The role of protein structure in chromatographic behavior.

Chromatographic retention is determined by a relatively small number of amino acids located in a chromatographic contact region on the surface of a polypeptide. This region is determined by the mode of separation and the amino acid distribution within the polypeptide. The contact area may be as small as a few hundred square angstroms in bioaffinity chromatography. In contrast, the contact region in ion exchange, reversed phase, hydrophobic interaction and the other nonbioaffinity separation modes is much broader, ranging from one side to the whole external surface of a polypeptide. Furthermore, structural changes that alter the chromatographic contact region will alter chromatographic properties. Thus, although immunosorbents can be very useful in purifying proteins of similar primary structure, they will be ineffective in discriminating between small, random variations within a structure. Nonbioaffinity columns complement affinity columns in probing a much larger portion of solute surface and being able to discriminate between protein variants.

High-performance liquid chromatography of biopolymers.

The ability to separate biological macromolecules with good resolution on liquid chromatographic columns has depended on the development of suitable packing materials. In size exclusion chromatography, molecules are separated by size on the basis of differential permeation of the packing. Ion exchange, hydrophobic interaction (or reversed-phase), and affinity chromatography are all surface-mediated separation methods, although they depend on different retention mechanisms. High-performance liquid chromatographic columns designed for biopolymers offer major advantages over conventional columns in both speed and resolving power. The exponential growth of literature on the high-performance separation of peptides and proteins in particular indicates that the technique will become the dominant form of column liquid chromatography.

Sex pheromone in the dog.

Methyl p-hydroxybenzoate has been identified in the vaginal secretions of female dogs in estrus. When small amounts of this compound were applied to the vulvas of anestrous or spayed females, males placed with these females became sexually aroused and attempted to mount them.

Chemical communication and "propaganda" in slave-maker ants.

Slave-maker ants of the Formica sanguinea group direct their raids by means of odor trails. Artificial trails made from whole-body extracts and extracts of Dufour's glands and hindguts can be used to guide columns of workers to selected target colonies and to initiate raids. In workers of F. pergandei and F. subintegra, members of the F. sanguinea group, the Dufour's glands are hypertrophied and contain large quantities of three acetates (decyl, dodecyl, and tetradecyl), which are discharged at defending workers during the slave raids. The acetates produce very efficient, long-lasting alarm signals that attract the slave-makers but disperse the defenders; in effect, therefore, they are "propaganda substances."

Identification of a ventral scent marking pheromone in the male Mongolian gerbil (Meriones unguiculatus).

Sebum from the ventral scent marking gland of the male Mongolian gerbil was fractionated and tested for its ability to elicit behavioral response in a conditioning task and in a stimulus preference situation. The active fraction was identified as phenylacetic acid; both it and a synthetic sample elicited the same behavioral response. Phenylacetic acid appears to be a major pheromone of the male Mongolian gerbil.

[Vaccine prevention in perinatal health care: parents, children and professionals]. / Vaccination en périnatalité : parents, enfants, professionnels.

Recent legislative texts have changed vaccinal policy and reinforced the role of midwives in vaccine prevention in perinatal healthcare. Quite as paediatricians and obstetricians-gynecologists, midwives can now prescribe and carry out, for the mothers, vaccines against rubella, tetanus, poliomyelitis, diphtheria, hepatitis B, influenza and whooping-cough and for the newborns vaccines against hepatitis B and tuberculosis. Concerning vaccinations, practitioners have to respect the vaccination calendar and a collaborative action is useful and necessary. These national guidelines are regularly updated when new vaccines and new recommendations come to light, for example for children (papillomavirus, tuberculosis, pneumococcus...), young adults (varicella, whooping-cough) and health professions in contact with very young children (varicella, measles, influenza and whooping-cough). The recent changes in tuberculosis prevention from routine vaccination of all newborn infants to selective vaccination lead to reinforce measures to detect the infants at higher risk, for them to be vaccinated before discharge at home. Midwives and nurses occupy a central place in family policy and become, with obstetricians-gynecologists and pediatricians, key actors for the effectiveness and the success of vaccine strategies in perinatal health.

Affinity-based screening of combinatorial libraries using automated, serial-column chromatography.

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.

Effects of chronic atrial fibrillation on gap junction distribution in human and rat atria.

OBJECTIVES: To elucidate the structural basis for the electrophysiologic remodeling induced by chronic atrial fibrillation (AF), we investigated connexin40 and connexin43 (Cx40 and Cx43) expression and distribution in atria of patients with and without chronic AF and in an animal model of AF with additional electrophysiologic investigation of anisotropy (ratio of longitudinal and transverse velocities). BACKGROUND: Atrial fibrillation is a common arrhythmia that has a tendency to become persistent. Since gap junctions provide the syncytial properties of the atrium, changes in expression and distribution of intercellular connections may accompany the chronification of AF. METHODS: Atrial tissues isolated from 12 patients in normal sinus rhythm at the time of cardiac surgery and from 12 patients with chronic AF were processed for immunohistology and immunoblotting for the detection of the gap junction proteins. The functional study of the cardiac tissue anisotropy was performed in rat atria in which AF was induced by 24 h of rapid pacing (10 Hz). RESULTS: Immunoblotting revealed that AF did not induce any significant change in Cx43 content in human atria. In contrast, a 2.7-fold increase in expression of Cx40 was observed in AF. Immunohistologic analysis indicated that AF resulted in an increase in the immunostaining of both connexins at the lateral membrane of human atrial cells. A similar spatial redistribution of the Cx43 signal was seen in isolated rat atria with experimentally-induced AF. In addition, AF in rat atria resulted in decreased anisotropy with slightly enhanced transverse conduction velocity. CONCLUSIONS: This experimental study showed that AF is accompanied by spatial remodeling of gap junctions that might induce changes in the biophysical properties of the tissue.

Sarcoplasmic reticulum Ca2+ATPase and phospholamban mRNA and protein levels in end-stage heart failure due to ischemic or dilated cardiomyopathy.

Abnormalities in intracellular Ca2+ handling play a crucial role in the pathogenesis of heart failure. The reduced capacity of failing human myocardium to restore low resting Ca2+ levels during diastole has been explained by the impairment of Ca2+ uptake into the sarcoplasmic reticulum (SR) via the SR Ca2+ATPase. It is unclear whether Ca2+ATPase function, protein levels, and mRNA steady-state levels correspond to one other, and whether the cause of heart failure, namely idiopathic dilated or ischemic cardiomyopathy, produces different changes. The present study examined SR Ca2+ATPase activity and both mRNA and protein levels of SR Ca2+ATPase, phospholamban, and Gi alpha 2 in left ventricular myocardium from eight nonfailing hearts, from eight hearts of patients with idiopathic dilated cardiomyopathy (DCM), and from six hearts from patients with ischemic cardiomyopathy (ICM). Compared to nonfailing myocardium, the activity of the SR Ca2+ATPase was significantly reduced in failing myocardium from patients with DCM (36%, P < 0.01) and from patients with ICM (37%, P < 0.001). Significantly lower levels of SR Ca2+ATPase mRNA levels (55% and -56%, P < 0.001 for DCM and ICM, respectively) and phospholamban mRNA (45%, P < 0.001 for DCM; 31%, P < 0.05 for ICM) were observed in failing than in nonfailing myocardium. In contrast, no significant changes were observed at the level of proteins, Gi alpha 2 mRNA and protein levels were both significantly increased in failing myocardium. There were no differences between idiopathic dilated and ischemic cardiomyopathy concerning the examined parameter. It is concluded that reduced SR Ca2+ATPase activity contributes to an altered intracellular Ca2+ handling by the SR in both dilated and ischemic cardiomyopathic hearts. However, changes in SR Ca2+ATPase and phospholamban steady-state protein levels do not contribute to these alterations. The dissociation between protein and mRNA levels provides evidence for a posttranscriptional or post-translational regulation of these proteins. The observed alterations are not dependent on the underlying cause of end-stage heart failure.

[131 iodine for the treatment of benign goiters]. / L'iode 131 comme traitement des goitres bénins.

INTRODUCTION: In order to evaluate the efficacy of 131 Iodine on goitre volume and on thyroid function, we studied a cohort of patients exhibiting a multinodular and toxic or non toxic goitre. METHODS: This retrospective study was conducted at the Marc Linquette clinic in Lille, in collaboration with the department of nuclear medicine. Thirty-eight patients treated with 131 Iodine were included from 1995 to 2001. Clinical examination and serum analyses including TSH, free T4 and T3, anti-thyroid peroxidase and anti-thyroglobulin antibodies and TSH-receptor antibodies measurements were conducted on inclusion and then at 3, 6, 12 and 72 months. The activity of 131 Iodine corresponded to a standard dose or was calculated according to Marinelli's method. We excluded patients who had not undergone assessment at the above-mentioned time schedules. RESULTS: The treatment was indicated in 30 patients presenting with a non compressive but toxic goitre, in 5 patients with a toxic compressive goitre and in 3 patients with a compressive but non-toxic goitre. Surgery had been excluded for all these patients because of their age, their cardiac status or because they had refused surgery after failure with prior partial thyroidectomy or medical treatment. Among the toxic goitres, TSH levels were low and T3 and T4 increased in 17 patients. In the 18 others, hyperthyroidism was manifested by an isolated decrease of TSH. The thyroid volume before treatment, assessed in 20 patients, was of 18 to 135 cm3 (mean: 53 cm3). Treatment consisted in administration of radioactivity of 3 to 30 mCi in 30 patients and standard activity of 20 to 25 mCi in 8. Functional efficacy with reduction in hyperthyroidism was noted after 3 months, and corrected in nearly all patients after 1 year, and morphological efficacy, with a mean decrease of 33.5% in the size of the goitres. No supplementary surgery was required, notably for the initially compressed goitres. Immediate and long term tolerance was satisfactory. CONCLUSION: Metabolic 131Iodine radiotherapy is effective for the functional and morphological treatment of goitres with good tolerance and few side effects. 131 Iodine is a reasonable alternative in cases with absolute or relative contraindication for surgery.

Rapid process monitoring in biotechnology.

Detection of protein variants in the production of recombinant DNA products is an important and complex task. Rapid acquisition of this information permits feedback control of the production process and continuous validation of the product. Much of the technology required for rapid process monitoring is currently available or under development.

Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life science.

Liquid chromatography and electrophoresis played a major role in the life-science revolution, most strikingly in protein purification, peptide fractionation and sequencing, amino acid analysis, and DNA sequencing. The objective of this article is to examine the potential role of separation systems in the continuing evolution of biochemistry, biotechnology and molecular biology. Very small chip-based systems may change how chemical analyses in biology, medical research and health care evolve over the next decade.

Excellent recovery after prolonged heart storage by preservation with coronary oxygen persufflation: orthotopic pig heart transplantations after 14-hr storage.

BACKGROUND: Improvement of heart preservation is still the greatest challenge in preservation research. The unchanged severe restriction of acceptable storage periods of heart grafts since the beginning of clinical heart transplantation indicates that technical innovations are necessary if a substantial improvement is to be achieved. METHODS: Here, we present the results of hypothermic preservation using the innovative technique of coronary oxygen persufflation (COP). COP simply adds gaseous oxygen to hypothermic graft storage and requires only a "valve guard" for reversible closure of the aortic valve. Fourteen-hr preservation was followed by orthotopic transplantation and evaluations of functional as well as metabolic recovery. Mature pig hearts, a model with restricted preservation tolerance similar to the human heart, were used to guarantee the clinical relevance of this study. RESULTS: After 14-hr hypothermic storage, COP-preserved hearts were able to recover within 2 hr of cardiopulmonary bypass to a steady cardiovascular function without mechanical or pharmacologic support. The left ventricular pressure amplitude of mHTK-COP-preserved hearts as well as energy charge potential recovered to pregrafting values and the ventricular power output to 66%. Hearts simply stored in University of Wisconsin (UW), modified Bretschneider's histidine-tryptophan-ketoglurate (mHTK), or Euro-Flush with glutathione (EFG) solution had only limited recovery, with significantly lower ventricular power output of 18%, 29% or 30% of pregrafting controls on average. CONCLUSIONS: Fourteen-hr oxygenated pig heart preservation using COP results in optimal recovery. Storage preservation in solutions containing hyaluronidase (mHTK and EFG) results in higher recoveries as compared to UW solution, an effect that may support the excellent recovery after mHTK-COP preservation.

Megakaryocytes and fibroblasts--interactions as determined in normal human bone marrow specimens.

An in vitro study was performed to investigate possible interactions between megakaryocytes and bone marrow fibroblasts, both obtained from healthy donors. We were able to demonstrate that the proliferation of fibroblasts increased significantly by co-culturing these cells with megakaryocytes for 6 days. Addition of neutralizing antibodies for PDGF and TGF beta 1, caused a significant reduction of fibroblast growth. Inhibition of cell to cell contacts via tissue culture inserts generated a conspicuous impairment of fibroblast proliferation compared with megakaryocyte-fibroblast co-cultures, where contact was allowed. Hence, our findings suggest that a close spatial relationship between megakaryocytes and fibroblasts is needed for the activation of growth in normal human bone marrow. Neighbouring of megakaryocytes and fibroblasts seems to be necessary in order to achieve a certain threshold of local growth factor concentration. Our results are in keeping with the assumption that PDGF and TGF beta 1, are secreted by normal human megakaryocytes in very low concentrations and promote significantly fibroblast proliferation.

Effects of angiotensin II and angiotensin-converting enzyme inhibitors on human myocardium.

Human myocardial angiotensin II receptors and the angiotensin AT1 and AT2 receptor subtypes were characterised using the partial angiotensin II receptor agonist [125I][Sar1,IIe8]angiotensin II and the selective antagonists losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'((1H-tetrazol-5-yl)biphen yl-4-yl)- methyl]imidazole) and PD 123177 (1-[(4-amino-3-methylphenyl)methyl]-5-(diphenyl-acetyl)- 4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid). The density of angiotensin II receptors was higher in atrial than in ventricular myocardium. Angiotensin AT2 receptors were predominant in atria and ventricles (80-85% of total angiotensin II receptors). Only in isolated, electrically driven atrial trabeculae but not in ventricular preparations, angiotensin II did produce a concentration-dependent positive inotropic effect, which was antagonized exclusively by the angiotensin AT1 receptor antagonist losartan and which amounted to about 20% of the positive inotropic effect of milrinone and isoprenaline. The application of the angiotensin-converting enzyme inhibitors captopril, enalaprilat and ramiprilat had no inotropic effect in either tissue. It is concluded that angiotensin AT1 receptors exclusively mediate direct positive inotropic effects in atrial myocardium. Since angiotensin-converting enzyme inhibitors do not produce any inotropic effect, tonic regulation of basal force of contraction by angiotensin II does not occur.

Evidence against a role of nitric oxide in the indirect negative inotropic-effect of M-cholinoceptor stimulation in human ventricular myocardium.

Nitric oxide (NO) has been reported to mediate several effects in response to muscarinic cholinergic stimulation in cardiovascular tissues. Recently, an attenuation of guinea pig cardiac myocyte contraction by NO has been described. The aim of the present study was to determine whether the indirect negative inotropic effect of M-cholinoceptor stimulation in human myocardium is in part due to an effect of endogenous NO. Therefore, the effect of carbachol was studied under control conditions and during inhibition of NO-synthase by pretreatment with NG-monomethyl-L-arginine (NMMA). Functional experiments were performed in isolated, electrically driven (1 Hz, 37 degrees C) left ventricular papillary muscle strips of human myocardium. Since cytokines have been reported to be increased in the serum of patients with heart failure and could induce NO-synthase activity in failing myocardium, we compared samples from nonfailing and terminally failing (classified as NYHA IV) hearts. The indirect negative inotropic effect of carbachol (10 mumol/l) was studied in the presence of the beta-adrenoceptor agonist isoprenaline (0.03 mumol/l). After stimulation with isoprenaline, carbachol significantly (P < 0.05) reduced force of contraction. This effect was diminished in failing myocardium compared to nonfailing, probably due to the diminished inotropic response most likely due to the lower cAMP levels in response to beta-adrenoceptor stimulation in the former condition. Pretreatment with NMMA (100 mumol/l) altered the antiadrenergic effect of carbachol neither in nonfailing nor in failing preparations. Furthermore, inhibition of guanylyl cyclase, the target enzyme of NO, by preincubation with methylene blue (10 mumol/l) for 30 min had no effect on the carbachol-induced decrease in force of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry.

Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the "drifting peak" phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the "drifting peak" phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.

Channel-specific coatings on microfabricated chips.

This paper reports channel-specific immobilization of fluorescein-5-isothiocyanate (FITC)-labeled bovine serum albumin and beta-galactosidase on microchips with a central channel and two crossing channels; referred to as a double cross channel configuration. Solvent wells at the termini of all channels were used to store reagents. Coatings were applied in multiple steps using electroosmotically driven flow to deliver reagents to specific channels in the chip. The first step in all coating reactions was derivatization of the capillary walls with an organosilane having a reactive pendant functional group. As the silylating reagent was transported from the reagent storage well to a specific waste well, capillary walls in the route of transport were silylated. Flow was maintained throughout a reaction. The route of transport, and thus the specificity of channel coating, were controlled by the well to which negative potential was applied. Flow in a multichannel network takes the shortest route between the electrodes delivering the motive potential. The second reagent in the reaction was delivered from a different well and took a different path through the channel network, as did other reagents. Only the channel being coated was in the flow path of all the reagents used in the coating process. The zone of immobilization in the case of FITC-labeled albumin was determined with confocal fluorescence microscopy. Enzyme activity of immobilized beta-galactosidase (beta-Gal) was monitored by following the hydrolysis of fluorescein mono-beta-D-galactopyranoside to fluorescein with laser-induced fluorescence.

Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography.

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.

Proteolysis of whole cell extracts with immobilized enzyme columns as part of multidimensional chromatography.

This paper describes the efficacy of immobilized trypsin columns in the digestion of cellular extracts that contained thousands of proteins. Effectiveness of proteolysis was evaluated with extracts of Escherichia coli by size-exclusion chromatography. Immobilized trypsin columns were operated in either the continuous-flow or stopped-flow mode at temperatures ranging from ambient to 37 degrees C with incubation times of 0-2 h. The results of these studies indicate that reduced and alkylated extracts of proteins from E. coli can be digested in 20 min when the enzyme column is operated at elevated temperature. The advantage of immobilized enzyme columns is that they can be easily incorporated into multidimensional separation systems for automated proteomics.