglycoproteins (ASG) are internalized by hepatocytes by ASG receptor (ASGR)-mediated endocytosis. We have shown previously that when a plasmid DNA, pAlb(9-12)CAT (expressing chloramphenicol acetyltransferase driven by an albumin promoter enhancer), was complexed with an ASG-polylysine conjugate and injected intravenously in rats, 80% of the DNA was internalized by the liver. In normal recipient rats, over 95% of the internalized DNA was degraded in 4 h; the plasmid was undetectable after 48 h. In contrast, when 66% hepatectomy was performed 20 min after DNA administration, the internalized DNA persisted for several weeks in cytoplasmic vesicles (Chowdhury, N. R., Wu, C. H., Wu, B. Y., Yerneni, P. C., Bommineni, V. R., and Chowdhury, J. R. (1993) J. Biol. Chem. 268, 11265-11271). Since microtubules are required for the translocation of ligand-containing endosomes to lysosomes, the site of ligand degradation, we hypothesized that persistence of the endocytosed DNA might be related to changes in microtubular structure and function. To test this hypothesis, we examined hepatocellular microtubules by immunofluorescence confocal microscopy. Liver from untreated rats or sham-operated controls showed a network of fibrillar microtubules throughout the cytoplasm. The extent of the microtubular network was substantially reduced 3-6 h after 66% hepatectomy. By 24 h, microtubules had regenerated. Intraportal infusion of cycloheximide (250 mg/kg body weight) 15 min before 66% hepatectomy, prevented microtubular disruption, indicating that protein synthesis is required for this process. Immunotransblot analysis showed that hepatic alpha-tubulin concentration remained unchanged through microtubular disassembly and subsequent reassembly, which is consistent with conservation and reutilization of tubulin released by depolymerization of microtubules.

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PubMed1993

Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo. Prolonged persistence in cytoplasmic vesicles after partial hepatectomy.

After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.

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PubMed1992

Prenatal diagnosis of bilirubin-UDP-glucuronosyltransferase deficiency in rats by genomic DNA analysis.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

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