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Biomedical subjects

M Rosenberg

Publications and source records attributed to M Rosenberg.

At least 19 recordsLinked to original sources
PubMed2002

Microarray-based identification of htrA, a Streptococcus pneumoniae gene that is regulated by the CiaRH two-component system and contributes to nasopharyngeal colonization.

Nasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniae arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrA and spoJ, that was specifically and strongly downregulated in a DeltaciaRH-null mutant. A S. pneumoniae strain lacking the htrA gene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the DeltaciaRH strain.

Animals↗
PubMed1992

Ferritin: a cytoprotective antioxidant strategem of endothelium.

Phagocyte-mediated oxidant damage to vascular endothelium is likely involved in various vasculopathies including atherosclerosis and pulmonary leak syndromes such as adult respiratory distress syndrome. We have shown that heme, a hydrophobic iron chelate, is rapidly incorporated into endothelial cells where, after as little as 1 h, it markedly aggravates cytotoxicity engendered by polymorphonuclear leukocyte oxidants or hydrogen peroxide (H2O2). In contrast, however, if cultured endothelial cells are briefly pulsed with heme and then allowed to incubate for a prolonged period (16 h), the cells become highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products. This protection is associated with the induction within 4 h of mRNAs for both heme oxygenase and ferritin. After 16 h heme oxygenase and ferritin have increased approximately 50-fold and 10-fold, respectively. Differential induction of these proteins determined that ferritin is probably the ultimate cytoprotectant. Ferritin inhibits oxidant-mediated cytolysis in direct relation to its intracellular concentration. Apoferritin, when added to cultured endothelial cells, is taken up in a dose-responsive manner and appears as cytoplasmic granules by immunofluorescence; in a similar dose-responsive manner, added apoferritin protects endothelial cells from oxidant-mediated cytolysis. Conversely, a site-directed mutant of ferritin (heavy chain Glu62----Lys; His65----Gly) which lacks ferroxidase activity and is deficient in iron sequestering capacity, is completely ineffectual as a cytoprotectant. We conclude that endothelium and perhaps other cell types may be protected from oxidant damage through the iron sequestrant, ferritin.

Animals↗
PubMed1992

Characterization of the transcription activator protein C1 of bacteriophage P22.

We cloned, expressed, and purified the positive regulatory protein C1 of the temperate phage P22 of Salmonella typhimurium. The purified protein was characterized as to its amino acid composition, protein sequence, molecular weight, and antigenicity. P22 C1 was shown to be a tetrameric protein composed of four identical subunits with M(r) = 10,000. Moreover, we identified and characterized two P22 C1-dependent phage promoters, P(RE) and Pa23, whose function was completely dependent on C1 both in vitro and in vivo. These two promoters share a common TTGCN6TTGC/T motif in their -35 regions, the same motif recognized by the analogous phage lambda transcription activator protein cII. P22 C1 protein bound selectively to this region and centered on the TTGC repeat motif. Binding and transcription experiments demonstrated that the two promoters respond coordinately to C1 activation. Last, in contrast to lambda cII, the C1 protein exhibited little cooperativity with Escherichia coli RNA polymerase for DNA binding, but because of its stronger inherent binding ability, achieved an overall promoter affinity similar to that observed for cII at its cognate promoter signals.

Amino Acid Sequence↗
PubMed1992

An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4.

A lambda phage expression methodology was adapted to dissect protein/ligand interactions efficiently through the creation and rapid screening of large numbers of mutants. Here we describe the method and its specific application to the interaction between the external envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, and the human cell surface protein CD4. Random substitutions were introduced throughout the gp120 binding region (amino acids 38-62) in the amino-terminal domain of CD4 by oligonucleotide mutagenesis. These mutations were expressed within phage plaques and directly screened for their effect on binding of gp120 using a modified phage plaque lift procedure. Plaques showing increased, decreased, and no effect on binding were identified and mutations were verified by sequence analysis. In this manner, 25 unique mutations were identified that altered CD4 binding to gp120. A new site was identified at which mutations reduced binding to gp120 and several novel amino acid substitutions were defined at sites previously implicated in binding. Of particular interest, this in vitro genetic approach identified a mutation which significantly increased binding to gp120. The phenotypes of several of these mutants were further characterized by quantitative measurement of their binding affinity. The results confirmed the accuracy of the phenotypic selection and demonstrated that the sensitivity of the system allowed detection of a 3-4-fold increase or decrease in affinity. In the context of the recently determined atomic structure of CD4, these results further implicate residues in the CDR2-like region and in an adjacent loop in recognition of gp120. This methodology should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.

Amino Acid Sequence↗
PubMed1992

Endometrial morphology and hormonal profiles in in vitro fertilization patients.

Endometrial biopsy was performed in 27 infertile women participating in the IVF program. Their mean age was 31.8 years, 33% of the women being over 35 years old. The average duration of infertility was 6.9 years. The superovulation protocol consisted of hMG/hCG in 5 cases, of combined GnRH analog/gonadotropin therapy in 20 women, and 2 patients received combined contraceptive pill/gonadotropin treatment. Judging by hormonal profiles, follicular growth rate and number of oocytes retrieved, the response to stimulation was normal. The mean estradiol (E2) levels increased from 132.7 pg/ml on day -5 (SEM = 9.67) to 1272 pg/ml (SEM = 103.7) on the day of hCG administration and to 1813 pg/ml (SEM = 209.6) 1 day later. One day before the hCG application, the mean progesterone and LH levels were 1.34 ng/ml and 8.38 IU/ml, respectively. Only one patient had clinical hyperstimulation syndrome. Ova were harvested in all women, the mean number of oocytes being 7.7 (SEM = 0.83) per patient. In all 27 cases lack of fertilization or faulty ovum cleavage were observed. Thus, an endometrial biopsy (EB) was performed 72 h after oocytes retrieval. The mean estrogen and progesterone levels on the EB day were 610.9 pg/ml (SEM = 78.44) and 45.4 ng/ml (SEM = 7.53), respectively. Histologic examination of the endometrium showed normal secretory endometrium consistent with day 16-17 of spontaneous ovulatory cycle. Two women who received combined contraceptive pills/gonadotropin therapy showed inactive endometrium with subnuclear vacuoles and decidual reaction in the stroma similar to that observed in women on estrogen-progestin birth control medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression.

In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.

Amino Acid Sequence↗
PubMed1992

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function.

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a more in vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%-h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used for in vitro research and testing and is also being tested in clinical applications.

Biological Dressings↗
PubMed1992

Developmental changes in the heterocellular epidermis of Pelobates syriacus integument.

Changes in characteristic components of the skin epidermis of the large tadpole of Pelobates syriacus were studied throughout its development. The fate of two specific cells in the skin epidermis was followed, from the young tadpole to the adult was studied. It was found that flask-shaped type cells in the tadpole epidermis which are PAS-positive, stain with peanut lectin (PNA). There is no detectable band 3 in the premetamorphosed stages, and mitochondria-rich cells are very rare. This pattern of staining changes completely upon metamorphosis: the PAS-positive cells, specific to the tadpole epidermis disappear, and the mitochondria-rich (MR) cells in the adult skin epithelium react with polyclonal anti-band 3 antibody. Western blot analysis showed the presence of a band 3-like protein of about 95 kDa, only in the adult epithelial extract, corroborating the immunocytochemical observations. The finding of the presence of band 3-like protein in the MR cells of Pelobates, is similar to the observations made in the skin of other amphibian species. On the other hand, the binding of peanut lectin to MR cells is species-specific, since it does not react with the MR cells in the skin epithelium of Pelobates syriacus.

Animals↗
PubMed1992

Patient-controlled analgesia.

Acute postoperative pain often is unrelieved by conventional intramuscular and intravenous analgesic therapy. Difficulties arise from determining severity of pain, appropriate dosing, dosing intervals, health professional attitudes towards opioid administration, and the pharmacodynamics and pharmacokinetics of analgesics themselves. Patient-controlled analgesia (PCA) is a safe, effective, and increasingly popular method used to treat postoperative pain that may be applicable for patients after major oral and maxillofacial surgical procedures.

Analgesia, Patient-Controlled↗
PubMed1992

Initial testing of a novel urine culture device.

The Diaslide urine culture device consists of a hinged case containing two opposing agar media separated by a sampler with a handle at one end and two bent sampler tips at the opposite end. The tips of the sampler are first dipped into the urine. The sampler is then pulled out through the casing, simultaneously inoculating both agar surfaces with a streaking dilution. As a result, individual colonies can be observed even when bacterial concentrations exceed 10(6) CFU/ml. The number of colonies on the Diaslide correlated linearly with CFU per milliliter as determined by dilution plating. The clinical performance of the Diaslide was compared with those of ordinary dipslides and conventional cultures with a sample of 473 prescreened hospital urine specimens. The sensitivity, specificity, and positive predictive value of Diaslide versus those of culture at the 10(4)-CFU/ml cutoff level were 97.5, 98.3, and 98.3%, respectively, compared with 98.8, 95.7, and 97.2%, respectively, for dipslide versus culture. Similar results were found at the 10(5)-CFU/ml cutoff level. Only 5.5% of the Diaslides required subculturing, compared with 14.7 and 9.4% of the dipslides and conventional cultures, respectively. The Diaslide proved more convenient than an ordinary dipslide for sampling low volumes of urine. These data suggest that the Diaslide is a simple, effective device for culturing of urine specimens.

Bacteria↗
PubMed1992

Acetazolamide and furosemide attenuate asthma induced by hyperventilation of cold, dry air.

We investigated the assumption that the efficacy of inhaled diuretics in asthma is dependent upon inhibition of the Na+/K+/2Cl- cotransporter. We compared the protective effect of acetazolamide, a diuretic without significant effect on the loop cotransporter, with the protection provided by inhaled furosemide in a cold, dry air hyperventilation model of asthma. Seven asthmatic subjects underwent a baseline bronchial challenge and then received a nebulized dose of 80 mg of furosemide or 500 mg of acetazolamide or saline placebo in a randomized, double-blind, placebo-controlled crossover design. Repeat challenges were performed immediately and at 2 and 4 h postnebulization. Acetazolamide caused a 47.2% increase in the amount of cold, dry air required to reduce the FEV1, by 20% (expressed in terms of respiratory heat loss as PD20RHL), from 0.79 multiplied or divided by (x/divided by) 1.13 kcal/min (geometric mean x/divided by geometric SEM) at baseline to 1.17 x/divided by 1.09 kcal/min postnebulization (p < 0.025). Furosemide increased the geometric mean PD20RHL by 53.9%, from 0.86 x/divided by 1.12 kcal/min to 1.33 x/divided by 1.12 kcal/min (p < 0.001). There was no significant change after placebo inhalation (0.81 x/divided by 1.15 kcal/min versus 0.87 x/divided by 1.10 kcal/min, NS). Airway responsiveness had returned to baseline by 2 h postnebulization on all 3 days. Furosemide also caused bronchodilatation, producing a 14.1% rise in the mean FEV1 (p < 0.005 versus prenebulization), whereas neither acetazolamide nor placebo altered airway tone significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed1992

Day-long reduction of oral malodor by a two-phase oil:water mouthrinse as compared to chlorhexidine and placebo rinses.

Few scientific investigations have addressed the ability of mouthrinses to reduce oral malodor for periods longer than 3 hours. In the present report, we have employed simple, recently described techniques to assess the day-long reduction in oral malodor of a novel 2-phase oil:water mouthrinse (TPM), as compared to a corresponding placebo rinse, and to a commercial 0.2% chlorhexidine mouthrinse. Sixty dental students were divided randomly into 3 groups, and instructed to use one of the rinses prior to bedtime and the following morning. Measurements carried out in the late afternoon, about 8 to 10 hours following rinsing, were compared with baseline measurements carried out in the late afternoon of the previous day. Volatile sulphide levels were measured using a portable industrial sulphide monitor. Microbial levels were estimated using a simple rinsing technique employing sterilized milk. These quantitative techniques were corroborated by organoleptic (hedonic) ratings of a single odor judge. Both TPM and chlorhexidine brought about significant decreases in volatile sulphides (P less than 0.05) as compared to the placebo group. These results were corroborated by the organoleptic data. Similarly, both chlorhexidine and TPM were highly effective in reducing microbial levels as measured by the rinsing technique, in comparison to the placebo group. Chlorhexidine appeared to be more effective than TPM in all measurement categories, although only in the case of microbial activity was there a significant (P less than 0.05) difference between the two groups.

Bacteria↗