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Biomedical subjects

M Levine

Publications and source records attributed to M Levine.

At least 235 records · Page 13Linked to original sources
PubMed1994

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.

Agglutination↗
PubMed1994

PCR-based analysis of herpes simplex virus type 1 latency in the rat trigeminal ganglion established with a ribonucleotide reductase-deficient mutant.

Competitive quantitative PCR and reverse transcriptase-PCR were used to quantitate DNA and RNA from an attenuated ribonucleotide reductase-deleted herpes simplex virus type 1 (HSV-1) mutant in the rat trigeminal ganglion after peripheral inoculation following corneal scarification. Amplification of ganglionic DNA with oligonucleotide primers specific for the HSV-1 glycoprotein B (gB) gene and for the latency-associated transcript (LAT) gene indicated that there were approximately 2 x 10(5) genome equivalents per ganglion at 2 days, 7 days, and 8 weeks after inoculation. Amplification of ganglionic RNA with primers specific for HSV-1 LAT indicated that the amount of LAT RNA was also stable over 8 weeks, with 10(7) LAT molecules per ganglion at 2 days and at 7 days postinoculation and 1.4 x 10(7) LAT molecules per ganglion at 8 weeks. In situ hybridization with a digoxigenin-labeled riboprobe specific for LAT detected an average of one to two LAT-positive cells in each positive 6-microns section of trigeminal ganglion. In situ PCR detection of HSV-1 genomes in similar sections, using digoxigenin-labeled nucleotides with primers specific for HSV-1 gB, identified as many as 120 genome-positive cells per section. These results indicate that there are approximately 50 LAT molecules per latent HSV-1 genome in the trigeminal ganglion, compared with 15 LAT molecules per latent HSV-1 genome in the central nervous system (R. Ramakrishnan, D. J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994), but that cells with detectable LATs by in situ hybridization represent only a small proportion of those ganglionic neurons containing HSV-1 genomes. The presence of latent HSV-1 genomes in a large number of neurons suggests that HSV-1 may be more efficient in establishing the latent state than would be anticipated from previous reports.

Animals↗
PubMed1994

Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.

Animals↗
PubMed1994

A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.

Animals↗
PubMed1994

Neurogenic expression of snail is controlled by separable CNS and PNS promoter elements.

The Drosophila snail (sna) gene is first expressed in cells giving rise to mesoderm and is required for mesoderm formation. sna is subsequently expressed in the developing nervous system. sna expression during neurogenesis evolves from segmentally repeated neuroectodermal domains to a pan-neural pattern. We have identified a 2.8 kb regulatory region of the sna promoter that drives LacZ expression in a faithful neuronal pattern. Deletion analysis of this region indicates that the pan-neural element is composed of separable CNS and PNS components. This finding is unexpected since all known genes controlling early neurogenesis, including the proneural genes (i.e. da and AS-C), are expressed in both the CNS and PNS. We also show that expression of sna during neurogenesis is largely independent of the proneural genes da and AS-C. The separate control of CNS and PNS sna expression and independence of proneural gene regulation add to a growing body of evidence that current genetic models of neurogenesis are substantially incomplete.

Animals↗
PubMed1993

Dif, a dorsal-related gene that mediates an immune response in Drosophila.

There are striking parallels between the regulation of gene expression along the dorsoventral (DV) axis of Drosophila embryos and lymphoid-restricted expression in the mammalian immune system. Both depend on regulatory factors containing rel domains (dorsal and NF-kappa B) that are controlled at the level of nuclear transport. A novel Rel-containing gene in Drosophila, Dif (dorsal-related immunity factor), provides a potential link between these seemingly disparate processes. Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae. Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury. Evidence is presented that once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes. These results suggest that mammalian and insect immunity share a common evolutionary origin.

Adipose Tissue↗
PubMed1993

Adjuvant radiotherapy for early breast cancer: patterns of practice in Ontario.

OBJECTIVE: To determine the number of different radiation schedules used in Ontario to treat women with node-negative breast cancer after lumpectomy and axillary dissection. DESIGN: Retrospective survey. SETTING: Princess Margaret Hospital, Toronto, and regional centres of the Ontario Cancer Treatment and Research Foundation (in Hamilton, London, Ottawa, Windsor and Thunder Bay). PATIENTS: A total of 551 of 1624 consecutive patients with node-negative breast cancer having undergone lumpectomy and axillary dissection who were eligible but did not participate in the Ontario Clinical Oncology Group randomized clinical trial and who received adjuvant breast irradiation between April 1984 and February 1989. OUTCOME MEASURES: Schedules of radiotherapy received. RESULTS: Forty-eight different radiotherapy schedules were identified. Total doses ranged from 4000 to 6600 cGy and the number of fractions from 15 to 30. Several different schedules were preferred: 322 patients (58.5%) received 4000 cGy in 15 or 16 fractions to the whole breast over 3 weeks plus a local boost of 1250 cGy to the primary site in 5 fractions over 1 week; 66 patients (12.0%) received 4000 cGy in 15 or 16 fractions over 3 weeks to the whole breast plus a local boost of 1000 cGy to the primary site in 4 or 5 fractions over 1 week; and 63 patients (11.5%) received 5000 cGy in 25 fractions to the whole breast in 5 weeks, without a boost. CONCLUSIONS: The practice of adjuvant radiotherapy for early breast cancer in Ontario varies. The optimal radiation regimen for patients after lumpectomy should be determined through randomized clinical trials.

Breast Neoplasms↗
PubMed1993

Ascorbic acid accumulation and transport in human fibroblasts.

As the initial step in the use of fibroblasts as a model system for 'in situ kinetics', ascorbic acid (vitamin C) accumulation in normal human fibroblasts was investigated for the first time. Ascorbic acid was transported into fibroblasts and accumulated against a concentration gradient up to 20-fold, as measured by h.p.l.c. with coulometric electrochemical detection. Ascorbic acid accumulation was mediated by two concentration-dependent transport activities. The first was a high-affinity activity with an apparent Km of 6 microM and an apparent Vmax. of 203 microM/h, and the second was a low-affinity activity with an apparent Km of 5 mM and an apparent Vmax. of 1.8 mM/h. Both activities were inhibited by metabolic inhibitors and inhibitors of ascorbic acid transport in human neutrophils. The low-affinity transporter could not be accounted for by diffusion. Although the high-affinity transport activity was comparable with that described for human neutrophils, the low-affinity transporter was different. These data provide the first evidence that two-component ascorbic acid transport may be a generalized mechanism for accumulation of this vitamin in humans.

2,4-Dinitrophenol↗
PubMed1993

Ascorbic acid recycling in human neutrophils.

Ascorbic acid (vitamin C) accumulation in activated human neutrophils is increased as much as 10-fold above the mM concentrations present in normal neutrophils. Internal concentrations as high as 14 mM are achieved when external vitamin is at physiologic concentration. The mechanism is by oxidation of external vitamin to dehydroascorbic acid, preferential transmembrane translocation of dehydroascorbic acid, and intracellular reduction to ascorbic acid within minutes. These data indicate that vitamin C accumulation is enhanced in activated human neutrophils and that human neutrophils utilize and recycle oxidized external vitamin C under physiologic conditions.

Adult↗
PubMed1993

Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen.

The dorsal (dl) morphogen gradient initiates the formation of the mesoderm, neuroectoderm, and dorsal ectoderm by setting different limits of regulatory gene expression along the dorsoventral axis of the early Drosophila embryo. In this paper, we show that low affinity dl-binding sites restrict target gene expression to the ventralmost regions (presumptive mesoderm), where there are peak levels of dl, while high affinity sites permit expression in ventrolateral regions (mesoderm and mesectoderm) containing intermediate levels of the morphogen. Activation by low levels of dl in lateral regions (the presumptive neuroectoderm) depends on cooperative DNA binding interactions between dl and bHLH proteins. The snail repressor blocks this interaction and restricts expression to the neuroectoderm. We discuss how enhancers serve as templates to bring weakly interacting regulatory factors into close proximity so that they can function combinatorially to activate and repress transcription.

Animals↗
PubMed1993

Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors.

In Drosophila, the dorsal (dl) morphogen gradient initiates the differentiation of the embryonic mesoderm and neuroectoderm by activating the expression of regulatory genes (e.g. twist and snail) in a concentration-dependent manner. dl also functions as a repressor that establishes the dorsal epidermis and amnioserosa by restricting regulatory genes such as dpp and zen to dorsal regions of the embryo. The ability of dl to function as both an activator and repressor distinguishes it from the bicoid morphogen, which appears to function solely as an activator. In an effort to determine how dl functions as a repressor we have performed a detailed characterization of a zen silencer element, called the VRE, which mediates ventral repression in response to the dl gradient. A minimal 110 bp VRE sequence is identified, which is able to silence the ventral expression of a heterologous promoter. This sequence contains two dl binding sites as well as binding sites for additional nuclear factors present in early embryos. Mutations in the latter binding sites convert the minimal VRE into an enhancer, which mediates transcriptional activation in ventral regions in response to dl. These results suggest that dl is intrinsically an activator, but is converted into a potent silencer when it interacts with neighboring corepressors.

Animals↗
PubMed1993

Coulometric electrochemical detection of hydroxyproline using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

A new method for detection of hydroxyproline has been developed. Hydroxyproline was derivatized using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; separation and detection were accomplished using HPLC with coulometric electrochemical detection. Derivatized hydroxyproline was initially detected using a 16 channel coulometric electrochemical array system. The assay was slightly modified for use with a simpler 2-channel coulometric electrochemical detector. Both detectors are sensitive into the upper fmol range. Derivatization of hydroxyproline occurs in 5 min and the derivative is stable for several hours at 0 degrees C. The techniques were used to quantitate hydroxyproline in purified type I collagen and other biological samples.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

A comparison of regularly dosed oral morphine and on-demand intramuscular morphine in the treatment of postsurgical pain.

A randomized, placebo-controlled, double-blind clinical trial was conducted to compare the use of regularly dosed po morphine and on-demand in morphine in 47 patients undergoing total hip arthroplasty. Patients were randomized to receive either 20 mg (initial dose) of regularly dosed morphine (every four hours po) plus breakthrough pain medication on-demand consisting of both 10 mg morphine po and placebo im, or an equivalent regularly dosed oral placebo (every four hours) with breakthrough pain medication consisting of oral placebo and 5-10 mg morphine im. Subsequent to each request for breakthrough pain medication, the next regularly dosed oral solution was increased by 5 mg (or equivalent volume of placebo) to a maximum of 40 mg po Q4H. Time-averaged pain scores were lower on both postoperative day 1 and 2 in the group receiving regularly dosed morphine po (P < 0.05). Fewer patients requested breakthrough pain medication on both days in the oral morphine group. The incidence of nausea and vomiting, and of decreased respiratory rates were similar in both groups. Regularly dosed oral morphine is inexpensive and should be compared to other methods of opioid delivery.

Administration, Oral↗
PubMed1993

Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine does not depress serologic responses to diphtheria, tetanus or pertussis antigens when coadministered in the same syringe with diphtheria-tetanus-pertussis vaccine at two, four and six months of age.

The safety and immunogenicity of a vaccine against Haemophilus influenzae type b consisting of purified polyribosylribitol phosphate conjugated to tetanus toxoid (PRP-T) were evaluated in 277 Chilean infants who were randomly assigned to one of three treatment groups: Group A, PRP-T mixed with diphtheria-tetanus-pertussis (DTP) vaccine in a single syringe and given as a single inoculation in one arm and placebo in the other arm; Group B, PRP-T given in one arm and DTP in the other arm; Group C, DTP given in one arm and placebo in the other. Infants were immunized at 2, 4 and 6 months of age and examined daily for 4 days after each immunization. Serum PRP antibodies; tetanus, diphtheria and pertussis antitoxin; pertussis agglutinins; and antibodies to Bordetella pertussis filamentous hemagglutinin were measured at baseline and 2 months after each dose. PRP-T was well-tolerated. After three doses of PRP-T vaccine 100% of infants attained PRP antibody concentrations > or = 0.15 micrograms/ml and 96 to 99% achieved high anti-PRP concentrations (> or = 1.0 micrograms/ml). The post-third dose anti-PRP geometric mean titer was high (6.94 micrograms/ml) in infants who were given PRP-T combined with DTP, although it was somewhat lower than the geometric mean titer of the group who received PRP-T in a separate arm (9.93 micrograms/ml) (P not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Bacterial↗
PubMed1993

Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila.

The dorsal (dl) morphogen has been implicated in the establishment of the embryonic mesoderm, neuroectoderm, and dorsal ectoderm in Drosophila. Here we show that the simultaneous reduction in the levels of dl and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are shown to be required for the formation of these embryonic tissues. Evidence is also presented that dl-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains. We discuss the striking parallels in mesoderm formation and sex determination.

Animals↗