Hydration study of homopolypeptides by (2)H NMR.

Deuteron T(1) and T(2) was studied as a function of hydration in homopolyglycine (PG) and homopolyproline (PP). Water deuteron relaxation rates in PG conform to a hydration model involving two types of primary hydration sites where water is directly bonded to the polymer. Once these sites are filled, additional water only bonds to water molecules at the primary sites and in so doing affect their dynamics. PP exhibits an anomalous T(1) and T(2) hydration dependence which has been interpreted in terms of a cooperative water molecule-PP molecule helical conformational rearrangement which occurs once a certain hydration level is reached. The proposal of a water-PP structure is tested using molecular dynamics simulations.

The determination of the size and shape of buried InAs/InP quantum dots by transmission electron microscopy.

Bright-field, diffraction-contrast imaging in the transmission electron microscope has been applied to the determination of the diameter and height populations of a single layer of buried, pure, InAs/InP quantum dots (QDs). Plan-view diffraction contrast from the QDs was observed to increase significantly when the sample was tilted away from the [001] growth direction to near the [111] zone-axis orientation. This added contrast was a result of contributions to the displacement of atoms in a direction perpendicular to the electron beam arising from strain in the growth direction. Since the strain in the growth direction was about an order of magnitude larger than the strain perpendicular to the growth direction, as the sample is tilted away from the [001] zone-axis condition, the larger strain component increases the projected strain thereby increasing the QD contrast in the image. For the sample studied, both of the populations for the QD diameter and the image contrast were observed to be multimodal with the seven peaks in the contrast distribution correlating with seven distinct populations of QDs each differing in height by one monolayer (ML), from 3 to 9MLs. An analysis of the theoretically expected and experimentally observed standard deviations in the Gaussian fits to the QD diameter and height distributions provided an additional constraint in the selection of the optimal model for the multimodal distributions.

A comparative investigation into the effect of chronic alcohol feeding on the myocardium of normotensive and hypertensive rats: an electrophoretic and biochemical study.

We investigated whether the imposition of chronic alcohol in hypertension leads to greater biochemical and cellular abnormalities of the myocardium than those arising in normotension. Fifteen-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were fed ethanol-containing diets for six weeks. Particular attention was focused on the composition of contractile proteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fractional rate of protein synthesis, and synthesis rates relative to RNA (RNA activity) or DNA (cellular efficiency). In addition, myocardial enzymes and adenine nucleotides were measured. In both SHR and WKY rats chronic ethanol caused a general decrease in the contents of all nine contractile proteins with myosin heavy chain predominantly affected. Fractional rates of mixed (i.e., total) and myofibrillary proteins remained unaltered in both WKY rats and SHR, as were cellular efficiencies. The RNA activity was significantly reduced in ethanol-treated SHR but not in WKY rats. In ethanol-treated SHR, cardiac creatine kinase (CK) and malate dehydrogenase (MDH) activities were increased, AMP levels were elevated, whilst ATP levels and the energy charge were reduced. In WKY rats, the only significant change related to increased aspartate aminotransferase activities in response to alcohol feeding. Although there were only subtle differences between the response of the normotensive and hypertensive rats due to ethanol dosage, the reduced ATP levels and increased CK and MDH activities in SHR may reflect a greater susceptibility to ischaemic damage. Reduced contractile protein content, particularly myosin heavy chain, may contribute to contractile defects, a common feature of subclinical and clinical alcoholic cardiomyopathy.

Cardiac protein abnormalities in dilated cardiomyopathy detected by two-dimensional polyacrylamide gel electrophoresis.

The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two-dimensional (2-D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p<0.01). The most prominent changes occurred in the contractile protein myosin light chain 2 and in a group of proteins identified as desmin. These changes do not appear to be artefactual degradation events occurring during sample processing. These proteins are not apparent in electrophoretic separations of vascular tissue or cultured endothelial cells, mesothelial cells or cardiac fibroblasts, which are clearly distinguishable from the 2-D protein patterns of whole heart and of isolated cardiac myocytes and do not appear to reflect variations in the cellular composition of biopsy samples. The different protein patterns observed in cardiomyopathy showed no obvious relationship with New York Heart Association (NYHA) functional class or haemodynamic parameters. The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function. These changes might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.

The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Construction of HSC-2DPAGE: a two-dimensional gel electrophoresis database of heart proteins.

The dissemination of information relating to the characterisation of proteins from two-dimensional electrophoresis (2-DE) gel databases is essential for their effective utilisation in the study of protein expression in cell biology. Since the inception of the World Wide Web and the pioneering development of SWISS-2DPAGE as a tool for retrieving information on proteins separated by 2-DE, the Internet has become the method of choice for disseminating and accessing information on 2-DE protein databases. At Harefield we have established HSC-2DPAGE which is an advanced interface for accessing protein database relating to heart disease. The Web site currently includes databases of proteins from human, dog and rat ventricular tissue and a human endothelial cell line. The databases are searchable individually or as a whole by remote keyword searches. Each database is represented by both synthetic (computer generated) and real (scanned gel) clickable images upon which characterised protein spots are highlighted by hyperlinked symbols. The database conforms to all the rules proposed for federated 2-DE protein databases and individual protein entries are linked to other protein databases such as SWISS-PROT by active cross-references. This paper describes the construction of HSC-2DPAGE, its maintenance, and access via the Internet.

Protein profiling in cardiac tissue in response to the chronic effects of alcohol.

An investigation was made into cardiac protein levels after chronic ethanol consumption to examine whether specific proteins are affected by alcohol. Ethanol was administered for six weeks to male Wistar rats which were fed a nutritionally complete liquid diet containing 35% of total calories as ethanol. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose; thus both groups had identical nutritional intakes, albeit differences in ethanol or carbohydrate. After six weeks' feeding, cardiac tissue was removed and analyzed by two-dimensional electrophoresis, where equal amounts of proteins were studied. Protein patterns were analyzed by computerized densitometry and characterized by comparison with a database of known cardiac proteins. Chronic alcohol feeding caused significant decreases in the relative amounts of various proteins, including several tentatively identified as heat shock protein (HSP) 60, HSP70, and desmin. The relative proportions of actin, vimentin, myosin light chain 1, myosin light chain 2, and albumin, remained unchanged. Examination of antibodies raised against HSP65 showed no overt differences in plasma levels following chronic alcohol consumption, and liver changes as assessed by histology were mild. In conclusion, chronic alcohol appears to have selective effects on particular proteins, and the effects were not directly ascribed to overt liver dysfunction or malnutrition. This may explain some of the functional and morphological characteristics observed in alcohol-induced heart muscle disease, including reduced contractility.

HSC-2DPAGE and the two-dimensional gel electrophoresis database of dog heart proteins.

A two-dimensional gel electrophoresis database of dog (Canis familiaris) proteins is presented. The database contains 1212 protein spots which have been characterised in terms of their pI and Mr. This database has been integrated into the HSC-2DPAGE database which is accessible on the Internet via the World Wide Web with the uniform resource location (URL): (http://www.harefield.nthames.nhs.uk/nhli/ protein/index.html). Identifications for 80 of the protein spots have been obtained by visual cross-matching with the human heart protein database in HSC-2DPAGE (42 spots), N-terminal microsequence analysis (25 spots) and peptide mass fingerprinting (20 spots). This database is being used in studies of alterations in protein expression in models of heart failure and heart disease.

Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis.

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.

Alcohol and the heart: biochemical alterations.

A considerable amount of attention has focused on the cardiovascular events associated with ethanol consumption. The available evidence suggests that moderate ethanol consumption is associated with reduced risk of coronary heart disease, i.e., vessel events. In contrast, this review is primarily concerned with ethanol and heart muscle damage. Clinical features of the consequences of prolonged and excessive ethanol consumption encompass defects in myocardial contractility and derangement of cellular architecture, including disarray of the contractile elements. Although the incidence of heart muscle abnormalities in alcohol misusers is generally higher than previously considered, the mechanisms are only just being elucidated. This process has been facilitated by laboratory based studies in which animals receive either a single dose of ethanol (acute studies) or a continuous supply of ethanol in their daily diets (chronic studies). Results from these models show that acute ethanol dosage causes a marked decrease in the synthesis of contractile proteins. This occurs in the absence of overt mitochondrial abnormalities: ATP concentrations are generally unaffected. Paradoxically, the synthesis of mitochondrial proteins is reduced. Use of metabolic inhibitors suggests that the deleterious effects of acetaldehyde contribute to these reductions in protein synthesis. In chronic studies, ethanol causes a reduction in the amount of contractile proteins, and two dimensional protein profiling implicates selective loss of individual myocardial proteins. The differential activities of lysosomal proteases may contribute to this patterned response. However, in chronic ethanol feeding, adaptive mechanisms also become important, as the synthesis of the myofibrillary proteins increases. Overall, the mechanisms inherent in these biochemical responses may contribute to the genesis of a distinct disease entity, alcoholic heart muscle disease.

The identification of MHC identical siblings without HLA typing.

By comparing genomic sequences of different MHC haplotypes, we defined highly polymorphic markers. After amplification, electrophoresis, and scanning with a laser, we have identified profiles that serve as signatures of the haplotype and its component alleles. One set of markers can be used to define the block that includes HLA-B and HLA-C, among other loci. Another set provides signatures for the entire HLA-DR and -DQ multigene cluster. By profile overlay, it is possible to identify siblings who share both haplotypes from HLA-C to HLA-DQ. Here we demonstrate the value of genomic analysis ("block matching") in selecting genotypically identical siblings prior to transplantation. Forty-six siblings from 10 families were genotyped by family analysis after meticulous HLA, C4, and Bf typing including molecular methods for HLA-DRB1. In 43 siblings, the haplotype assignments were unequivocal. Twenty-two identical sibling pairs could then be compared with 77 nonidentical pairs. Independent genomic analysis yielded entirely concordant results. In three siblings, the possibility of parental recombination was considered but could not be defined by the conventional typing. By genomic analysis, however, it was clear that recombination had indeed occurred in one case. In the remaining two cases, additional, more telomeric markers will be necessary to resolve the issue. This simple, cost-effective method has immediate application to the identification of matched pairs (HLA-C to HLA-DQ) for bone marrow and renal transplantation.

Coelectrophoresis of cardiac tissue from human, dog, rat and mouse: towards the establishment of an integrated two-dimensional protein database.

We have investigated the feasibility of identifying homologous proteins in whole tissue protein extracts of dog, mouse and rat hearts by comparison with our human heart two-dimensional (2-D) database. Samples of ventricular myocardial tissue from each of these species were coelectrophoresed with a human tissue sample. Gels were silver stained and patterns were analysed using PDQUEST. The number of proteins comigrating with human proteins was 301, 201 and 356 for the dog, mouse and rat, respectively. In the dog pattern, 33 of these comigrating proteins were tentatively identified from the similarity between their migration properties and those of known human proteins. Twenty-nine such proteins were identified in the mouse pattern while 30 comigrating rat proteins were identified. While these tentative identifications require confirmation, we feel that this technique offers a useful shortcut in the characterisation of proteins present in similar tissue samples from different species and avoids the necessity for duplicating laborious procedures, such as protein microsequencing, otherwise used in the identification of these proteins in each species.

Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.

The human myocardial two-dimensional gel protein database: update 1994.

An updated human heart protein two-dimensional electrophoresis (2-DE) database is presented. The database, which contains some 1388 protein spots characterised in terms of M(r) and pI, has been analysed further by Western immunoblotting and protein sequencing. From a total of 103 protein spots analysed, 49 have been identified by immunoblotting and 32 have been identified by protein sequencing. A further six proteins have tentatively been assigned by comparison with the human heart 2-DE protein database of Jungblut et al. (Electrophoresis) 1994, 15, 685-607). This database is being used in studies of alterations in protein expression in the diseased and transplanted human heart.

Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: an interlaboratory comparison.

An intra- and interlaboratory comparison of positional reproducibility of protein spots in two-dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG-DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4-8, for the first dimension and homogeneous SDS-PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well-resolved spots distributed across the 2-D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG-dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along the x-axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation approximately 1.5 mm). Nevertheless, IPG-DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.

Needle exchange programs and social policy.

The specter of AIDS will continue to dominate the concerns of clinicians, policy-makers, and social scientists into the next century. In addition to being a biological issue, HIV disease is a political issue. As a result of this, interest groups have mobilized to restrict certain interventions aimed at stopping the spread of HIV. Among those restricted interventions is the exchange of sterile needles and syringes for "dirty" needles and syringes with injection drug users (IDUs). Increasing the availability of clean equipment by removing the laws restricting their availability, and/or by funding needle exchange programs, would appear to be a much needed and rational public health policy. However, needle exchange programs have been viewed as fostering drug addiction or enabling drug addicts, thus marginalizing it as an early stage of treatment for addicts and as a demonstrated public health intervention. There is no empirical evidence to support this conclusion. In the absence of better knowledge about how to prevent the use of illicit injection drugs and how to effectively treat IDUs, we believe that needle exchange programs (NEPs) need to be implemented for several key reasons. First, they can help slow the spread of HIV infection. Second, they can be cost-effective when compared to the higher health care costs that result without needle exchange programs. Third, they can act as a precursor to treatment, or recovery, for addicts.