Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

SPAG11/isoform HE2C, an atypical anionic beta-defensin-like peptide

A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded beta-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates. An approximately 6kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography. By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C. Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase. This was confirmed by N-terminal sequencing. In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells. Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide. Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay. Purified recHE2C strongly bound to Escherichia coli DH5alpha and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria. Different from the previously described beta-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of -1. Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.     

The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Increased albumin plasma efflux contributes to hypoalbuminemia only during early phase of sepsis in rats

The mechanisms leading to hypoalbuminemia in sepsis were explored by measuring plasma volume, albumin distribution, plasma albumin transcapillary escape rate (TER), and efflux (TER x albumin intravascular pool). These parameters were quantified in infected rats, injected intravenously with live Escherichia coli, and pair-fed and well-fed rats using an injection of (35)S-albumin and measuring plasma and whole body albumin concentrations. Animals were studied on days 1, 6, and 10 after infection. In pair-fed rats, neither albumin distribution nor exchange rate between the intra- and extravascular compartments was modified. The increase of plasma volume after infection partly explained hypoalbuminemia. Infection resulted in a reduction of the total albumin pool of the body all along the experimental period, indicating a net loss of the protein. Albumin TER (%/day) was significantly increased 1 and 6 days after infection, but the absolute efflux was increased only on day 1. Normal values were observed on day 10. Therefore, an accelerated plasma efflux contributes to hypoalbuminemia only during the early period of sepsis. During this phase, the protein was retained in the extravascular space where it was probably catabolized. Later on, other factors are probably involved.     

Increased platelet-activating factor-induced periventricular brain microvascular constriction associated with immaturity

Oxidant stress contributes to the pathogenesis of hypoxic-ischemic encephalopathies. Platelet-activating factor (PAF) is generated during oxidant stress. We studied the vasomotor mode of actions of PAF on periventricular (PV) microvessels of fetal ( approximately 75% of term), newborn (1-3 days), and adult pigs. PAF constricted PV microvessels from fetal (29.27 +/- 2.6%) and newborn (22.14 +/- 3.2%) pigs but was ineffective in adults (<2.5%). Specific [(3)H]PAF binding was greater in fetus and newborn than in adults; a concordant developmental PAF-induced inositol phosphate formation was observed. PAF-induced vasoconstriction was abrogated by thromboxane A(2) (TXA(2)) synthase and receptor inhibitors, calcium channel blockers, and by removal of endothelium; vasoconstriction to TXA(2) mimetic U-46619 did not differ with age. Immunoreactive TXA(2) synthase expression and PAF-evoked TXA(2) formation revealed a fetus> newborn>adult profile. Thus the greater PAF-induced PV microvascular constriction in younger subjects seems attributable to greater PAF receptor density and mostly secondary to TXA(2) formation from endothelium. The resulting decrease in blood flow may contribute to the increased vulnerability of the PV brain regions to oxidant stress-induced injury in immature subjects.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster

In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.