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Biomedical subjects

R Mahalingam

Publications and source records attributed to R Mahalingam.

At least 19 recordsLinked to original sources
PubMed2001

Acute, chronic, and recurrent varicella zoster virus neuropathy without zoster rash.

The authors report three patients with acute, chronic, and recurrent neuropathy associated with varicella zoster virus (VZV) infection but without zoster rash. CSF of all three patients contained VZV immunoglobulin G antibody, but not herpes simplex virus. In two patients, serum/CSF ratios of VZV immunoglobulin G were reduced compared to normal ratios for immunoglobulin G and albumin, and one patient also had VZV immunoglobulin M in CSF. All three patients received antiviral therapy and improved. The diagnosis of nervous system infection by VZV may be confirmed by the presence of antibody to VZV in CSF even without detectable VZV DNA.

Acute Disease↗
PubMed2001

Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays.

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced-PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced-PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for approximately 1,000 stress-induced and -repressed Arabidopsis genes.

DNA Transposable Elements↗
PubMed2001

The DNA sequence of the simian varicella virus genome.

In nonhuman primates, simian varicella virus (SVV) causes a natural disease which is clinically similar to human varicella-zoster virus (VZV) infections. The SVV and VZV genomes are similar in size and structure and share extensive DNA homology. This report presents the complete DNA sequence of the SVV genome. SVV DNA is 124,138 bp in size, 746 bp shorter than VZV DNA, and 40.4% G + C. The viral genome includes a 104,104-bp unique long component bracketed by 8-bp inverted repeat sequences and a short component composed of a 4904-bp unique short region bracketed by 7557-bp inverted repeat sequences. A total of 69 distinct SVV open reading frames (ORFs) were identified, including three that are duplicated within the inverted repeats of the short component. Each of the SVV ORFs shares extensive homology to a corresponding VZV gene. The only major difference between SVV and VZV DNA occurs at the leftward terminus. SVV lacks a VZV ORF 2 homolog. In addition, SVV encodes an 882-bp ORF A that is absent in VZV, but has homology to the SVV and VZV ORF 4. The results of this study confirm the relatedness of SVV and VZV and provide further support for simian varicella as a model to investigate VZV pathogenesis and latency.

Animals↗
PubMed2001

Simian varicella virus infects ganglia before rash in experimentally infected monkeys.

Monkeys experimentally infected with simian varicella virus (SVV) develop rash 10-14 days later. However, the route and the time of ganglionic infection are unknown. Using PCR, we analyzed DNA extracted from tissues of 13 monkeys 5 to 60 days after either intratracheal or intravenous inoculation with SVV. SVV DNA was detected in ganglia from four of five monkeys sacrificed 6 to 7 days after intratracheal inoculation. Further, analysis of ganglia from monkeys sacrificed at 10 days revealed that intravenous inoculation produced a higher proportion of SVV DNA-positive ganglia (63%) than that after intratracheal inoculation (13%), pointing to the role of hematogenous spread in ganglionic infection. Like other organs, monkey ganglia become infected with SVV before the appearance of rash.

Animals↗
PubMed2001

Presence of VZV and HSV-1 DNA in human nodose and celiac ganglia.

Polymerase chain reaction (PCR) revealed herpes simplex virus (HSV) and varicella zoster virus (VZV) DNA in human nodose and celiac ganglia. This is the first detection of VZV DNA in ganglia of the human autonomic nervous system. The ability of reactivated VZV to produce serious, sometimes fatal neurological disease in the absence of rash, raises the possibility that VZV reactivation from autonomic ganglia might be involved in visceral disease.

Aged↗
PubMed2001

An animal model of varicella virus infection.

Varicella-zoster virus (VZV) causes chickenpox in children; establishes latency in cranial nerve, dorsal root, and autonomic ganglia; and reactivates decades later to produce zoster. VZV produces disease only in humans. Although attempts to produce disease and study VZV latency in experimentally infected animals have resulted in virus in trigeminal or dorsal root ganglia, no clinical signs of infection or reactivation developed. In contrast, simian varicella virus (SVV) produces a naturally occurring exanthematous disease in non-human primates that mimics human varicella. Experimental inoculation of non-human primates causes similar, if not identical, clinical and pathological changes observed in monkeys naturally infected with SVV. Like VZV, SVV becomes latent in ganglia and reactivates, often with entire body rash. SVV and VZV encode antigenically related polypeptides. Both virus genomes have been sequenced and shown to be colinear, sharing up to 75% DNA homology. During latency, an SVV homolog of one of the five VZV genes transcribed in latently infected human ganglia has been detected in monkey ganglia. Preliminary studies in which monkeys were inoculated intratracheally with SVV revealed the presence of viral DNA and RNA in multiple tissues, including blood mononuclear cells, months after experimental infection. These findings differed from the expected restricted localization of the virus DNA to ganglia only and the expected limited viral gene expression, and probably reflect the high virus load delivered intratracheally compared to natural SVV infection in monkeys. Nevertheless, clinical, pathological, and molecular similarities between SVV and VZV indicate that SVV infection in non-human primates has considerable potential as an animal model for human varicella.

Animals↗
PubMed2000

Solidification and stabilization of asbestos waste from an automobile brake manufacturing facility using cement.

Currently, the generated brake lining waste dust, which contains asbestos as its major component, is disposed of into a secure landfill without any additional treatment. As an alternative to this, solidification/stabilization (S/S) disposal of the dust was investigated using Portland cement alone and Portland cement mixed with activated carbon (AC), as the binders. Toxicity Characteristics Leaching Procedure (TCLP) results on the solidified matrix showed that cement was able to immobilize the heavy metals, Ba, Zn, Cr, Pb, Cu and Fe, to within the limits set by the US EPA for TCLP. Addition of AC to the cement reduced the leaching of heavy metals by an additional 4-24% compared to cement alone. The pH of the TCLP leachate extracted from virgin cement, and from dust treated with cement with or without AC was found to increase to 10.9-12.5 as opposed to an initial value of 4.93 for the TCLP extract for the untreated dust. Results of ANS 16.1 (modified) leach protocol revealed that Ba in cement-treated samples showed the highest leach rate, followed by Zn, Pb, Cr, Cu and Fe. The leach rate of heavy metals decreased with progress in time. Cement mixed with AC exhibited similar leach characteristics, however, the leach rate was lower. The linear relationship between the cumulative fraction leached (CFL) and the square root of leaching time in all cement-based samples indicate that a diffusional process is the controlling transport mechanism for the leaching of the heavy metals. The obtained Leachability Indices (L(i)) of 7.6-9.1 and 8.3-9.5 for cement and cement with AC, respectively, were low but exceeded the guidance value of 6, which clearly indicates that all the heavy metals studied are retained well within solid matrices. Cement-based S/S hardening times increased from 30 to 96 h as the dust content increased from 40 to 70 wt.%. The resulting solid matrices exhibited a compressive strength ranging from 1 to 12 MPa, which was well above the specified limit of 414 kPa for such matrices. An economic analysis indicates that the disposal costs for the dust in the only available secure landfill would increase by 40.3% if one were to go for the cement S/S option. Addition of AC to the cement would escalate this by an additional 43.8%. Although the S/S of brake lining dust using cement effectively immobilized the heavy metals of concern, cost considerations may hinder the commercial adaptation of this technique for waste disposal unless new regulatory demands are implemented.

Asbestos↗
PubMed2000

Sequence analysis of the leftward end of simian varicella virus (EcoRI-I fragment) reveals the presence of an 8-bp repeat flanking the unique long segment and an 881-bp open-reading frame that is absent in the varicella zoster virus genome.

Simian varicella virus (SVV) causes varicella (chickenpox) in nonhuman primates, becomes latent in cranial and dorsal root ganglia, and reactivates to produce zoster (shingles). Because the clinical and molecular features of SVV closely resemble those of varicella zoster virus (VZV) infection of humans, SVV infection of primates has served as an experimental model of VZV pathogenesis and latency. The SVV genome has been completely mapped, but attempts to clone the 3600-bp EcoRI fragment located at the leftward end of the virus genome have hitherto been unsuccessful. Herein, we report the cloning and the complete nucleotide sequence of this region. Comparison of the SVV and VZV sequences in this region revealed an 8-bp inverted repeat sequence flanking the unique long segment of the SVV genome; an 879-bp open-reading frame (ORF) A in SVV that is absent in VZV but has 42% amino acid identity to SVV ORF 4 and 49% to VZV ORF 4; a 342-bp ORF B in SVV with 35% amino acid identity to a 387-bp ORF located to the left of ORF 1 on the VZV genome; and a 303-bp ORF in SVV with 27% amino acid identity to VZV ORF 1. No homologue of VZV ORF 2 was detected. Transcripts specific for ORFs A and B were present in SVV-infected cells in culture and in acutely infected monkey ganglia. Overall, there are more than 2000 bp of DNA in the SVV genome that are absent in the VZV genome.

Amino Acid Sequence↗
PubMed2000

Identification of simian varicella virus gene 21 promoter region using green fluorescent protein.

Clinical, pathological, immunological and virological features of simian varicella virus (SVV) infection in primates closely resemble those of varicella zoster virus (VZV) infection in humans. In ganglia infected latently of humans and monkeys, gene 21 of VZV and SVV is transcribed, respectively. We determined the nucleotide sequence of the intragenic region between SVV genes 20 and 21 to identify the putative promoter region for SVV gene 21. A recombinant clone was prepared in which the gene encoding green fluorescent protein (GFP) was inserted ten base pairs upstream of the predicted translational start site for SVV gene 21. SVV-infected monkey kidney cells transfected with the recombinant clone showed the presence of green fluorescence, whereas transfection of these cells with a construct containing the GFP gene in the opposite orientation, produced no fluorescence. The recombinant clone containing GFP flanked by SVV sequences can be used to prepare a SVV mutant in which the virus gene 21 promoter drives GFP. Such a mutant will be useful in analyzing varicella pathogenesis and latency in experimentally infected animals, studies not possible in humans.

Animals↗
PubMed1999

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

Animals↗
PubMed1999

Polygalacturonase and polygalacturonase inhibitor protein: gene isolation and transcription in Glycine max-Heterodera glycines interactions.

The cell wall acts as the first line of defense during pathogen invasion. Polygalacturonases (PGs) are a class of cell-wall-modifying enzymes with precise temporal and organ-specific expression. A 350-bp fragment with high homology to PGs was identified by differential display (DD) analysis of soybean cyst nematode (SCN) race 3 resistant PI 437654 and susceptible cultivar Essex. The fragment was strongly expressed in Essex, 2 days after inoculation (DAI). Complete coding sequences of two PG cDNAs, PG1 and PG2, were isolated by 3' and 5' rapid amplification of cDNA ends polymerase chain reaction (RACE PCR). PI 437654 and Essex had identical PG1 and PG2 sequences. A transversion from A to C created a PstI restriction site in the PG2 cDNA that was used to distinguish the two PG cDNAs by cleaved amplified polymorphic sequence (CAPS) analysis. A cDNA encoding a polygalacturonase-inhibitor protein (PGIP) that is 89% identical to the Phaseolus vulgaris PGIP was isolated from soybean roots by reverse transcription (RT)-PCR. Steady-state levels of PG and PGIP were investigated by RNA gel blot analysis in roots 1 to 5 DAI and in hypocotyls and leaves. Differences in the constitutive levels of PG mRNAs were observed in roots of different soybean genotypes. Steady-state levels of PG mRNAs were enhanced during compatible interactions with SCN and reduced in incompatible interactions and in mechanically wounded roots. Enhanced PGIP transcription was observed in response to mechanical wounding in both PI 437654 and Essex, but only in compatible interactions with SCN, suggesting uncoupling of PGIP functions in developmental and stress cues. Constitutive expression in incompatible interactions shows PGIP is not a factor in SCN resistance. Thus, the up-regulation of endogenous PG transcription in soybean roots early after SCN infection could facilitate successful parasitism by SCN.

Amino Acid Sequence↗
PubMed1998

Profound cerebrospinal fluid pleocytosis and Froin's Syndrome secondary to widespread necrotizing vasculitis in an HIV-positive patient with varicella zoster virus encephalomyelitis.

Demonstration of the direct involvement of cranial blood vessels by varicella zoster virus (VZV) is facilitated by immunohistochemistry (IHC), in situ hybridization (ISH) and polymerase chain reaction (PCR) techniques. The extent to which an inflammatory vasculitis serves as the pathogenic mechanism for VZV encephalomyelitis (VZVE) is still, however, debated. Most VZVE patients are immunocompromised and show little inflammation, either pre-mortem in cerebrospinal fluid (CSF) or at autopsy. We describe an HIV-positive patient with a moderately depressed CD4 count (304) who presented with massively elevated CSF protein (1800 mg/dl), bloody CSF and pleocytosis (1300 white blood cells (WBC)/mm3). His CSF was positive for VZV DNA by PCR. He was treated with acyclovir and foscarnet, but died. At autopsy, an unusually widespread, inflammatory, transmural vasculitis caused by VZV affected meningeal vessels at virtually all brain stem and spinal cord levels, causing multiple subpial hemorrhages and necrosis. Virus DNA in multiple areas of brain, brainstem and spinal cord was readily revealed by PCR, but not by the presence of viral inclusions, IHC or ISH. This case, with a clinically confusing presentation for VZVE, illustrates the extensive, albeit infrequent, degree of necrotizing vasculitis and CSF abnormalities that VZV is capable of producing. Antiviral therapy may have inhibited VZV genome replication and subsequent antigen production, resulting in negative ISH and IHC studies, but generated increased VZV genomic fragments that were detectable by the more sensitive PCR technique.

AIDS-Related Opportunistic Infections↗
PubMed1998

Selected nuclear LINE elements with mitochondrial-DNA-like inserts are more plentiful and mobile in tumor than in normal tissue of mouse and rat.

The nuclear DNA of normal and tumor mouse and rat tissue was examined for mitochondrial-DNA-like inserts by means of the Southern blot technique. The two probes were 32P-labeled cloned mitochondrial DNA. KpnI, which doesn't cut either mitochondrial DNA, was one of the restriction enzymes, while the enzymes that fragment mitochondrial DNA were for mouse and rat PstI and BamHI, respectively. When KpnI alone was used in the procedure a nuclear LINE family whose elements had mitochondrial-DNA-like insertions was selected. Such elements were much more abundant in tumor than in normal tissue. The results with PstI alone and BamHI alone and each combined with KpnI indicated that there were mobile LINE elements with mitochondrial-DNA-like inserts in the nuclear genome of tumor. The mouse tissues were normal liver and a transplantable lymphoid leukemic ascites cell line L1210 that had been carried for 40 years. The rat tissues were normal liver and a hepatoma freshly induced by diethylnitrosoamine in order to minimize the role of 40 years of transplantation. Our unitary hypothesis for carcinogenesis of 1971, which suggested these experiments, has been augmented to include mobile nuclear elements with inserts of mitochondrial-DNA-like sequences. Such elements have been related to diseases of genetic predisposition such as breast cancer and Huntington's disease.

Animals↗