Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro

Extracellular calcium/polyvalent cation-sensing receptors (CaR) couple to G proteins and contain highly conserved extracellular cysteine residues. Immunoblotting of proteins from rat kidney inner medullary collecting duct endosomes with CaR-specific antibodies reveals alterations in the apparent molecular mass of CaR depending on protein denaturation conditions. When denatured by SDS under nonreducing conditions, CaR migrates as a putative dimeric species of 240-310 kDa. This is twice the predicted molecular mass of the CaR monomer observed after SDS denaturation in the presence of sulfhydryl-reducing agents. In sucrose density gradients, Triton X-100-solubilized CaR sediments as a 220-kDa complex, not explainable by binding of G proteins to CaR monomers. Treatment of Triton-soluble CaR with divalent (Ca2+, Mg2+) and trivalent (Gd3+) metal ion CaR agonists, but not monovalent ions (Na+), partially shifts the electrophoretic mobility of CaR under reducing conditions from a predominantly monomeric to this putative dimeric species on immunoblots in a manner similar to their rank order of functional potency for CaR activation (Gd3+ > Ca2+ > Mg2+). This Ca2+ effect is blocked by pretreatment with N-ethylmaleimide. We conclude that disulfide bonds present in CaRs mediate formation of dimers that are preserved in Triton X-100 solution. In addition, CaR exposure to Ca2+ induces formation of additional disulfide bonds within the Triton-soluble CaR complex.     

Efficacy and acceptability of two monophasic oral contraceptives containing ethinylestradiol and either desogestrel or gestodene

In this study, we have examined the effects of authentic nitric oxide (NO), NO+ (NOBF4), glutathione (GSH), glutathione disulphide (GSSG), and S-nitrosoglutathione (GSNO) in the presence and absence of Cu2+, which thermally releases NO from S-nitrosothiols on the transport of L-arginine into the human platelet. The K(M,apparent) was unaffected by NO, NO+, GSH, and GSNO. However, Cu2+ lowered K(M,apparent) by approximately 2.85-fold. Cu2+-dependent lowering of K(M,apparent) was also observed, albeit to a smaller extent when this ion was mixed with GSH (approximately 1.9-fold lower) and GSNO (approximately 2.0-fold). GSSG also lowered K(M,apparent) by approximately 1.5-fold. The Vmax,apparent of L-arginine uptake was unaffected by NO, NO+, GSH, and Cu2+. Vmax,apparent was stimulated by to the largest extent by GSNO (approximately 2.28-fold) and GSNO plus Cu2+ (approximately 2.7-fold). GSSG and GSH plus Cu2+ also increased Vmax,apparent by approximately 1.9-fold. When these parameters are expressed in terms of transport efficiency (Vmax/K(M)) the largest effect of nearly 4.7-fold (over controls) was obtained by a combination of GSNO plus Cu2+. These results suggest that platelet L-Arg transport is not affected either by NO or NO+ but by a thiol-disulphide exchange reactions on the platelet L-Arg transporter, brought about by GSNO and GSSG. Based on these results, a GSNO/GSSG/Cu2+ dependent regulatory mechanism for the uptake of L-arginine in human platelets has been proposed.     

Synthesis and structure-activity data of some new epibatidine analogues

The high-pressure Diels-Alder reaction of N-carbomethyoxypyrroles and phenyl vinyl sulfone affords versatile intermediates for the palladium-catalyzed preparation of new epibatidine analogues. Structure-activity relationships of new epibatidine analogues are presented. High affinities of Ki = 0.81 and 2.6 nM for the [3H]-cytisine rat brain nicotinic acetylcholine binding sites were found for the 5-pyrimidinyl and the 5-(2-amino)-pyrimidinyl epibatidine analogues, respectively.     

The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals

The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism. The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group. Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B. However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes. E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine. E(Pc) is expressed ubiquitously in Drosophila embryogenesis. E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites. Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter. This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function. Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans. Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C. elegans and humans. The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.     

Early mortality after surgical repair of postinfarction ventricular septal rupture: importance of rupture location

BACKGROUND: The aim of this study was to identify factors influencing early outcome after surgical treatment of postinfarction ventricular septal rupture. We investigated the influence of proximal or distal rupture location. METHODS: Between 1980 and 1992 109 patients were treated surgically for ventricular septal rupture using a standardized technique. A division in time periods was made. The rupture was categorized according to its anterior or posterior site and proximal or distal location. RESULTS: The 30-day mortality rate was 27.5%. Multivariate logistic regression analysis identified preoperative shock (p = 0.0007) and right atrial oxygen saturation less than 60% (p = 0.021) as predictors for early death; the risk for early death declined over the time periods from 50% to 12.8% (p = 0.0007). Proximal ventricular septal rupture location (p = 0.0092) and interval between infarction and ventricular septal rupture less then 1 day (p = 0.034) were risk factors for the occurrence of preoperative shock. CONCLUSIONS: Proximal ventricular septal rupture location was the main determinant of preoperative cardiogenic shock, which in turn was the strongest predictor of early mortality. Over the time periods a decrease in early mortality was reached.     

Acute appendicitis following laparoscopic inguinal hernioplasty. Coincidence or complication?]

The Authors report two cases of renal leiomyosarcomas with atypical clinical features. Despite a malignant histological picture, nephron-sparing surgery was performed. The two patients are alive and disease-free at six years and fifteen months respectively. Specific radiologic findings, indications and rationale for conservative treatment are discussed.     

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.     

Matrix metalloproteinases contribute to the blood-brain barrier disruption during bacterial meningitis

In this study, we investigated the involvement of matrix metalloproteinases (MMPs) in the pathophysiology of bacterial meningitis. By using an enzyme immunoassay, high concentrations of MMP-9 were detected in the cerebrospinal fluid (CSF) of adult patients with bacterial meningitis but not in controls, and in patients with Guillain-Barré syndrome. Moreover, we observed significantly elevated concentrations of the tissue inhibitor of metalloproteinase-1 (TIMP-1) in the CSF of patients with bacterial meningitis, compared with controls. In a rat model of meningococcal meningitis, intracisternal injection of heat-killed meningococci caused a disruption of the blood-brain barrier (BBB), an increase in intracranial pressure, and CSF pleocytosis paralleled by the occurrence of MMP-9 activity in the CSF 6 hours after meningococcal challenge. The MMP inhibitor batimastat (BB-94) significantly reduced the BBB disruption and the increase in intracranial pressure irrespective of the time of batimastat administration (15 minutes before and 3 hours after meningococcal challenge) but failed to significantly reduce CSF white blood cell counts. In conclusion, our results suggest that MMPs are involved in the alterations of BBB permeability during experimental meningococcal meningitis.     

Donor factors affecting outcome after pancreas transplantation

In this study, we demonstrated that Px grafts from donors older than 45 years are associated with an increased risk of developing poor glycemic control and premature loss of Px function. Previous studies corroborate our finding that age of the donor is the principal donor characteristic impacting postoperative Px survival. Whereas prior studies also implicated hyperamylasemia as a factor which contributes adversely to outcome, we were unable to demonstrate a significant influence of donor hyperamylasemia on long-term graft survival, although it did correlate with the degree of immediate postoperative pancreatitis and with the need for oral hypoglycemic agents. Similarly, elevated blood glucoses in the donor, which can be a result of many other factors unrelated to the quality of the graft, did not predict a poor outcome in the recipient. NHB donor pancreata did as well as HB pancreata with regards to all postoperative functional parameters. A marginally increased risk of developing major complications was associated with older donors. Despite the frequent use of non-ideal donors, including older and NHB donors, excellent overall Px graft survival can be achieved. Although the quality of the pancreas graft was not directly addressed in this study, we believe irrespective of hyperglycemia or hyperamylasemia, subjective assessment of organ quality by an experienced transplant surgeon is the most important determinant of suitability.