A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics

We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains. Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall. Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin. In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins. Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains. Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation.     

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.     

Dinosaurs and other tetrapods in an Early Cretaceous bauxite-filled fissure, northwestern Romania

The bauxite mine at Cornet near Oradea in northwestern Romania produced thousands of bones in an excavation in 1978, mainly from ornithopod dinosaurs and rarer pterosaurs. Bird specimens reported previously from this fauna are equivocal. The fossils are disarticulated bones in good condition which occur highly concentrated in lenses within bauxite clays, which are dated as Berriasian (earliest Cretaceous). The bauxite represents detrital material washed into deep fissures and caves formed within a karst of uplifted Tithonian (latest Jurassic) marine limestones. The bones are generally uniform in size and shape, and they are abraded, evidence for considerable transport and for winnowing of the deposit. The area was one of several islands on the northern shore of Tethys, and it was inundated by the sea later in the Early Cretaceous. There is evidence for insular adaptations in the dinosaur faunas. The ornithopod dinosaurs may include several taxa, but they are smaller on average than an assemblage of typical Wealden ornithopods, perhaps because of dwarfing on the island. In addition, sauropods are absent and theropods are barely represented in the fauna. The fauna is geographically significant since it shows relationships with western Europe and with Asia.     

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Z _max=2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291–D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Z _max=3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.     

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.     

A simplified 2-D electrophoresis protocol with the aid of an organic disulfide

2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.     

Experimental studies on bacterial product cantastim derived from Pseudomonas aeruginosa. VII. Activation of immune cells of healthy controls and cancer patients

CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa with beneficial effects related to enhancing the immune responses in conditions such as chronic viral and bacterial infections, immunodeficiencies and cancer immunosuppression. The aim of this study was to determine the capacity of this biological product to stimulate in vitro human leukocytes in whole blood. Blood samples from healthy donors and cancer patients were incubated with CS for 24 h and leukocytes were assessed for induction of inflammatory cytokines (TNF-alpha and IL-1beta) by ELISA and expression of early activation marker CD69 by flow-cytometry. For both groups of investigated subjects, the levels of TNF-alpha and IL-1beta in the supernatants of whole blood culture stimulated with CS were significantly higher than in unstimulated cultures, although lower than in LPS-stimulated samples. Stimulation of whole blood cultures with CS increased both the frequency and the expression of CD69 on the surface of T lymphocytes and NK cells. Importantly, this was noticed not only for healthy controls, but also for cancer patients. These data demonstrate the capacity of bacterial immunomodulator CS to activate human leukocytes of healthy subjects and cancer patients.     

Common polymorphisms of calpain-10 and the risk of polycystic ovary syndrome in Tunisian population: a case–control study

Recent studies have suggested that calpain-10 (CAPN10) gene polymorphisms play a role in the susceptibility to polycystic ovary syndrome (PCOS). The aim of the present study was to evaluate the possible association between three single nucleotide polymorphisms (SNPs) in CAPN10 gene: UCSNP-43 (rs3792267), UCSNP-19 (rs3842570), and UCSNP-63 (rs5030952) and PCOS in Tunisian cases and control women. Study subjects included 127 women with PCOS (mean age 29.8 ± 4.7 year) and 150 healthy women (mean age 33.5 ± 5.6 year). CAPN10 genotyping was carried-out by direct PCR and PCR–RFLP. Linkage disequilibrium pattern in the genomic region explored was determined by HAPLOVIEW 4.2 while reconstruction of haplotypes was done using PHASE 2.1. The phylogenetic distribution of haplotypes in the population was determined by ARLEQUIN 2.000. Six haplotypes were observed. None of SNPs associated with PCOS or its components while the haplotype H4 associated with the phenotype PCOS-obese (P < 0.025). Moreover the pair of haplotypes H1/H4 strongly associated with high blood-pressure (OR = 14.4, P < 0.012). This work confirms the association of CAPN10 gene with metabolic components in PCOS and highlights the role of haplotypes as strong and efficient genetic markers.     

Defect in insulin receptor phosphorylation in erythrocytes and fibroblasts associated with severe insulin resistance

The insulin receptor is a tyrosine-specific protein kinase. Upon binding of the hormone, the kinase is activated resulting in autophosphorylation of the receptor. This kinase activity has been postulated to be an early step in the transmembrane signaling produced by insulin. To evaluate the physiologic relevance of receptor phosphorylation, we have studied insulin binding and autophosphorylation properties using cells from an individual with a variant of the Type A syndrome of severe insulin resistance and acanthosis nigricans. Erythrocytes and cultured fibroblasts from this individual exhibited normal or near normal 125I-insulin binding. Receptors extracted from erythrocytes with Triton X-100 also exhibited normal 125I-insulin binding and competition curves. Despite this, receptors extracted from both erythrocytes and fibroblasts showed a 50% decrease in insulin-stimulated autophosphorylation. Partially purified receptors from the patient's fibroblasts also exhibited a 40% decrease in their ability to phosphorylate exogenous substrates. These data suggest that the insulin resistance in this syndrome is due to a genetic abnormality which impairs insulin receptor phosphorylation and kinase activity and further support the possible role of receptor phosphorylation and kinase activity in insulin action.