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Biomedical subjects

B Herbert

Publications and source records attributed to B Herbert.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of 2-fluoro- and 6-fluoro-(2S,3R)-(3,4-dihydroxyphenyl)serine as potential in vivo precursors of fluorinated norepinephrines.

The title compounds were prepared by the aldol condensation of 3,4-dibenzyloxy-2-fluorobenzaldehyde and 4,5-dibenzyloxy-2-fluorobenzaldehyde with the oxazolidinone 2, a chiral glycine equivalent. Removal of the chiral auxiliary and blocking groups produced the target amino acids 2-fluoro- and 6-fluoro-(2S,3R)-(3,4-dihydroxyphenyl)serine (1b and 1c) in >98% ee.

Adrenergic Agonists↗
PubMed2001

The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes.

The last few years have brought dramatic improvements for sample preparation and solubilization of protein for electrophoretic analyses. The use of reagents such as thiourea and novel sulfobetaine surfactants increases the total number of proteins able to be visualized from a complex mixture such as a cell lysate and also allows more hydrophobic membrane proteins to be resolved. As the red blood cell (RBC) contain no organelles, it is an ideal source of relatively pure plasma membrane for protein solubilization studies. In addition, there are a number of diseases related to abnormalities of RBCs proteins, thus it is of medical relevance as well as a test sample for technology development. However, the procedure for purifying RBC membranes is rather time-consuming and is normally carried out under almost physiological conditions, which can be conducive to proteolytic degradation of the membrane proteins. Significant differences in two-dimensional (2-D) patterns with and without protease inhibitors in sample preparation are demonstrated. In addition, is shown that preparation of RBC membranes with sodium carbonate, pH 11, leads to multimeric complexes of hemoglobin and causes hemoglobin to be irreversibly attached to the membrane. When using immobilized pH gradients (IPG) as the first dimension, it is demonstrated that the spectrins (large, filamentous proteins of 280 kDa) are lost from the 2-D map unless active, instead of passive, sample hydration into the IPG strip is adopted.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.

Alkylation↗
PubMed2001

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

Alkylation↗
PubMed2001

Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.

Although it is highly recommended that reduction and alkylation of free -SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA). The present matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free -SH group of Cys in proteins at alkaline pH values (pH 8.5-9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the -SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.

Alkylation↗
PubMed2001

Proteomic analysis of a developmentally regulated secretory vesicle.

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion.

Animals↗
PubMed2000

Synthesis of (-)-7-epiaustraline and (-)-1-epicastanospermine.

Highly efficient and selective syntheses of the title compounds are described. The cornerstone of the synthetic plan is the tandem inter [4 + 2]/inter [3 + 2] cycloaddition process. These syntheses differ from previous applications of this strategy in that they incorporate an alkylation in the hydrogenolysis step to close the second ring of the azabicyclic systems. Notable features of the sequence are (1) the highly regio- and stereoselective [3 + 2] cycloaddition of nitronate 15 with siloxymethyl (Z)-beta-silylvinyl ketone (Z)-22b and (2) the highly selective reduction of the resulting ketone 24a with L-Selectride. A single-crystal X-ray structure analysis of synthetic (-)-7-epiaustraline confirmed that the targeted structure was successfully synthesized. This stimulated a reexamination of the structural assignment of the natural product. (-)-1-Epicastanospermine was synthesized in four steps from the common intermediate 27a. The absolute configuration of (-)-1-epicastanospermine was assured by single-crystal X-ray structure analysis of intermediate (-)-27a. Thus, the sign of the optical rotation had to be revised. The overall efficiency of these syntheses were 9 steps and 23% yield for (-)-7-epiaustraline and 10 steps and 20% yield for (-)-1-epicastanospermine

Alkylation↗
PubMed2000

Syntheses of (R)- and (S)-2- and 6-fluoronorepinephrine and (R)- and (S)-2- and 6-fluoroepinephrine: effect of stereochemistry on fluorine-induced adrenergic selectivities.

Several routes to the enantiomers of fluoronorepinephrines (1) and fluoroepinephrines (2) were explored. A catalytic enantioselective oxazaborolidine reduction and a chiral (salen)Ti(IV) catalyzed asymmetric synthesis of silyl cyanohydrins proved efficacious in the key stereo-defining steps of two respective routes. Binding studies of the catecholamines with alpha(1)-, alpha(2)-, beta(1)-, and beta(2)-adrenergic receptors were examined. The assays confirmed that fluorine substitution had marked effects on the affinity of (R)-norepinephrine and (R)-epinephrine for adrenergic receptors, depending on the position of substitution. Thus, a fluoro substituent at the 2-position of (R)-norepinephrine and (R)-epinephrine reduced activity at both alpha(1)- and alpha(2)-receptors and enhanced activity at beta(1)- and beta(2)-receptors, while fluorination at the 6-position reduced activity at the beta(1)- and beta(2)-receptors. The effects of fluorine substitution on the S-isomers were less predictable.

Adrenergic beta-Agonists↗
PubMed2000

A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes.

Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis. This was demonstrated with different samples. In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE. Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma. With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.

Acrylamides↗
PubMed1999

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death.

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2'-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Antineoplastic Agents↗
PubMed1999

Effect of nutrient limitation on adhesion characteristics of Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes a variety of diseases in humans including lung and ocular infections. Infections of the cornea are usually associated with wearing contact lenses and can result in loss of vision. This study aimed to determine the effect of carbon or nitrogen limitation on the adhesion to contact lenses of a strain of Ps. aeruginosa isolated from contact lens-related corneal inflammation. Cells were grown in a continuous culture apparatus in varying levels of glucose or ammonia to effect nutrient limitation. Adhesion to contact lenses was measured as total counts and viable counts. The cell surface hydrophobicity and charge were measured using adhesion to surface-modified Sepharose. Changes in lipopolysaccharide were determined using 1D SDS-PAGE and changes in cell-surface proteins were measured using 2D gel electrophoresis. The more the cultures were nitrogen limited, the greater the increase in adhesion to unworn hydrogel contact lenses 0.3 x 10(3) - 2.2 x 10(3) cells/mm2 on Etafilon A lenses. Cells that were carbon limited showed a greater increase in adhesion to contact lenses when the lenses had been coated in artificial tears. It appeared that lipopolysaccharide may have been involved in the constitutive adhesion to unworn lenses that occurred during C-limitation, whereas changes in the outer membrane proteins contributed to the increased adhesion under nitrogen limitation, or the change in adhesion that occurred to carbon-limited cells using contact lenses coated in artificial tears. Nine cell-surface proteins appeared during nitrogen limitation with kDa/pI of 75/4.8, 4.9, 5.0; 62/5.6; 89/6.5; 38/6.4; 28/1.5; 18/6.4; 12/4.5. Any or all of these may have been involved in the increased adhesion and further experiments are underway to examine this possibility.

Bacterial Adhesion↗
PubMed1998

[Nutritional status of participants in the Giessen Senior Long-Term Study with respect to antioxidant vitamins and selenium].

Within the longitudinal study on nutrition and health status in an aging population of Giessen, Germany, baseline measurements with regard to antioxidant status were obtained in 85 free living elderly persons. The subjects were at least 60 years old, non-smoking, feeding themselves, and not supplementing any vitamins or minerals. Dietary intake of nutrients was assessed by means of a 3-day estimated food record, which was especially developed and validated for this study. Antioxidant status was evaluated by fasting plasma concentrations of vitamin C, E, beta-carotene, and selenium. The results show that in this study group self-determined nutrition provides enough nutrients to yield a good antioxidant status in the advanced age. Although no differences could be detected in dietary intake of vitamin C, E, and beta-carotene between males and females, significantly higher plasma concentrations of all substances investigated were found in females when compared to males.

Aged↗
PubMed1997

Ultrastructure and cytochemical detection of alkaline phosphatase in long-term cultures of osteoblast-like cells from rat calvaria.

Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation.

Alkaline Phosphatase↗
PubMed1997

Domestic violence and mental health: correlates and conundrums within and across cultures.

Gender-based violence, only recently emerging as a pervasive global issue, contributes significantly to preventable morbidity and mortality for women across diverse cultures. Existing documentation suggests that profound physical and psychological sequelae are endemic following intimate partner violence. The presentation of domestic violence is often culture specific. A new lexicon, prompted by the expansion of human rights analysis, describes particular threats to local women including dowry deaths, honor murder, saiti, and disproportional exposure to HIV/AIDS as well as globally generic perils including abuse, battering, marital rape, and murder. While still fragmentary, accruing data reveal strengthening associations between domestic violence and mental health. Depression, stress-related syndromes, chemical dependency and substance (ab)use, and suicide are consequences observed in the context of violence in women's lives. Emerging social, legal, medical, and educational strategies, often culture specific, offer novel local models to promote social change beginning with raising the status of women. The ubiquity, gravity, and variability of domestic violence across cultures compel additional research to promote the recognition, intervention, and prevention of domestic violence that are both locally specific and internationally instructive.

Cross-Cultural Comparison↗
PubMed1997

Violence-related injuries in a pediatric emergency department.

BACKGROUND: Since the 1980s, violence has emerged as a leading public health concern in the United States. Recent studies have begun to address the impact of interpersonal violence specifically on young children. The purpose of this study was to describe the epidemiology of violence-related injuries (VRI) in an urban pediatric emergency department (ED). METHODS: A six-month retrospective chart review was conducted. The records of 11,000 patients 17 years of age and younger who were seen in an urban pediatric ED were accessed for VRI. VRI were defined as purposefully inflicted with intent to harm. Patients discharged from the ED with VRI were compared to patients admitted with VRI. RESULTS: Three hundred seventeen (3%) of patients were categorized as having VRI during the six-month study period. Eighty-three percent were discharged, and 17% were admitted. The majority of injuries was the result of interpersonal conflict with a friend or acquaintance. Patients discharged were more likely to involve: younger children, female victims, and blunt instruments. Thirteen percent of discharged patients, however, involved potentially lethal weapons (knife or firearm). CONCLUSIONS: The vast majority of patients with VRI are discharged from the ED. Females and young children were frequently evaluated for VRI in the pediatric ED. Identification of these patients can be used to initiate service protocols directed at violence prevention.

Adolescent↗