Element- and enantiomer-selective visualization of molecular motion in real-time.

Ultrafast optical-domain spectroscopies allow to monitor in real time the motion of nuclei in molecules. Achieving element-selectivity had to await the advent of time resolved X-ray spectroscopy, which is now commonly carried at X-ray free electron lasers. However, detecting light element that are commonly encountered in organic molecules, remained elusive due to the need to work under vacuum. Here, we present an impulsive stimulated Raman scattering (ISRS) pump/carbon K-edge absorption probe investigation, which allowed observation of the low-frequency vibrational modes involving specific selected carbon atoms in the Ibuprofen RS dimer. Remarkably, by controlling the probe light polarization we can preferentially access the enantiomer of the dimer to which the carbon atoms belong.

Weight gain adequacy and pregnancy outcomes in gestational diabetes: a meta-analysis.

The Institute of Medicine updated guidelines for gestational weight gain in 2009, with no special recommendations for gestational diabetes. Our objectives were to describe the prevalence of weight gain adequacy and their association with adverse pregnancy outcomes in gestational diabetes. We searched MEDLINE, EMBASE, COCHRANE and SCOPUS. We calculated the pooled prevalence of gain adequacy and relative risks for pregnancy outcomes within Institute of Medicine categories. Thirty-three studies/abstracts (88,599 women) were included. Thirty-one studies provided data on the prevalence of weight gain adequacy; it was adequate in 34% (95% CI: 29-39%) of women, insufficient in 30% (95% CI: 27-34%) and excessive in 37% (95% CI: 33-41%). Excessive gain was associated with increased risks of pharmacological treatment, hypertensive disorders of pregnancy, caesarean section, large for gestational age and macrosomic babies, compared to adequate or non-excessive gain. Weight gain below the guidance had a protective effect on large babies (RR: 0.71; 95% CI: 0.56-0.90) and macrosomia (RR 0.57; 95% CI 0.40-0.83), and did not increase the risk of small babies (RR 1.40; 95% CI 0.86-2.27). Less than recommended weight gain would be beneficial, while effective prevention of excessive gain is of utmost importance, in gestational diabetes pregnancies. Nevertheless, no ideal range for weight gain could be established.

Role of rotational wave packets in strong field experiments.

Complex revival features of rotational wave packets are obtained from the interplay of a molecular rotational distribution and a measured physical observable. The analysis of the measured temporal behavior can be used to retrieve either one or both quantities. We show here the first observation of high order fractional revival (up to 1/12 in CO2) using time-of-flight measurements of ion yields leading to the information required for full reconstruction of the rotational wave packet. We further show via an analysis of higher order fractional revivals in high harmonic generation that new information on the participating ionic channels can be clearly identified, showing the general implication of our results.

Localization and functional characterization of the human NKCC2 isoforms.

AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. CONCLUSION: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.

Effect of oral organic nitrates on expression and activity of vascular soluble guanylyl cyclase.

BACKGROUND AND PURPOSE: The regulation of vascular soluble guanylyl cyclase (sGC) expression by nitric oxide (NO) is still under discussion. In vitro, NO has been shown to downregulate the expression of sGC but it is unclear if this mechanism is operative in vivo and occurs during nitrate treatment. EXPERIMENTAL APPROACH: We investigated whether high dose isosorbide mononitrate (ISMN) or pentaerythrityl tetranitrate (PETN) treatment changes vascular sGC expression and activity in vivo. New Zealand White rabbits received a standard diet, 2 or 200 mg ISMN kg(-1) d(-1) for 16 weeks, and C57BL/6 mice received a standard diet, 6, 60 or 300 mg PETN kg(-1) d(-1) for four weeks. Absorption was checked by measuring the plasma levels of the drug/metabolite. KEY RESULTS: Western blots of rabbit aortic rings showed similar protein levels of sGC alpha1- (P=0.2790) and beta1-subunits (P=0.6900) in all groups. Likewise, ANOVA showed that there was no difference in the expression of sGC in lungs of PETN-treated mice (P=0.0961 for alpha1 and P=0.3709 for beta1). The activities of isolated sGC in response to SNAP (1 microM-1 mM) were identical in aortae of ISMN-treated rabbits (P=0.0775) and lungs of PETN-treated mice (P=0.6348). The aortic relaxation response to SNAP slightly decreased at high ISMN but not at high PETN. CONCLUSIONS AND IMPLICATIONS: These data refute the hypothesis that therapeutic treatment with long acting NO donors has a significant impact on the regulation of vascular sGC expression and activity in vivo.

The C-terminus of complement regulator Factor H mediates target recognition: evidence for a compact conformation of the native protein.

The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C-terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C-terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N-terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme-linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C-terminal recognition domain of Factor H.

Reporter gene recombination in juxtaglomerular granular and collecting duct cells by human renin promoter-Cre recombinase transgene.

To assess the feasibility of using the renin promoter for expressing Cre recombinase in juxtaglomerular (JG) cells only, we generated five independent transgenic mouse lines (designated hRen-Cre) expressing Cre recombinase under control of a 12.2-kb human renin promoter. In the kidneys of adult mice Cre mRNA (RT-PCR) was found in the renal cortex, with Cre protein (immunohistochemistry) being localized in afferent arterioles and to a lower degree in interlobular arteries. Cre mRNA levels were regulated in a renin-typical fashion by changes in oral salt intake, water restriction, or isoproterenol infusion, indicating the presence of key regulatory elements within 12.2 kb of the 5'-flanking region of the human renin gene. hRen-Cre mice were interbred with both the ROSA26-EGFP and ROSA26-lacZ reporter strains to assess renin promoter activity from Cre-mediated excision of a floxed stop cassette and subsequent enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-gal) detection. In adult mice, beta-gal staining and EGFP were observed in afferent arterioles and interlobular arteries, overlapping with Cre protein expression. In addition, intense beta-gal staining was found in cortical and medullary collecting ducts where Cre expression was minimal. In embryonic kidneys, beta-gal staining was detected in the developing collecting duct system beginning at embryonic day 12, showing substantial activity of the human renin promoter in the branching ureteric bud. Our data indicate that besides its well-known activity in JG cells and renal vessels the human renin promoter is transiently active in the collecting duct system during kidney development, complicating the use of this approach for JG cell-specific excision of floxed targets.

Vasoconstrictor and vasodilator effects of adenosine in the mouse kidney due to preferential activation of A1 or A2 adenosine receptors.

The present experiments in mice were performed to determine the steady-state effects of exogenous adenosine on the vascular resistance of the whole kidney, of superficial blood vessels, and of afferent arterioles. The steady-state effect of an intravenous infusion of adenosine (5, 10, and 20 microg/min) in wild-type mice was vasodilatation as evidenced by significant reductions of renal and superficial vascular resistance. Resistance decreases were augmented in adenosine 1 receptor (A1AR) -/- mice. Renal vasodilatation by the A2aAR agonist CGS 21680A [2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamido-adenosine hydrochloride] (0.25, 0.5, and 1 microg/kg/min) and inhibition of adenosine-induced relaxation by the A2aAR antagonist ZM-241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol] (20 mg/kg) suggests that the reduction of renovascular resistance was largely mediated by A2aAR. After treatment with Nomega-nitro-L-arginine methyl ester (L-NAME) adenosine was unable to alter superficial blood flow and resistance significantly indicating that adenosine-induced dilatation is NO-dependent. Absence of a dilatory effect in endothelial nitric-oxide synthase (NOS) -/- mice suggests endothelial NOS as the source of NO. When infused into the subcapsular interstitium, adenosine reduced superficial blood flow through A1AR activation. Adenosine (10(-7) M) constricted isolated perfused afferent arterioles when added to the bath but not when added to the luminal perfusate. Luminal adenosine caused vasoconstriction in the presence of L-NAME or the A2AR antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Our data show that global elevation of renal adenosine causes steady-state vasorelaxation resulting from adenosine 2 receptor (A2AR)-mediated generation of NO. In contrast, selective augmentation of adenosine around afferent arterioles causes persistent vasoconstriction, indicating A1AR dominance. Thus, adenosine is a renal constrictor only when it can interact with afferent arteriolar A1AR without affecting the bulk of renal A2AR at the same time.

Detection of the CCR5-Delta32 HIV resistance gene in Bronze Age skeletons.

A mutant allele of the chemokine receptor CCR5 gene (CCR5-Delta32), which confers resistance to HIV-1 infection, is believed to have originated from a single mutation event in historic times, and rapidly expanded in Caucasian populations, owing to an unknown selective advantage. Among other candidates, the plague bacillus Yersinia pestis was implicated as a potential source of strong selective pressure on European populations during medieval times. Here, we report amplifications of the CCR5-Delta32 DNA sequence from up to 2900-year-old skeletal remains from different burial sites in central Germany and southern Italy. Furthermore, the allele frequency of CCR5-Delta32 in victims of the 14th century plague pandemic in Lubeck/northern Germany was not different from a historic control group. Our findings indicate that this mutation was prevalent already among prehistoric Europeans. The results also argue against the possibility of plague representing a major selective force that caused rapid increase in CCR5-Delta32 gene frequencies within these populations.

Diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis.

OBJECTIVE: To assess the additional diagnostic and clinical value of the second test generation of anti-cyclic citrullinated peptide antibodies (CCP2) compared with rheumatoid factor isotypes (IgG-RF, IgA-RF, IgM-RF) in patients with rheumatoid arthritis. METHODS: This was a prospective study on 715 patients: rheumatoid arthritis (n = 295), degenerative or other inflammatory joint disease (n = 163), connective tissue disease or vasculitis (n = 103), and healthy controls (n = 154). Sera from each subject were tested for CCP2 and RF isotypes by enzyme linked immunosorbent assay (ELISA). Agreement with clinical indices such as disease activity, joint destruction, disease duration, and other laboratory tests was assessed. Sensitivity and specificity of the tests were evaluated taking the clinical diagnosis as the gold standard. RESULTS: Highest sensitivity was found for IgM-RF (66.4%) and CCP (64.4%). Highest specificity was achieved by CCP (97.1%) and IgG-RF (91.0%). In rheumatoid patients with high disease activity or severe joint damage, CCP was more often present (81.4% and 83.6%) than all RF isotypes. Of special diagnostic value was the detection of positive CCP in 34.5% of all patients with rheumatoid arthritis when all measured RF isotypes (IgG-RF, IgA-RF, and IgM-RF) were negative. CONCLUSIONS: As a screening method for rheumatoid arthritis the IgM-RF and the CCP assays are superior to other RF isotypes. Positivity in the highly specific CCP ELISA supports the diagnosis of rheumatoid arthritis. CCP proved to be a powerful diagnostic tool, especially in ambiguous cases or RF negative patients with rheumatoid arthritis.

Introital ultrasound of the lower genital tract before and after colposuspension: a 4-year objective follow-up.

OBJECTIVE: To assess the topography of the bladder neck by introital ultrasound before and after open colposuspension. METHODS: Three hundred and ten women with urodynamically proven stress urinary incontinence were included in this long-term study to investigate the position and function of the bladder neck at rest and during straining. Height (H), distance (D), and urethrovesical angle of the bladder neck (beta) were measured by means of preoperative and postoperative introital ultrasound. Women were followed up; 152 of them (49%) completed 48 months of follow-up. RESULTS: At the 6-month follow-up examination, 90.0% of the women were continent (279/310), 3.5% (11/310) showed voiding difficulties, 3.5% (11/310) had urgency, and 1.6% (5/310) had developed de novo urge incontinence. At the 48-month follow-up, 76.8% of the patients were still continent. All postoperative measurements yielded significantly lower values for angle beta at rest and during straining compared with the preoperative results (P < 0.0001). The median linear movement of the bladder neck during straining decreased from 18.0 mm before surgery to 6.4 mm at the 48-month follow-up (P < 0.0001). The median level of ventrocranial elevation of the vesicourethral junction was 14.3 mm immediately after surgery, 9.9 mm after 6 months and 6.6 mm after 48 months. The degree of surgical bladder-neck elevation was associated with postoperative urgency/de novo urge incontinence (P < 0.0001) and voiding difficulty (P < 0.0001). CONCLUSIONS: The colposuspension procedure reduces angle beta at rest and during straining, restricts linear movement with straining, and elevates the bladder neck. Perioperative introital ultrasound improves understanding of this surgical procedure and might help to prevent postoperative complications.

Clinical significance of anti-dsDNA antibody isotypes: IgG/IgM ratio of anti-dsDNA antibodies as a prognostic marker for lupus nephritis.

The objective of this paper is to investigate the association between patterns of anti-dsDNA antibody isotypes and specific clinical manifestations (categorized in renal, musculoskeletal, cutaneous, hematological, pulmonary, neurological and cardiac). Sera of 202 systemic lupus erythematosus (SLE) patients, 33 patients suffering from other autoimmune diseases and 115 healthy blood donors were analysed for anti-dsDNA antibodies by IgG-, IgA- and IgM-specific ELISA, Farr-assay and CLIF. A subset of 24 SLE patients was investigated in a longitudinal study over a period of one to six years. Disease activity of 105 SLE patients was measured according to the ECLAM score. In the cohort of SLE patients 63% were positive for the IgG class, 40% for the IgA and 57% for the IgM class specific anti-dsDNA ELISA. Sensitivity (79%) and specificity (99%) for the diagnosis of SLE appeared to be highest for the ELISA measuring all isotypes of anti-dsDNA antibodies. The concentrations of anti-dsDNA isotypes showed a strong correlation with disease activity. Analysing the relationship between IgG, IgA and IgM anti-dsDNA antibody isotypes and clinical manifestation, we found a significant association of the IgM isotype with cutaneous involvement and of the IgG isotype with lupus nephritis. The IgG/IgM ratio of anti-dsDNA antibodies represented a significant parameter to distinguish patients with lupus nephritis from those without renal involvement. In the longitudinal study, a continuous ratio under 0.8 was associated with absence of renal involvement throughout the investigated period. In conclusion, the evaluation of anti-dsDNA isotypes provides a diagnostic tool to define subsets within SLE patients with different clinical manifestations. In particular, the IgG/IgM ratio of anti-dsDNA antibodies could be used as a prognostic marker for lupus nephritis during the course of the disease.

Attachment of the soluble complement regulator factor H to cell and tissue surfaces: relevance for pathology.

Complement is a central element of innate immunity and this vital defense system initiates and coordinates immediate immune reactions which attack and eliminate microbes, foreign particles and altered self cells. Newly generated activation products are extremely toxic and consequently, activation is highly restricted in terms of time and space. The initial activation of the alternative complement pathway occurs continuously and the early phase acts indiscriminatoryl and forms on any surface. However, the system discriminates between self and foreign, and therefore allows activation on foreign surfaces e.g. microbes, and restricts activation on host cells. Consequently, self cells and tissues are protected from the harmful activation products. This protection is mediated by specific regulators or inhibitors, which exist in the fluid phase and/or in membrane-bound forms. Here we review a novel mechanism, i.e. the attachment of the soluble complement regulator factor H to the surface of self cells. This attachment, which is demonstrated experimentally by means of immunofluorescense microscopy and by flow cytometry, increases the inhibitory potential at the cell surface and mediates protection by reducing the local formation of toxic inflammatory products. This attachment is highly relevant and has pathophysiological consequences in several human diseases, including Factor H-associated hemolytic uremic syndrome (FH-HUS), membrano-proliferative glomerulonephritis type II, recurrent microbial infections and chronic inflammation, e.g. rheumatoid arthritis and immune evasion of tumor cells. Defects of this safeguard activity have been recently understood in patients with FH-HUS. Point mutations in the Factor H gene occurring in the C-terminus of the protein result in impaired cell binding capacity of Factor H and, consequently, during an inflammatory insult endothelial cells are not properly protected and are damaged.

Polarized function of thick ascending limbs of Henle cells in osmoregulation.

BACKGROUND: Organic osmolytes are necessary for osmoregulation in mammalian kidney. Since renal epithelial cells in many cases possess specific mechanisms both for uptake and osmotically regulated release, we investigated their localization in polarized cells. METHODS: An immortalized epithelial cell line derived from the thick ascending limb of Henle's loop (TALH) was used to examine the transport characteristics of the apical and basolateral plasma membranes for osmotic regulation of organic osmolytes. Cells were cultured on filters in a two-compartment chamber. RESULTS: In culture under hypertonic conditions the TALH cells accumulated in the following balance: sorbitoverline> betaine = myo-inositoverline> glycerophosphoryl choline (GPC). When extracellular osmolarity was decreased, then sorbitol was released on the apical side, whereas betaine and myo-inositol efflux occurred on the basolateral side. GPC release showed no preference of either side. Taurine did not seem to be necessary for osmoregulation under these conditions. Osmotically regulated myo-inositol and betaine uptake was located on the apical side, and choline uptake took place on both sides equally. CONCLUSION: These results show that in renal epithelial cells, both osmotically induced release and the uptake of organic osmolytes are divided between the apical and the basolateral sides. This might be important for volume regulation.

Evidence for a sorbitol transport system in immortalized human renal interstitial cells.

Sorbitol plays a major role in the maintenance of cell volume and functional integrity of several renal cells. Sorbitol synthesis takes place in inner collecting duct cells, whereas sorbitol dehydrogenase activity, which catalyzes the degradation of sorbitol to fructose, could mainly be detected in renal inner medullary interstitial cells. Therefore, we supposed that interstitial cells would require a sorbitol transport into the cells. However, such a transport system has not yet been described. Therefore, we have characterized the uptake of sorbitol in immortalized interstitial TK-173 cells, which were derived from human renal fibroblasts. Comparable to fresh isolated renal fibroblasts of the rat, immortalized TK-173 cells have a high sorbitol dehydrogenase activity. In this report, a temperature-dependent sorbitol uptake with saturation kinetics could be detected in immortalized TK-173 cells. The transport is characterized by a high velocity (Vmax 84 mmol/l x h) and an apparent Km of 10 mmol/l. The sorbitol uptake is independent of membrane potential, sodium, and chloride. Altogether, the physiological characteristics of this sorbitol transport are different from those of the osmotically regulated sorbitol efflux from epithelial cells. These results provide evidence that TK-173 cells derived from renal fibroblasts have a specific sorbitol transport. Furthermore, these data suggest a cooperation between epithelial and interstitial cells concerning osmoregulation.

Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization.

The CC chemokine receptor CCR5 mediates chemotaxis of leukocytes and serves as a principal co-receptor for macrophage-tropic human immunodeficiency virus type 1. To identify determinants on the CCR5 carboxyl-terminal domain that regulate receptor signaling and internalization, we generated several CCR5 mutants, which were progressively shortened from the COOH terminus or had carboxyl-terminal serine, cysteine, or leucine residues substituted by alanine and expressed them in RBL-2H3 cells. Using fluorescence resonance energy transfer between beta-arrestin and CCR5 tagged with cyan and yellow variants of green fluorescent protein, we show that high affinity association of the two molecules in living cells requires intact carboxyl-terminal serine phosphorylation sites. Phosphorylation-deficient truncation or Ser/Ala replacement mutants of CCR5 mediated a sustained calcium response and enhanced granular enzyme release in RANTES-stimulated cells. Carboxyl-terminal serine residues are critically involved in CCR5 endocytosis and a dileucine motif, similar to that implicated in the regulation of CXCR2 and CXCR4, contributes to the internalization of CCR5 in a phosphorylation-independent manner. Despite their prominent role in receptor desensitization and internalization, beta-arrestins are dispensable for the CCR5-mediated stimulation of mitogen-activated protein kinase pathways in RBL-2H3 cells. We also show that CCR5 is palmitoylated on carboxyl-terminal cysteine residues. Inhibition of CCR5 palmitoylation by alanine mutagenesis of cysteines or treatment with a palmitate analogue inhibitor profoundly reduces phorbol 12-myristate 13-acetate- and RANTES-induced receptor phosphorylation, homologous desensitization, and internalization. Alanine mutagenesis of serine, cysteine, or leucine residues or the limited carboxyl-terminal truncation of CCR5 did not impair chemokine-stimulated migration of RBL-2H3 cells. Together these results indicate that post-translational modifications of carboxyl-terminal serine and cysteine residues have a significant impact on receptor deactivation and internalization.

Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5Delta32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5Delta32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n=346) and in CD patients (n=235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5Delta32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy-Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, Delta=1 9.6%, P=0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, Delta=7.3%, P=0.136) in carriers of the mutant CCR5Delta32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility.

Identification of proteins in human prostate tumor material by two-dimensional gel electrophoresis and mass spectrometry.

Protein patterns in cells collected from benign prostatic tissues and prostate carcinomas were analyzed using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Polypeptide expression was evaluated by computer-assisted image analysis (PDQUEST). Proteins expressed by prostate tumors were identified via in-gel digestion and subsequent matrix-assisted laser desorption/ionization mass spectrometry. In addition to cytoskeletal and mitochondrial proteins, a 40-kDa protein was identified as prostatic acid phosphatase (PAP). PAP expression decreased approximately twofold between benign and malignant tissue. Increased expression of heat shock protein 70 and decreased expression of tropomyosin 1 were also observed in the malignant tissue. The analysis of prostate material by two-dimensional gel electrophoresis and mass spectrometry shows that particular proteins are of interest as markers of disease.

Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling.

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor G(i alpha) inhibitory to G(beta gamma) stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of G(beta gamma)-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655-662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949--953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, beta-arrestin, and G(beta), in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or G(beta) antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with G(beta) antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, beta-arrestin, and G(beta) has varying consequences on their ability to associate. As a result, increased availability of and signaling via G(beta gamma) could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing G(beta gamma) signaling and opioid tolerance formation.