PubMed Health⌕ Search

Biomedical subjects

M Wisniewski

Publications and source records attributed to M Wisniewski.

At least 19 recordsLinked to original sources
PubMed2004

The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves.

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.

Acclimatization↗
PubMed2004

Molecular interactions in collagen and chitosan blends.

Molecular interactions between collagen and chitosan (CC) have the potential to produce biocomposites with novel properties. We have characterised the molecular interactions in CC complexes by viscometry, wide angle X-ray scattering and Fourier transform infrared spectroscopy. It was found that CC are miscible at the molecular level and exhibit interactions between the components; X-ray diffraction of CC blends indicate that the collagen helix structure is lost in CC films with increasing chitosan content. Non-linear viscometic behaviour with decreasing chitosan content is interpreted as evidence of a third structural phase formed as a complex of CC. The blending of collagen with chitosan gives the possibility of producing new bespoke materials for potential biomedical applications.

Biocompatible Materials↗
PubMed2001

The Y181C mutant of HIV-1 reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors alters the size distribution of RNase H cleavages.

We investigated the effects of the nonnucleoside reverse transcriptase inhibitor-resistant mutant Y181C on RNA 5'-end-directed RNase H cleavage by HIV-1 reverse transcriptase, using an RNA.DNA hybrid in which a radiolabeled RNA 5' end was recessed. Y181C produced a higher ratio of secondary (9 nucleotide long) to primary (18 nucleotide long) products than wild type. When the RNA was 3'-end-labeled, Y181C generated a long product, which results when secondary cleavage precedes the primary. When using an RNA.DNA hybrid in which the labeled RNA 5' end and DNA 3' end were flush, formation of secondary product by both enzymes was inhibited. Under these conditions, Y181C cleaved closer to the RNA 5' end than wild type. Studies with this substrate labeled at the RNA 3' end showed that Y181C is no more likely than wild type to cleave toward the RNA 3' end. Thus, Y181C RT has a strong preference to cleave in the direction of the RNA 5' end even when secondary cleavage is prevented, resulting in a disruption of the normal sequence of primary followed by secondary cleavages.

3' Untranslated Regions↗
PubMed2001

A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance.

Lithium acetate transformation and electroporation were applied to the biocontrol yeast, Candida oleophila. The hygromycin B resistance gene, flanked by the phosphoglycerate kinase promoter and terminator of Candida tropicalis, served as the genetic selection marker. The transformation efficiency of electroporation was almost 400 times more efficient than that of the lithium acetate method. While incorporation of DNA, flanked by a sequence endogenous to C. oleophila, transpired apparently by homologous recombination, the integration of DNA (that did not contain C. oleophila DNA) occurred at random. Whereas transformants were observed with a linear segment of the plasmid, none were detected with the undigested plasmid. This system provides both a tool for the molecular analysis of the biocontrol mechanism of C. oleophila and a means of tagging C. oleophila for field studies.

Anti-Bacterial Agents↗
PubMed2001

Improving inpatient antibiotic prescribing: insights from participation in a national collaborative.

BACKGROUND: Increasing rates of antimicrobial resistance and the role antibiotic overuse plays in contributing to this problem have been widely documented and have prompted appeals to change prescribing practices. How to actually achieve such changes represents a major challenge. As part of the Institute for Healthcare Improvement (IHI) Breakthrough Series project Improving Prescribing Practices (IPP), in 1997-1998, Cook County Hospital (Chicago) worked with other institutions that chose antibiotics as their focus in this national collaborative. PRACTICAL SUGGESTIONS: Practical suggestions are offered within six categories--adopting a general approach to improving antibiotic prescribing (marshalling credible evidence, addressing physician concerns and skepticism, and removing barriers to make it easier for prescribers to change); rethinking guidelines (providing syndrome-based guidance, revising national guidelines for local use, defining scenarios in which drug(s) can be safely withheld, offering alternatives, prospectively resolving conflicts over drug of choice and empiric regimens, and defining situations in which immediate treatment is and is not critical); getting the message out and changes implemented (using antibiotic order forms, computer order entry, and infectious disease specialist consultation); building viable linkages to leverage change (bridging disciplines); improving measurement; and promoting nondrug strategies and patients' roles in treating and preventing infection. CONCLUSION: Antimicrobials are unique, being the only class of drug therapy that affects not only the patient to whom it is prescribed but other current and future patients as well. Institutions therefore have a special responsibility to ensure their efficient and judicious use. It is often easier to prescribe antibiotics than to exercise restraint.

Academies and Institutes↗
PubMed2001

Use of a long-acting inhaled beta2-adrenergic agonist, salmeterol xinafoate, in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a condition in which continuous bronchodilation may have clinical advantages. This study evaluated salmeterol, a beta-agonist bronchodilator with a duration of action substantially longer than that of short-acting beta-agonists, compared with ipratropium, an anticholinergic bronchodilator, and placebo in patients with COPD. Four hundred and five patients with COPD received either salmeterol 42 microg twice daily, ipratropium bromide 36 microg four times daily, or placebo for 12 wk in this randomized, double-blind, parallel-group study. Patients were stratified on the basis of bronchodilator response to albuterol (> 12% and > 200-ml improvement) and were randomized within each stratum. Bronchodilator response was measured over 12 h four times during the treatment period. Salmeterol provided similar maximal bronchodilatation to ipratropium but had a longer duration of action and a more constant bronchodilatory effect with no evidence of bronchodilator tolerance. Both active treatments were well tolerated. Salmeterol was an effective bronchodilator with a consistent effect over this 12-wk study in patients with COPD, including those "unresponsive" to albuterol. The long duration of action of salmeterol offers the advantage of twice daily dosing compared with the required four times a day dosing with ipratropium.

Adrenergic beta-Agonists↗
PubMed2000

The sequential mechanism of HIV reverse transcriptase RNase H.

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.

Base Sequence↗
PubMed2000

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H.

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5' end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5' end of the RNA to make a secondary cut creating two products 8-9 nt long. RT then binds the new 5' end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3' end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.

Base Sequence↗
PubMed2000

Inorganic lead and calcium interact positively in activation of calmodulin.

Calmodulin is a ubiquitous calcium-binding protein that mediates many of the intracellular actions of Ca2+ ions. The calcium-binding sites of calmodulin consist of four EF-hand motifs; full activation of calmodulin normally occurs when all four sites are occupied by Ca2+. Inorganic lead (PY2+) has been shown to activate calmodulin at total lead concentrations similar to the concentrations of Ca2+ required for activation (Goldstein and Ar, 1983; Habermann et al., 1983), but the free Pb2+ concentrations required for calmodulin activation have not been determined. In addition, it is possible that activation may occur with different sites occupied by different divalent cations, for example Ca2+ and Pb2+. We investigated the ability of free Pb2+, alone or in combination with Ca2+, to activate calmodulin. In aqueous media, N-phenyl-1-naphthylamine (NPN) and 8-anilino-1-naphthalenesulfonate (ANS) show increased fluorescence when bound to hydrophobic regions of proteins. This increased fluorescence has been used to monitor the conformational change that occurs during calmodulin activation (LaPorte et al., 1980). In the presence of calmodulin, both Ca2+ and Pb2+ stimulated increased fluorescence of NPN and ANS. Threshold and EC50 free metal concentrations were approximately 100 nM and 450-500 nM, respectively, for Ca2+ and 100 pM and 400-550 pM, respectively, for Pb2+. Fluorescence was enhanced by combinations of low concentrations of free Ca2+ and Pb2+; for example, as little as 20 pM free Pb2+ enhanced fluorescence in combination with 200 nM free Ca2+. The activity of the PDE1 isoform of cyclic nucleotide phosphodiesterase is stimulated by Ca2+/calmodulin (Wang et al., 1990). In the presence of calmodulin, we found that Ca2+ and Pb2+ activated calmodulin-stimulated PDE activity, with threshold and EC50 free metal concentrations of approximately 200 nM and 1200 nM, respectively, for Ca2+ and 300 pM and 430 pM, respectively, for Pb2+. PDE activity was stimulated by combinations of Ca2+ and Pb2+. For example, with 100 nM free Ca2+, as little as 50 to 100 pM free Pb2+ further stimulated PDE activity; with 1000 nM free Ca2+, 20 to 50 pM free Pb2+ further stimulated PDE activity. Isobolographic analysis indicated that stimulation of PDE by Ca2+ and Pb2+ was additive. These results show that concentrations of free Pb2+ as low as 100 to 300 pM activate calmodulin and that, in the presence of physiological concentrations of free Ca2+, Pb2+ can activate calmodulin at concentrations below 50 pM. The intracellular free Ca2+ concentration in Ca2+ "hot spots," for example near sites of influx through Ca2+-permeable plasma membrane channels, can reach dozens of pM, with the free Ca2+ concentration decreasing rapidly with distance from the source of the hot spot. Our results suggest that picomolar concentrations of intracellular free Pb2+ should expand both the effective amplitude and volume of Ca2+ hot spots with respect to calmodulin activation, and thus may amplify intracellular Ca2+ signaling in lead-exposed cells.

Calcium↗
PubMed1999

Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity.

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.

Base Pair Mismatch↗
PubMed1998

Control of initiation of viral plus strand DNA synthesis by HIV reverse transcriptase.

Human immunodeficiency virus reverse transcribes its single-stranded RNA genome making a DNA copy. As synthesis proceeds, the RNA is simultaneously degraded to oligomers; one of these, the polypurine tract, primes synthesis of a plus strand DNA. The viral reverse transcriptase (RT) degrades all of the non-polypurine tract oligomers. We show that unlike other DNA polymerases the retroviral RT can bind either end of an annealed RNA primer, the 5'-end for degradation and the 3'-end for synthesis. The competition between the two binding modes at any primer determines whether it will be extended or degraded. The 5'-end binding can be suppressed in at least two ways. The sequence of the primer can be such that a region at the 5'-end is unannealed or a DNA primer can be annealed just adjacent to the 5'-end of the RNA primer. This promotes binding of RT to the RNA 3'-end, allowing a primer that would normally be degraded to be extended. Implications for human immunodeficiency virus replication and antiviral therapy are discussed.

Binding Sites↗
PubMed1997

Mutations within the primer grip region of HIV-1 reverse transcriptase result in loss of RNase H function.

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3'-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5'-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5'-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3'-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5'-RNA-directed RNase H catalysis.

Binding Sites↗
PubMed1997

Effects of near-lethal heat stress on bud break, heat-shock proteins and ubiquitin in dormant poplar (Populus nigra Charkowiensis x P. nigra incrassata).

We assessed the effects of near-lethal heat stress on bud break, heat-shock proteins (HSPs) and ubiquitin in hybrid poplar (Populus nigra (L.) Charkowiensis x P. nigra (L.) incrassata). Shoots, with 10-15 buds each, were collected from September to March and exposed to temperatures between 20 and 60 degrees C for 2 h. Shoots were then placed in a greenhouse at 18-22 degrees C with supplemental light and cumulative bud break was recorded over a 4-week period. Samples of bud tissues were collected during and up to 96 h after heat treatment for protein analysis. De novo synthesis of proteins was monitored by exposing excised buds to [(35)S]-methionine for 3 h before, during, or after heat treatment. Heat treatments of 40-45 degrees C resulted in both a release from endodormancy and a decrease in thermal units needed for bud break during ecodormancy. The response to near-lethal heat stress was complex and was affected by intrinsic thermal sensitivity. Heat treatments were least effective during August and became progressively more effective as endodormancy progressed. In the later stages of ecodormancy, a heat treatment of 45 degrees C either inhibited bud break or killed the buds. Although temperatures of 42.5 to 45 degrees C inhibited incorporation of [(35)S]-methionine into proteins for at least 48 h, several HSPs were synthesized in response to temperatures of 40-45 degrees C. Immunoblots indicated that one of the heat-induced proteins was immunologically related to HSP70. Increases in free and conjugated forms of ubiquitin were also observed in response to heat treatment. Production of HSPs and ubiquitin, however, was not consistently associated with the heat treatments that induced the highest percentage of bud break. The roles of heat-induced protein degradation, HSPs, and ubiquitin in overcoming dormancy by near-lethal heat stress are discussed.

Journal Article↗
PubMed1996

Misincorporation by HIV-1 reverse transcriptase promotes recombination via strand transfer synthesis.

Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.

DNA Primers↗
PubMed1996

Fluticasone propionate aqueous nasal spray compared with terfenadine tablets in the treatment of seasonal allergic rhinitis.

BACKGROUND: Comparative studies with topical corticosteroids and antihistamines for treatment of allergic rhinitis have not always demonstrated clear distinctions between the two on the basis of therapeutic efficacy. OBJECTIVE: This study was designed to compare the efficacy and tolerability of fluticasone propionate aqueous nasal spray with those of terfenadine in the treatment of seasonal allergic rhinitis. METHODS: Three hundred forty-eight patients with allergic rhinitis were given fluticasone propionate aqueous nasal spray (200 micrograms once daily), terfenadine tablets (60 mg twice daily), or placebo for 4 weeks in a multicenter, randomized, double-blind, double-dummy, parallel-group study. RESULTS: Clinician-rated total nasal symptom scores after 1, 2, 3, and 4 weeks of therapy and patient-rated total nasal symptom scores throughout treatment were significantly (p <0.05) lower in the fluticasone propionate group compared with the terfenadine group or the placebo group. Terfenadine was not statistically different from placebo on the basis of clinician-related nasal symptom scores, except for sneezing. Total nasal airflow, measured by rhinomanometry, significantly (p <0.05) improved in the fluticasone propionate group compared with the terfenadine group or the placebo group. More fluticasone propionate-treated patients compared with placebo-treated patients had reduced nasal mucosal eosinophil counts after 4 weeks of therapy (p <0.05). No serious or unusual drug-related adverse events were reported. Morning plasma cortisol concentrations after 4 weeks of therapy did not differ among groups. CONCLUSION: Fluticasone propionate aqueous nasal spray is more effective than terfenadine tablets for treatment of seasonal allergic rhinitis.

Administration, Intranasal↗
PubMed1996

The effect of prior dural puncture on cisternal cerebrospinal fluid morphine concentrations in sheep after administration of lumbar epidural morphine.

Combined spinal epidural anesthesia has become increasingly popular as a method of providing rapid onset of analgesia or surgical block with access for further administration of analgesics or anesthetics. No in vivo studies have evaluated the relationship between dural puncture and drug transfer from the epidural space to the cerebrospinal fluid (CSF). To determine whether morphine administered in the epidural space adjacent to a dural puncture results in increased CSF concentrations at the cisterna magna (CM), 12 adult ewes were studied. Each animal was assigned to one of three groups. Animals in Group 1 served as a control and received no dural puncture. Animals in Group 2 received a dural puncture with a 25-gauge (G) Whitacre needle, while Group 3 animals received a dural puncture with an 18-G Tuohy needle. One hour after dural puncture, each animal was given epidural morphine, 0.2 mg/kg. Six hours after the administration of epidural morphine, CSF from the CM was sampled and analyzed by gas chromatography-mass spectrometry for morphine concentration. The mean morphine concentration at the CM for Group 1 (control) was 22 +/- 12 ng/mL, whereas animals with 25-G and 18-G dural punctures had concentrations of 154 +/- 32 ng/mL and 405 +/- 53 ng/mL, respectively (P = 0.0005). These data demonstrate that a significant increase in CSF morphine concentration at the brainstem will occur when lumbar epidural morphine is administered adjacent to a dural puncture. Furthermore, the increase in CSF morphine concentration is positively correlated with the size of the needle producing the dural puncture. These findings highlight the potential for delayed respiratory depression when epidural opiate administration follows a dural puncture.

Analgesics, Opioid↗
PubMed1996

Beneficial effect of proteases on allograft arteriosclerosis in a rat aortic model.

Recently it has been shown that protease therapy ameliorates certain immune-mediated diseases. Thus we studied the effect of administration of a protease mixture on aortic transplant arteriosclerosis in rats. Segments of abdominal aorta from SHR strain were transplanted orthotopically into WKY recipients. Two groups of allografted rats were used. One group (n = 8) was treated with daily intraperitoneal injections of 12 mg of a protease formulation containing trypsin, bromelain and rutosid, and another group (n = 8) with placebo. Eight WKY rats were transplanted with syngenic aortas and treated with placebo. After 8 weeks, structural changes of the grafted segment were evaluated by morphometric analysis of formalin-fixed sections with specific stains. In untreated allografts there was a marked intimal thickening, medial necrosis with disruption of elastic fibres, and inflammatory infiltrates in the adventitia. Administration of proteases inhibited formation of neointima by 59.0% when cross-sectional areas were compared (80+/-11 versus 195+/-11 microm2, P<0.01; protease-versus placebo-treated allograft recipients respectively) and decreased medial injury as estimated by the integrity of elastic fibres and smooth-muscle cell density. Thus, in an experimental model of rat aortic allograft, protease administration ameliorates rejection-induced arterial wall remodelling.

Animals↗