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Biomedical subjects

A M Roy

Publications and source records attributed to A M Roy.

13 recordsLinked to original sources
PubMed2001

Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclear periphery.

A critical phase of the influenza virus life cycle is the regulated translocation of genomic ribonucleoproteins (vRNPs) from the nuclear interior, across the nuclear envelope, and into the cytoplasm. Two viral proteins, M1 and NS2, have previously been implicated as mediators of vRNP export. We show here that vRNP nuclear export is prevented by leptomycin B (LMB), an inhibitor of the cellular factor CRM1. In LMB-treated cells, vRNPs were found in a peripheral nuclear location that localized with the nuclear lamina. vRNPs were not colocalized with either M1 or NS2. In situ extraction of cells late in infection also revealed a peripheral localization of nuclear vRNPs, whereas early in infection vRNPs were dispersed throughout the nuclear interior. We believe that vRNPs at the nuclear periphery represent a novel intermediate in the influenza virus nuclear export pathway.

Active Transport, Cell Nucleus↗
PubMed2000

Upstream flanking sequences and transcription of SINEs.

SINEs, short interspersed repeated DNA elements, undergo amplification through retroposition and subsequent integration into a new location in the genome. Each new SINE insertion will be located in a new chromosomal environment, with different flanking sequences. Modulation of transcription by different flanking sequences may play an important role in determining which SINE elements are preferentially active in a genome. We evaluated the ability of upstream flanking sequences to regulate the transcription of three different SINEs (Alu, B2 and ID) by constructing chimeric constructs with known 5' flanking sequences of RNA polymerase III-transcribed genes. Upstream sequences from the 7SL RNA gene, U6 RNA gene, vault RNA gene, and BC1 gene increase transcription of Alu, B2 and BC1 in transient transfections of NIH3T3, HeLa, Neuro2a and C6 glioma cell lines. The 7SL sequence proved most efficient in increasing SINE transcription. The 7SL upstream fused to the BC1 RNA gene (an ID element) was used to create a transgenic mouse line. In contrast to the tissue-specific endogenous BC1 transcription, BC1 transgene transcripts were detected in all tissues tested. However, expression was much higher in those tissues that express the endogenous gene, demonstrating both transcriptional and post-transcriptional regulation. The BC1 RNA was detected in a similar ribonucleoprotein complex in the different tissues.

Alu Elements↗
PubMed2000

Early stages of influenza virus entry into Mv-1 lung cells: involvement of dynamin.

Viruses generally have one of two mechanisms for entry and uncoating. They can enter the cell either by endocytosis or by direct fusion at the plasma membrane. We have established a novel mink lung (Mv-1) cell line that expresses a dominant-interfering form of dynamin-1 (K44A) under the control of a tetracycline-responsive element and studied the early events in influenza infection using these cells. We found that influenza virus binds equally to both induced and uninduced cells, but in K44A-expressing cells, electron microscopy showed viruses trapped in deep coated pits and irregular-shaped tubular structures that contain discrete coated regions. We also show by immunofluorescence and confocal microscopy that entry of incoming virus into the nucleus is blocked in K44A-expressing cells. Virus replication was assayed by immunofluorescence microscopy and was strongly inhibited at both early and late times postinfection in K44A-expressing cells. Virus infectivity was inhibited by approximately 2 log units in cells expressing K44A dynamin when analyzed by influenza plaque assay. Overall these data show that dynamin is required for efficient influenza virus entry, presumably due to its function in release of vesicles from coated pits.

Animals↗
PubMed2000

Potential gene conversion and source genes for recently integrated Alu elements.

Alu elements comprise >10% of the human genome. We have used a computational biology approach to analyze the human genomic DNA sequence databases to determine the impact of gene conversion on the sequence diversity of recently integrated Alu elements and to identify Alu elements that were potentially retroposition competent. We analyzed 269 Alu Ya5 elements and identified 23 members of a new Alu subfamily termed Ya5a2 with an estimated copy number of 35 members, including the de novo Alu insertion in the NF1 gene. Our analysis of Alu elements containing one to four (Ya1-Ya4) of the Ya5 subfamily-specific mutations suggests that gene conversion contributed as much as 10%-20% of the variation between recently integrated Alu elements. In addition, analysis of the middle A-rich region of the different Alu Ya5 members indicates a tendency toward expansion of this region and subsequent generation of simple sequence repeats. Mining the databases for putative retroposition-competent elements that share 100% nucleotide identity to the previously reported de novo Alu insertions linked to human diseases resulted in the retrieval of 13 exact matches to the NF1 Alu repeat, three to the Alu element in BRCA2, and one to the Alu element in FGFR2 (Apert syndrome). Transient transfections of the potential source gene for the Apert's Alu with its endogenous flanking genomic sequences demonstrated the transcriptional and presumptive transpositional competency of the element.

Alleles↗
PubMed1999

Cytotoxicity of [125I]iodoHoechst 33342: contribution of scavengeable effects.

PURPOSE: The incubation of the DNA minor-groove binder [125I]iodoHoechst 33342 (125IH) with plasmid DNA leads to the production of one double-strand break (dsb) per decay, both in the presence and absence of dimethylsulfoxide (DMSO). In contrast, when 125I is incorporated into mammalian cell DNA as an iodinated pyrimidine base, DMSO decreases the dsb yield and enhances survival. Because these variations in radioprotective effects may be due either to the location of 125I vis-à-vis the DNA helix or to differences in DNA architecture, the toxicity of 125IH and its modification by DMSO were examined in mammalian cells. METHODS: Uptake and retention of 125IH in V79 cells were measured, and survival was determined after accumulation of 125I decays at 0.3 degrees C +/-10% DMSO. RESULTS: A linear-quadratic survival curve was obtained both in the absence [D37 = 114+/-36 decays/cell, alpha = (5.39 1.17) x10(-3) cell/decay] and presence [D37 = 211+/-65 decays/cell, alpha = (1.27+/-0.52) x10(-3) cell/decay] of DMSO. The dose modification factor for the linear component of the survival curve was 4.25+/-1.97, indicating the predominance of indirect mechanisms. This value is similar to that obtained with DNA-incorporated 125I (4.05+/-1.72) and for the initial slope (alpha) of 137Cs gamma-rays (4.43+/- 1.41). CONCLUSIONS: Cytotoxicity resulting from the decay of the Auger electron emitter 125I in the mammalian cell nucleus is caused mainly by indirect mechanisms.

Animals↗
PubMed1999

Recently integrated human Alu repeats: finding needles in the haystack.

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution.

Animals↗
PubMed1998

[125I/127I/131I]Iodorhodamine: synthesis, cellular localization, and biodistribution in athymic mice bearing human tumor xenografts and comparison with [99mTc]hexakis(2-methoxyisobutylisonitrile).

The synthesis of halogenated rhodamine (Rh) derivatives was carried out by controlling the stoichiometry of the halogenating agents, bromine and iodine monochloride. In the no-carrier-added synthesis of radioiodinated rhodamine 123, direct labeling of rhodamine 123 (Rh 123) with Na125I/Na131I required the presence of the oxidant peracetic acid. 125I/131I-Rh 123 was synthesized in modest yields (40-45%). HPLC purification separated Rh 123 from its mono- and diiodo derivatives. Monohalogenation of Rh 123 did not alter the compound's ability to permeate viable cells and localize in mitochondria. 125I/131I-Rh 123 was stable in serum in vitro but rapidly metabolized after intravenous injection into mice. Consequently, scintigraphy and biodistribution data reveal poor targeting of subcutaneously growing human tumor xenografts. The results are compared to those obtained following the administration of [99mTc]hexakis(2-methoxyisobutylisonitrile) which also did not image human tumor xenografts in nude mice.

Animals↗
PubMed1998

Recent B2 element insertions in the mouse genome.

B2 elements are a family of short interspersed repeats that have amplified within rodent genomes. Recent mobility of only two individual B2 elements has been reported to date. We identified an additional recent B2 insertion occurring within intron 4 of the murine beta-glucuronidase gene (Gus-s) of the BalbC strain of mouse by analyzing orthologous loci of a nonrandomly selected B2 element. The basis of selection for the B2 element was its high level of sequence identity to the B2 consensus. The selected B2 element was amplified by the polymerase chain reaction (PCR) using primers to the unique flanking sequences from genomic DNA of several species and laboratory strains of mice. Our results demonstrated the presence of the selected B2 element only in the genome of Mus musculus BalbC strain. Cloning and sequencing of a representative sample of the products obtained confirmed the absence of the B2 element within this intron in addition to other variations in the sequence. The detection of the B2 element only in the BalbC strain suggests that the element recently inserted within this mouse population when the initial laboratory colony was formed. Sequence comparison of the two previously identified recent B2 inserts also shows a low divergence in relation to the B2 type II consensus. The data presented confirms that recently inserted B2 elements closely match their consensus sequence, potentially allowing for their identification.

Animals↗
PubMed1997

cDNAs derived from primary and small cytoplasmic Alu (scAlu) transcripts.

We have isolated and sequenced twenty-six cDNAs derived from primary Alu transcripts. Most cDNAs (22/26) sequenced end in multiple T residues, known to be at the termination for RNA polymerase III-directed transcripts. We conclude that these cDNAs were derived from authentic, RNA polymerase III-directed primary Alu transcripts. Sequence alignment of the cDNAs with Alu consensus sequences show that the cDNAs belong to different, previously described Alu subfamilies. The sequence variation observed in the 3' non-Alu regions of each of the cDNAs led us to conclude that they were derived from different genomic loci, thus demonstrating that multiple Alu loci are transcriptionally active. The subfamily distribution of the cDNAs suggests that transcriptional activity is biased towards evolutionarily younger Alu subfamilies, with a strong selection for the consensus sequence in the first 42 bases and the promoter B box. Sequence data from seven cDNAs derived from small cytoplasmic Alu (scAlu) transcripts, a processed form of Alu transcripts, also have a similar bias towards younger Alu subfamilies. About half of these cDNAs are due to processing or degradation, but the other half appear to be due to the formation of a cryptic RNA polymerase III termination signal in multiple loci. Using our sequence data, we have isolated a transcriptionally active genomic Alu element belonging to the Ya5 subfamily. In vitro transcription studies of this element suggest that its flanking sequences contribute to its transcriptional activity. The role of flanking sequences and other factors involved in transcriptional activity of Alu elements are discussed.

Base Sequence↗
PubMed1994

Mutations in firA, encoding the second acyltransferase in lipopolysaccharide biosynthesis, affect multiple steps in lipopolysaccharide biosynthesis.

The product of the firA (ssc) gene is essential for growth and for the integrity of the outer membrane of Escherichia coli and Salmonella typhimurium. Recently, Kelly and coworkers (T. M. Kelly, S. A. Stachula, C. R. H. Raetz, and M. S. Anderson, J. Biol. Chem., 268:19866-19874, 1993) identified firA as the gene encoding UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the third step in lipid A biosynthesis. We studied the effects of six different mutations in firA on lipopolysaccharide synthesis. All of the firA mutants of both E. coli and S. typhimurium examined had a decreased lipopolysaccharide synthesis rate. E. coli and S. typhimurium strains defective in firA produced a lipid A that contains a seventh fatty acid, a hexadecanoic acid, when grown at the nonpermissive temperature. Analysis of the enzymatic activity of other enzymes involved in lipid A biosynthesis revealed that the firA mutations pleiotropically affect lipopolysaccharide biosynthesis. In addition to that of UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the enzymatic activity of the lipid A 4' kinase (the sixth step of lipid A biosynthesis) was decreased in strains with each of the firA mutations examined. However, overproduction of FirA was not accompanied by overexpression of the lipid A 4' kinase.

Acyltransferases↗
PubMed1993

Synchronization of motor-unit firings in several human muscles.

1. Synchronization of concurrently active motor-unit firings was studied in six human muscles performing isometric constant-force contractions at 30% of the maximal level. The myoelectric signal was detected with a quadrifilar needle electrode and was decomposed into its constituent motor-unit action-potential trains with the Precision Decomposition technique, whose accuracy has been proven previously. 2. Synchronization was considered as the tendency of two motor units to fire at fixed time intervals with respect to each other more often than would be expected if the motor units fired independently. A rigorous statistical technique was used to measure the presence of peaks in the cross-interval histogram of pairs of motor-unit action-potential trains. The location of the center of peak as well as their width and amplitude were measured. A synch index was developed to measure the percentage of firings that were synchronized. The percentage of concurrently active motor-unit pairs that contained synchronized firings was measured. 3. Synchronization of motor-unit firings was observed to occur in two modalities. The short-term modality was seen as a peak in the cross-interval histogram centered about zero-time delay (0.5 +/- 2.9 ms, mean +/- SD) and with an average width of 4.5 +/- 2.5 ms. The long-term modality was seen as a peak centered at latencies ranging from 8 to 76 ms. On the average, the peaks of the long-term synchronization were 36% lower but had approximately the same width as the peaks for the short-term synchronization. Short-term synchronization was seen in 60% of the motor-unit paris, whereas long-term synchronization was seen in 10% of the pairs. 4. Short-term synchronization occurred in bursts of consecutive firings, ranging in number from 1 to 10, with 91% of all synchronized firing occurring in groups of 1 or 2; and the bursts of discharges appeared at sporadic times during the contraction. 5. The amount of synchronization in motor-unit pairs was found to be low. In the six muscles that were tested, an average of 8.0% of all the firings were short-term synchronized, and an average of 1.0% were long-term synchronized. The synch index was statistically indistinguishable (P = 0.07-0.89) among the different muscles and among 9 of the 11 subjects tested. 6. Sixty percent of concurrently active motor-unit pairs displayed short-term synchronization, 10% of the pairs displayed long-term synchronization, and 8% displayed both modalities.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1984

Airborne contact urticaria due to sodium benzoate in a pharmaceutical manufacturing plant.

Three workers exposed to airborne contact with sodium benzoate (SB) in a pharmaceutical plant developed transient urticaria related to skin contamination with SB. Patch test responses to SB and benzoic acid (BA), without occlusion, were similar to those of three previously unexposed controls in keeping with the nonimmunologic nature of the reaction. Sweating, which lowers skin pH and increases topical BA concentration, appeared to increase the susceptibility to urticaria in two of the three workers. Ventilation and hygiene control methods designed to reduce SB skin contamination eliminated the problem in the workplace.

Adult↗