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Biomedical subjects

L Weiner

Publications and source records attributed to L Weiner.

At least 37 records · Page 2Linked to original sources
PubMed1998

A spectrophotometric assay for allicin and alliinase (Alliin lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.

Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure alliinase activity and for the activity of the enzyme in crude garlic extracts.

Carbon-Sulfur Lyases↗
PubMed1998

Liposome-catalyzed unfolding of acetylcholinesterase from Bungarus fasciatus.

The kinetics of thermal inactivation of acetylcholinesterase from the venom of the snake, Bungarus fasciatus, were studied at 45-54 degrees C. An Arrhenius plot reveals an activation energy of 113 kcal/mol. The thermally denatured enzyme displays the spectroscopic characteristics of a partially unfolded 'molten globule' state. The rate of thermal denaturation is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, the energy barrier for the transition being lowered from 113 to 52 kcal/mol. In contrast to our findings for partially unfolded Torpedo californica acetylcholinesterase [Shin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2848-2852], the thermally denatured snake enzyme does not remain bound to the liposomes but is released after unfolding and subsequently aggregates. The liposomes thus serve as catalysts for unfolding of the snake enzyme, and its rate of unfolding in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 8 x 10(-7) M). The phospholipid vesicles display a catalytic turnover number of kcat approximately 4 min-1, assuming 15 binding sites per vesicle for the snake acetylcholinesterase.

Acetylcholinesterase↗
PubMed1998

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

Alcohol Dehydrogenase↗
PubMed1998

Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.

Basonuclin, a zinc-finger protein, is found in stratified squamous epithelia and hair follicles. In the basal keratinocytes of mouse epidermis, basonuclin is detected mainly in the cytoplasm. During the development of murine hair follicles, this protein concentrates in the nuclei of the basal cells that form the primary hair germs. As follicle morphogenesis proceeds, the epithelial cells possessing nuclear basonuclin invade the dermis and surround the follicular papilla. In mature anagen follicles, nuclear basonuclin is principally restricted to the basal layers of the outer root sheath and bulbar matrix; these regions are known to contain cells capable of proliferation, and to lack the features of terminal differentiation. During catagen, the compartment of cells containing nuclear basonuclin regresses, and in telogen, only a small number of these cells remain to form the secondary hair germ at the follicle base. During the next anagen, this basonuclin-containing population expands and regenerates the hair-producing portion of the follicle. It is concluded that in all hair cycles, the transient segment of the follicle originates from germinative cells possessing nuclear basonuclin.

Animals↗
PubMed1997

Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase.

Cases of familial amyotrophic lateral sclerosis (fALS; a neurodegenerative disorder) have been reported in which the gene for Cu/Zn superoxide dismutase (CuZnSOD) was mutated. Several studies with the fALS mutant CuZnSOD in transgenic mice and cells showed that the fALS mutations act through an as yet undefined dominant gain-of-function mechanism. Wild-type CuZnSOD catalyzes the dismutation of superoxide (O(2)(-).) but also produces hydroxyl radicals (.OH) with H(2)O(2) as substrate. Two laboratories have recently demonstrated that the .OH production ability was preferentially enhanced by the fALS mutant CuZnSOD, suggesting that this might be the function gained in fALS. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of wild-type CuZnSOD was also associated with increased .OH production and impaired muscle function. Enhanced formation of .OH was detected, by spin trapping, in brain and muscle extracts of the Tg-CuZnSOD mice. Three independently derived Tg-CuZnSOD lines showed muscle abnormalities, reflected by altered electromyography (EMG) and diminished performance in the rope grip test. After treatment with paraquat (PQ), a widely used herbicide and O(2)(-).-generating compound, muscle disability significantly deteriorated in Tg-CuZnSOD mice but not in control mice. The results indicate that elevated levels of CuZnSOD cause indigenous long-term oxidative stress leading to impairment of muscle function. These findings may provide valuable clues about the concurred role of indigenous oxidative stress and exogenous agents in the etiology of sporadic ALS and several other neurodegenerative diseases in which a specific subset of neurons is affected.

Animals↗
PubMed1997

Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer.

Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a "molten globule" upon mild thermal denaturation or upon chemical modification of a single non-conserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.

Acetylcholinesterase↗
PubMed1997

Vertical transmission of HIV in New York State: a basis for statewide testing of newborns.

Infants (n = 313) of HIV-infected mothers were enrolled (mean age 1.9 weeks, range 0-8 weeks) in a 3-year prospective study of vertical transmission. Fifty-six infants (17.9%) had laboratory and clinical evidence of HIV infection. Polymerase chain reaction (PCR) provided early and reliable identification of infected infants. Thirty-one of the 56 infected infants had specimens submitted when the infants were 4 weeks of age or less and 30 (97%) tested PCR positive. This percentage increased to 100% by 8 weeks of age when 51 of the 56 infected infants had specimens tested for that time period. Immune complex dissociation (ICD) antigen testing was a sensitive method for diagnosis of infection but only in infants older than 1 month. p24 antigen testing, although free of false positives, is less sensitive than either of the other methods. Among surrogate markers of HIV infection, elevation of soluble CD8 levels precedes an increase in immunoglobulin levels or a decline in CD4 T lymphocytes. Vertical transmission is significantly lower in Central and Western New York State than other regions. Transmission is significantly higher in low birthweight babies and in infants whose mothers have CD4 counts < 500. This study provided the basis for establishing a Pediatric HIV PCR Testing Service for the early diagnosis of HIV infection in neonates.

Blotting, Western↗
PubMed1996

Two partially unfolded states of Torpedo californica acetylcholinesterase.

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.

Acetylcholinesterase↗
PubMed1996

Hypertensive, hypervolemic, hemodilutional therapy for aneurysmal subarachnoid hemorrhage. Is it efficacious? No.

Many neurosurgeons routinely use hypertensive, hypervolemic, hemodilutional, or hyperdynamic therapy (HT) in some form to prevent or to treat vasospasm. Despite the widespread use of this therapy during the past 20 years, however, there are no randomized, prospective, controlled clinical studies demonstrating that HT improves the short- or long-term neurologic outcome or survival after subarachnoid hemorrhage. Guidelines need to be developed to standardize the clinical application of HT, and well-controlled, prospective, randomized clinical trials must be conducted before HT can become an accepted treatment for vasospasm.

Blood Pressure↗
PubMed1996

Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments.

Upon exposure to oxygen, Escherichia coli increases the expression of enzymes essential for aerobic respiration, such as components of the TCA cycle and terminal oxidase complexes. This increase requires the elimination of repression mediated by the Arc regulatory system under anaerobic conditions. Coordinately, the synthesis of enzymes that function in anaerobic processes such as fermentation decreases, partly due to the inactivation of the transcription factor Fnr. E. coli is thus able to adjust the levels of respiratory enzymes to fit its environmental circumstances, and in this case, reduces the production of the less energy efficient fermentation enzymes in favor of the aerobic pathways. In contrast to the advantage in energy production, aerobiosis brings a disadvantage to E. coli: the production of reactive oxygen species (ROS), i.e. superoxide anion radical (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (.OH). These byproducts of aerobic respiration damage many biological molecules, including DNA, proteins, and lipids. To alleviate the toxicity of these compounds, E. coli induces the synthesis of protective enzymes, such as Mn-dependent superoxide dismutase (SodA) and catalase I (HP I), and this induction is controlled by the regulatory proteins SoxRS, OxyR, and ArcAB. Thus, ArcAB, Fnr, SoxRS, and OxyR function in concert so that E. coli can optimize its energy production and growth rate. Fnr and SoxRS are cytoplasmic, DNA-binding proteins, and these regulatory systems utilize iron-sulfur clusters as cofactors which may directly sense the redox environment. OxyR is also a cytoplasmic, DNA-binding protein, and appears to respond to redox potential through the oxidation state of a specific cysteine residue. In the ArcAB system (which belongs to the family of two-component regulatory systems), ArcB, a membrane protein, functions as the sensor, and ArcA, a DNA-binding protein, directly controls target gene expression. Under anaerobic conditions, ArcB undergoes autophosphorylation and transphosphorylates ArcA, stimulating ArcA's DNA-binding activity. During aerobic growth, the transphosphorylation of ArcA does not occur. In this signal transduction mechanism, the ArcB C-terminal or "receiver" domain plays a critical role; that is, it stimulates or abolishes the transphosphorylation depending on the metabolic state of the cell, which in turn is influenced by the availability of oxygen. E. coli thus employs at least four global regulatory systems which monitor the cellular oxidative/metabolic conditions, and adjust the expression of more than 70 operons to give the organism a better aerobic life.

Adaptation, Biological↗
PubMed1996

Identification, nucleotide sequence, and characterization of PspF, the transcriptional activator of the Escherichia coli stress-induced psp operon.

The phage shock protein (psp) operon (pspABCE) of Escherichia coli is strongly induced in response to a variety of stressful conditions or agents such as filamentous phage infection, ethanol treatment, osmotic shock, heat shock, and prolonged incubation in stationary phase. Transcription of the psp operon is driven from a sigma54 promoter and stimulated by integration host factor. We report here the identification of a transcriptional activator gene, designated pspF, which controls expression of the psp operon in E. coli. The pspF gene was identified by random miniTn10-tet transposon mutagenesis. Insertion of the transposon into the pspF gene abolished sigma54-dependent induction of the psp operon. The pspF gene is closely linked to the psp operon and is divergently transcribed from one major and two minor sigma 70 promoters, pspF encodes a 37-kDa protein which belongs to the enhancer-binding protein family of sigma54 transcriptional activators. PspF contains a catalytic domain, which in other sigma54 activators would be the central domain, and a C-terminal DNA-binding domain but entirely lacks an N-terminal regulatory domain and is constitutively active. The insertion mutant pspF::mTn10-tet (pspF877) encodes a truncated protein (PspF delta HTH) that lacks the DNA-binding helix-turn-helix (HTH) motif. Although the central catalytic domain is intact, PspF delta HTH at physiological concentration cannot activate psp expression. In the absence of inducing stimuli, multicopy-plasmid-borne PspF or PspF delta HTH overcomes repression of the psp operon mediated by the negative regulator PspA.

Amino Acid Sequence↗
PubMed1995

Analysis of the proteins and cis-acting elements regulating the stress-induced phage shock protein operon.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly induced by adverse environmental conditions. Expression is controlled principally at the transcriptional level, and transcription is directed by the sigma factor sigma 54. PspB and PspC are required for high-level psp expression during osmotic shock, ethanol treatment and f1 infection, but heat-induced expression is independent of these proteins. We report here that the promoter region contains an upstream activation sequence (UAS) that is required for psp induction and has the enhancer-like ability to activate at a distance. A DNA-binding activity is detected in crude protein extracts that is dependent on the UAS and induced by heat shock. We further show that integration host factor (IHF) binds in vitro to a site between the UAS and sigma 54 recognition sequence. In bacteria lacking IHF, psp expression is substantially reduced in response to high temperature and ethanol. During osmotic shock in contrast, psp expression is only weakly stimulated by IHF, and IHF mutants can strongly induce the operon. The dependence of psp expression on IHF varies with the inducing condition, but does not correlate with dependence on PspB and PspC, indicating distinct, agent-specific activation mechanisms.

Bacterial Proteins↗
PubMed1995

Physician awareness of fetal alcohol syndrome: a survey of pediatricians and general practitioners.

OBJECTIVE: To determine the knowledge, clinical experience and perceived needs for resource materials of Saskatchewan physicians in regard to fetal alcohol syndrome (FAS) and alcohol-related birth defects. DESIGN: Mailed survey. SETTING: Saskatchewan. PARTICIPANTS: All 48 pediatricians and half (394) of the family physicians (FPs) and general practitioners (GPs) practising in Saskatchewan received a questionnaire. The numbers of physicians who completed it were 24 and 249 respectively. RESULTS: The pediatricians were more likely than the other physicians to be aware of FAS and to have diagnosed at least one case of FAS. Among the FPs and GPs, the year of graduation from medical school was a significant factor in their knowledge of FAS and their diagnostic practices. Those who graduated before 1974, the year FAS was first described in the medical literature, were less likely than the more recent graduates to be aware of FAS and to ask their patients about alcohol use during pregnancy but were more likely to feel comfortable discussing alcohol-related issues in families. All of the groups reported a need for more information about FAS and for resources on alcohol-related issues in general. CONCLUSIONS: Saskatchewan physicians are aware of FAS but have expressed a need for more information about FAS, particularly for parents, as well as physician training materials and information about where to refer patients with FAS and parents with alcohol-related problems.

Adult↗
PubMed1995

Irreversible thermal denaturation of Torpedo californica acetylcholinesterase.

Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.

Acetylcholinesterase↗
PubMed1995

Community mobilization to reduce point-of-purchase advertising of tobacco products.

This project was designed to address the problem of point-of-purchase tobacco advertising through media advocacy and community mobilization. Precampaign assessment revealed a considerable amount and density of tobacco advertising and promotions in more than 100 stores sampled in San Jose, California. After sharing the results with community activists and other residents, a community mobilization campaign was instigated to capitalize on an existing sign control ordinance that limits store window coverage and sidewalk signs. Through presentations and media advocacy efforts, community residents were mobilized to file complaints with the city's code enforcement office when neighborhood stores were shown to be noncompliant with ordinance provisions. Relative to the baseline, significant reductions in campaign-related tobacco advertising variables were seen in the San Jose stores after the sign law campaign. No changes were seen in four smaller reference communities. Differences were noted between stores close to and farther away from schools. These results demonstrate that mobilization of community residents to activate enforcement of laws originally designed for other purposes can have a significant impact on one aspect of tobacco point-of-purchase advertising.

Advertising↗
PubMed1995

Metaphyseal dissociation fractures of the proximal tibia. An analysis of treatment and complications.

A study was done of 44 metaphyseal dissociation fractures of the proximal tibia in 42 patients (27 men and 15 women, aged 22 to 77 years; mean, 42 years). Follow-up ranged from 6 months to 4 years. There were 2 study groups: a retrospective group (group 1, 22 fractures) given a variety of treatments ranging from casts to dual plates, and a prospective group (group 2, 22 fractures) treated by combining external fixation and optional minimal internal fixation. There were 12 comminuted fractures in group 1 and 20 in group 2 (P < 0.01). All fractures eventually healed, with an average healing time in group 1 of 3.8 months, and 5.3 months in group 2. There was one delayed union in group 2. Results were graded from poor to excellent, based on pain, range-of-motion, and malunion. There were 6 poor and 4 fair results in group 1, and no poor and 3 fair results in group 2. Complications included 6 deep infections, 5 in group 1 (1 requiring a free-flap procedure); and 1 pin-tract infection resulting in septic arthritis in group 2. There were 7 gastrocnemius flaps required in group 1, and 1 in group 2. The results of this study suggest that patients treated with external fixation had better results with less infection and soft-tissue complications than those treated with conventional internal fixation.

Adult↗
PubMed1994

A metastable state of Torpedo californica acetylcholinesterase generated by modification with organomercurials.

Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.

Acetylcholinesterase↗