Magnetic affinity cell sorting to isolate transiently transfected cells, multidrug-resistant cells, somatic cell hybrids, and virally infected cells.
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Animals↗
Biomedical subjects
R Padmanabhan
Publications and source records attributed to R Padmanabhan.
K-ras and p53 point mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced hamster lung tumors.
Lung tumors were induced in Syrian golden hamsters by s.c. injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). After 40 weeks lung tumor tissue was isolated. Administration of the NNK and exposure of the animals to an atmosphere of 65% oxygen resulted in a statistically significant reduction in tumor size but did not alter the histological tumor type or tumor incidence when compared with carcinogen treated animals maintained under ambient air. Histologically, lung tumors had the morphologic features of adenomas and adenocarcinomas with approximately 15% being squamous cell carcinomas. Lung tumors were examined for mutations in the Ki-ras oncogene and the p53 tumor suppressor gene by direct sequencing. The Ki-ras mutation frequency in RNA isolated from pooled tumors and in DNA isolated from individual tumors were found to be identical. Activated Ki-ras alleles were detected in 77-94% of tumors. All mutations observed (from a total of 65) except one were GC-AT. The Ki-ras mutations resulted in amino acid substitutions at either codons 12 or 13. No mutations were detected at the 61st codon. Examination of the same tumors for p53 mutations showed only one point mutation. We conclude that the NNK treatment in Syrian golden hamsters results in a distinctive mutation pattern in the Ki-ras gene whereas p53 gene mutations may not play a major role at this stage in hamster lung tumorigenesis.
Animals↗
Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.
Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.
Amino Acid Sequence↗
ATP and GTP as alternative energy sources for vinblastine transport by P-170 in KB-V1 plasma membrane vesicles.
Purified plasma membrane vesicles isolated from multidrug-resistant human KB-V1 cells accumulate [3H]vinblastine in an energy-dependent manner. The accumulation of [3H]vinblastine in the presence of ATP is a saturable process. ATP can be replaced by other purine nucleotide triphosphates, of which GTP is the most efficient.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5.
Processing of dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5 could be mediated by the catalytically active NS3 protease domain and NS2B in trans at the dibasic sites NS3-NS4A and NS4B-NS5. Subcellular localization of the unprocessed precursor NS3-NS4A-NS4B-NS5 showed that it was confined to a distinct subcellular organelle in the cytoplasm, which was distinct from the distribution of the mature NS5.
Animals↗
Detection of stable secondary structure at the 3' terminus of dengue virus type 2 RNA.
The 3'-terminal sequences of flavivirus genomes within approx. 100 nucleotides (nt) have been suggested to have a highly conserved secondary structure, as based on the known nt sequence data and free-energy calculations using computer programs. To test the existence of a secondary structure in solution, we devised a strategy to generate truncated RNA molecules from about 0.3-1.4 kb in length, having the same polarity and nt sequence as dengue virus type 2 (DEN-2) RNA (New Guinea-C strain). When these labeled RNA molecules were digested by RNase A, and analyzed by denaturing polyacrylamide-gel electrophoresis, three resistant fragments of 16, 20 and 23 nt in length were reproducibly obtained. To examine whether these RNase A-resistant (RNaseR) fragments emerged from a stable secondary structure formed in solution consisting of 3'-terminal sequences, hybridization of the RNaseR fragments to four chemically synthesized oligodeoxyribonucleotides (oligos), complementary to nt 1-24, 25-48, 49-72, and 73-96 from the 3' terminus of DEN-2 RNA, followed by RNase H digestion were carried out. Oligos complementary to nt 25-48 and 49-72 from the 3' end of DEN-2 RNA were sufficient to render all three RNaseR fragments susceptible to RNase H digestion. These data indicate that a stable secondary structure is formed in solution involving nt 18-67 from the 3' terminus. The potential use of these unique transcripts to identify the viral and/or host proteins which might interact at the 3' terminus of DEN-2 RNA during initiation of replication is discussed.
Base Composition↗
Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells.
Adenovirus (Ad) DNA polymerase (AdPol) and the preterminal protein (pTP) form a complex that is involved in the in vitro initiation of Ad DNA replication. Recombinant vaccinia viruses (vv) were constructed in which the genes encoding AdPol and pTP were cloned into a vaccinia/T7 hybrid expression-based vector downstream from the T7 promoter (pT7)/encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR). HeLa cells infected with the recombinant vv-AdPol or vv-pTP or a mixture of both, together with the vv expressing T7 RNA polymerase produced significant levels of pTP and AdPol which were biologically active in the in vitro initiation of Ad DNA replication. These amounts of pTP and AdPol were only about two-fold less than the levels produced in insect cells infected with the recombinant baculovirus constructs expressing AdPol and pTP.
Adenoviruses, Human↗
Magnetic-affinity cell sorting of human multidrug-resistant cells.
We have developed a method for isolating multidrug-resistant cells from a heterogeneous cell population using the magnetic-affinity cell-sorting system. Human KB carcinoma cell lines expressing different amounts of P-glycoprotein have been selected by use of the monoclonal antibody MRK-16 coupled to magnetic particles. This specific, rapid, and sensitive method allows the selection of viable and clonable drug-resistant cells from various drug-resistant human and murine cell populations. This method may prove useful in isolating drug-resistant cells from tumors with heterogeneous P-glycoprotein expression for further analysis.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.
The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Synthesis of biologically active adenovirus preterminal protein in insect cells using a baculovirus vector.
A DNA fragment encoding the polyhedrin promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV strain) was constructed using overlapping oligodeoxyribonucleotides (oligos), which included the 5'-untranslated leader sequence of the polyhedrin-encoding gene. This DNA fragment was cloned into an intermediate transfer vector (pKX105) providing a unique BamHI site for the insertion of foreign genes. The Escherichia coli lacZ gene was first cloned at the BamHI site of pKX105 and the XhoI-KpnI fragment containing the lacZ gene was transferred to another plasmid vector (pEI) consisting of flanking AcMNPV sequences (pEI-lacZ). The E. coli beta-galactosidase that was produced in the infected insect cells using the recombinant virus constituted about 10% of the total cytoplasmic proteins. The pKX105 plasmid was also modified to give rise to pTT-lacZ which consisted of the lacZ gene under the control of the Rous sarcoma virus long terminal repeat promoter to facilitate rapid screening of the baculoviral recombinants in which the gene of interest was cloned under the control of the polyhedrin promoter. The efficiency of these transfer vectors was verified by obtaining high levels of expression of the adenovirus(Ad)-encoded preterminal protein (pTP) which is involved as a protein primer in the initiation of Ad DNA replication. The baculovirus-produced pTP was immunoprecipitable using rabbit polyclonal antibodies raised against a hydrophilic domain of pTP. The pTP protein was localized in the nucleus of the infected insect cells, and was biologically active in the in vitro Ad type 2 (Ad2) replication initiation assay.
Adenoviruses, Human↗
Is exencephaly the forerunner of anencephaly? An experimental study on the effect of prolonged gestation on the exencephaly induced after neural tube closure in the rat.
Exencephaly is said to precede anencephaly resulting from failure of the rostral neuropore closure. In order to verify if the exencephaly induced after neural tube closure would also lead to anencephaly, exencephaly was induced in rat fetuses by maternal administration of a single dose (15 mg/kg) of cyclophosphamide on day 12 of gestation and pregnancy was prolonged by uterine ligation until postconception (PC) day 24. Fetal death was found to increase with prolongation of gestation and no sign of recovery from growth retardation was observed. Alizarin red-S-stained skeletal preparations substantiated the persistence of skull malformations in the exencephalic fetuses. Histological observations of the mesenchyme and the brain indicated degenerative changes that were intensifying with time. The ventricular system expanded progressively; the ependyma was denuded and neural mass lay free in the ventricle. The choroid plexus appeared to be elaborate and extensive. The haemorrhagic capillary network around the brain tissue was highly proliferative and appeared to penetrate the former from the exterior. Tissue necrosis seemed to progress unabated. Cystic spaces appeared beneath the base of the brain and became progressively large and it appeared as if the brain was being pushed out of the shallow cranial fossae. By PC day 24, most of the brain tissue had degenerated, thus giving clearly the appearance of the anencephalic condition.
Anencephaly↗
Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals.
Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
Adenoviridae↗
Mutational analysis of single-stranded DNA templates active in the in vitro initiation assay for adenovirus DNA replication.
Three distinct domains, A, the minimal origin, as well as B and C, the binding sites for the host nuclear factors, are required for efficient initiation of adenovirus (Ad) DNA replication at the termini. The initiation reaction was examined using partially purified nuclear extracts and various single-stranded oligomers as DNA templates. We observed that single-stranded oligomers containing Ad2 minimal origin (Ori) sequences (bp 1-18) from the I-strand of the Ad2 genome supported preterminal protein-dCMP complex formation in vitro. Using oligomers containing point mutations in the Ad2 minimal Ori sequence, six positions were identified as important to the function of the Ad2 minimal Ori sequence. Point mutations at position 7, 8, or 11 virtually abolished the ability of the oligomer to support the initiation reaction. Point mutations at position 4, 9, or 17 were found to decrease the ability of the oligomers to support the initiation reaction to 33, 67, and 58% of control, respectively. An oligomer complementary to the I-strand of the Ad2 minimal Ori was found to block initiation on minimal Ori template. A number of randomly selected nonspecific oligomers did not, in general, serve as templates for initiation with the exception of two oligomers, one of which was found to be about threefold more active than the control minimal Ori template. The biological significance of the in vitro initiation of Ad2 DNA replication on single-stranded DNA templates is discussed.
Adenoviruses, Human↗
Characteristics of the limb malformations induced by maternal exposure to cadmium in the mouse.
Single doses of 2,3,4,6,8,10, and 15 mg/kg of cadmium chloride were administered (SC) to groups of MF1 mice on one of days 7 to 12 of gestation. Fetuses collected on day 18 were observed for limb malformations, and alizarin red-S stained skeletons were examined for their skeletal bases. Ectrodactyly, postaxial polydactyly, syndactyly, brachydactyly, adactyly, phocomelia, meromelia, and malrotation of the limbs were detected in a significant number of fetuses. Days 7 to 10 were greatly susceptible for induction of these malformations. Postaxial ectrodactyly was more frequent in the forepaws, and sidedness was not significant. Preaxial ectrodactyly preferentially affected the left hindpaws in the 9th-day treatment group. Postaxial polydactyly was predominently right sided and mostly involved the forepaws. Day 8 was particularly susceptible for induction of adactyly. Malrotation of the limbs together with edema and caudal narrowing resulted in a 'penguin-like' appearance. Ossification of the long bones, the carpals, tarsals, and phalanges were affected. Even those limbs that were not externally malformed had skeletal dysgenesis. Limb buds examined histologically at midgestation showed scanty and poorly organized mesenchyme, extensive elaboration of marginal sinsus, reduced thickness of the apical ectodermal ridge (AER), and discontinuous basement membrane. It is speculated that such histologic alterations at early stages of development could have contributed to the defective morphogenesis of limbs in this animal model.
Abnormalities, Drug-Induced↗
Electron-microscopic studies on the pathogenesis of exencephaly and cranioschisis induced in the rat after neural tube closure: role of the neuroepithelium and choroid plexus.
Exencephaly was induced in Wistar rat fetuses by the administration of a single dose of cyclophosphamide (15 mg/kg) in saline, after neural tube closure. The neuroepithelium (NE) and the choroid plexus were studied electron-microscopically in sections taken from a few hours after treatment to day 19 of gestation. The reduction in polyribosomes and condensation of the nucleus and cytoplasm were followed by cell death and fragmentation in the NE. Such cellular debris were phagocytosed and digested by the apparently normal neuroblasts. Cell proliferation was inhibited. The progressive loss of cells and lack of neuropil arborisation resulted in the expansion of the extracellular space and reduced intercellular contacts. The internal and external limiting membranes became weak. The vascular endothelium was attenuated. There were no obvious discontinuities of endothelium, but clusters of extravascular red blood cells, particularly in the vicinity of capillaries, in the cavitations in the NE and in the ventricular lumen were prominent by day 15. Subsequently, the cavities in the NE frankly communicated with the ventricle internally and subcutaneous blebs externally. The choroid plexus of exencephalic embryos was more extensive than that of the age-matched controls. Hydropic vacuoles, dense bodies, distended mitochondria, clusters of vesicles in basal cytoplasm and lakes of monoparticulate glycogen progressively increased in the plexus cells. Pericapillary oedema was obvious in the core of the plexus. These observations suggest that, in addition to cell death and reduced cell proliferation, haemorrhage, oedema and enhanced cerebrospinal fluid production contribute to reopening of the closed neural tube in this model.
Abnormalities, Severe Teratoid↗
Scanning-electron-microscopic studies on the pathogenesis of exencephaly and cranioschisis induced in the rat after neural tube closure.
Following a single-dose (15 mg/kg) administration of cyclophosphamide on day 12 of gestation to Wistar rats, fetuses were collected at various intervals of time and studied under the scanning electron microscope. Comparisons were made with age-matched controls. Disorganization of the neuro-epithelium (NE) was characterized by expansion of the intercellular space (ICS), appearance of abundant cellular debris, extravascular red blood cells and formation of haemorrhagic cysts in the subependymal zone. Proliferation of the choroid plexus and its surface modifications parallelled the changes in the NE. The ventricular system dilated progressively. Supra-ependymal elements appeared earlier and in greater abundance in the experimental than in the control embryos. The mesenchymal cells of the cyclophosphamide-treated embryos were scanty, poorly organized, had less numerous cytoplasmic processes than the corresponding controls and failed to develop into the primordium of the skull vault. Degenerative changes in the NE, ventricular dilatation and the absence of a supporting skull vault primordium appeared to underlie the reopening of the closed neural tube.
Animals↗
Effect of linoleic acid hydroperoxide on adenovirus DNA replication.
The effect of linoleic acid hydroperoxide on replication of adenovirus type 2 DNA in HeLa cells was examined. The level of the DNA was extremely reduced when the cells immediately after the infection with adenovirus type 2 were incubated for 24 h in the presence of 1 nmol/ml (measured by the thiobarbituric acid reaction and expressed in terms of malondialdehyde) of the hydroperoxide. The levels of the messenger RNA for adenovirus type 2 DNA polymerase and the expressed polymerase were equally reduced. The inhibition of DNA replication by lipid hydroperoxides in animal cells was discussed in relation to degenerative disorders.
Adenoviruses, Human↗
Ultrastructural studies on the placentae of streptozotocin induced maternal diabetes in the rat.
Following induction of diabetes by a single injection of (IP) streptozotocin (STZ) to pregnant Wistar rats on days 2, 4 and 6 to 12 of gestation, fetuses and placentae were collected on day 20. The controls were either untreated or vehicle treated; alternatively following STZ injection, 2-6 IU of insulin was administered (sc) daily until term. The placentae were fixed in a glutaraldehyde and paraformaldehyde mixture and ultrathin sections were examined under the electron microscope. The structure of the vehicle treated control resembled that of the untreated control. The insulin control group had pathological changes similar to those of the diabetic group but with considerably less frequency. The giant cells in the basal zone of STZ group were numerous; they had abundant dilated cisternae of rough endoplasmic reticulum, intracytoplasmic fibrinoid and nuclear inclusions. The trophospongial cells presented numerous clear vacuoles, lysosomes and myelin bodies. Enlarged vacuoles often impinged deeply on the nucleus. The glycogen cells disintegrated resulting in cyst formation. In the labyrinthine zone, layer I trophoblast revealed increased number of large pores through which layer II trophoblast projected into the maternal sinusoid. Layer II had abundant glycogen, lipid droplets and lysosomes. Layer III had imbibed much fluid and appeared foamy with swollen organelles. Fibrinoid substance was produced by the giant cells, basophils and the trophoblast bordering the maternal sinusoids. Cyst development was preceded by degeneration of glycogen cells in the basal zone and of the trophoblast in the labyrinthine zone. Pronounced development of gonadotropin/somatotropin granule-like 'secretory granules' and smooth endoplasmic reticulum associated lipid droplets also characterised the labyrinthine trophoblast. The observed placental pathology appears to correlate well with the intrauterine growth retardation and fetal malformations recorded in this animal model.
Animals↗