Isolation of Extracellular Recombinant Fragment of Rat Connexin-43

Analysis of membrane topology of connexin-43 (Cx-43) made it possible to determine one of its extracellular fragments (E2): Q173-1208. The nucleotide sequence of this fragment was cloned into pCBDQ and pHPML vectors containing the sequences of calmodulin-binding domain (CBD) and HPML-domain of Ca-ATPase of human hPMCA4b cells plasma membrane. This yields two chimeric proteins with N-terminal 6-histidine motif containing the extracellular fragment Cx43 E2 and one of hPMCA4b domains (Cx43-CBD and Cx43-HPML). The latter were inserted into the recombinant polypeptide to improve solubility and enhance immunogenicity of the product. Affinity-purified on Ni-NTA agarose recombinant Cx43-CBD was used for immunization of mice and obtaining of monoclonal antibodies. Primary selection of clones was carried out by solid-phase IEA with immobilized Cx43-HPML and by immunoblotting with Cx43-HPML. The positive clones were tested immunohistochemically on rat brain sections. This two-stage testing made it possible to select two hybridomas, which produced monoclonal antibodies to Cx43 in native conformation. The resultant antibodies can be used in vitro and in vivo for immunophenotyping of various Cx43-positive cells.     

Identification of active variants of PARF in human pathogenic group C and group G streptococci leads to an amended description of its consensus motif

Certain streptococcal M proteins bind collagen via an octapeptide motif that is located in their hypervariable N-terminal region. The interaction with this extracellular matrix protein enhances adhesion to the host tissue and thereby facilitates infection. Moreover, it has the side effect of eliciting collagen autoimmune responses, a phenomenon which is also observed in patients with acute rheumatic fever. Therefore, the octapeptide motif was named peptide associated with rheumatic fever (PARF). Only a comprehensive characterization of the collagen-binding M proteins and their collagen-binding motifs will allow the investigation of their associations with certain streptococcal infections and their sequelae. Therefore, a collection of Streptococcus dysgalactiae equisimilis strains that were isolated from infected humans was examined, in order to identify collagen-binding proteins and motifs. Strains that bound collagen independent of a hyaluronic acid capsule belonged to 7 distinct types of the emm gene, which codes for the M protein (emm types). Only one of these emm types was previously described as collagen-binding. Five possessed a PARF sequence. The other 2 emm types stC2sk.0 and stG2574 had PARF-like motifs that diverged from the previously described consensus sequence AXYLZZLN but were able to induce collagen autoimmunity when injected into mice. The results led to the amended PARF consensus (A/E/T)XYLXXLN. Moreover, they demonstrate a predictive power regarding collagen-binding and elicitation of collagen autoimmunity, indicating that PARF may be one of the markers for strains that cause collagen-dependent acute rheumatic fever.     

Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors

To develop a collagen-based wound targeting repair system, we introduced two collagen-binding domains (CBDs) into the human basic fibroblast growth factor (bFGF). Three expression vectors were constructed: the first one (named V-bFGF) contained bFGF and the CBD WREPSFCALS derived from von Willeband's factor (vWF); the second (named C-bFGF) contained bFGF and the CBD TKKTLRT derived from collagenase; the third (named bFGF) was bFGF as a control. The recombinant proteins of V-bFGF and C-bFGF were demonstrated to retain both growth factor activity and collagen-binding activity. We found that C-bFGF possessed higher collagen-binding ability than V-bFGF. The targeted repair systems consisting of collagen scaffolds and CBD-bFGFs were assembled in vitro and then implanted subcutaneously. Results showed that C-bFGF promoted vascularization at the implanted sites more effectively than V-bFGF. Histological analysis showed more cells migrated into collagen scaffolds incorporated with C-bFGF than those with V-bFGF. These data suggested that the higher collagen-binding ability the CBD-bFGF possessed, the more significant vascularization, and cellularization were observed. In summary, CBD-bFGF/collagen system could be used as a targeted repair system with beneficial effects of the restriction of bFGF diffusion, the prolonging of bFGF activity, and the targeted promotion of vascularization and cellularization.     

Somatic mutations of the KEAP1 gene in common solid cancers

Yoo N J, Kim H R, Kim Y R, An C H & Lee S H
(2012) Histopathology  60, 943–952 Somatic mutations of the KEAP1 gene in common solid cancers Aims: KEAP1 inhibits nuclear factor erythroid 2‐related factor 2 (NRF2)‐induced cytoprotection, and is considered to be a candidate tumour suppressor. Somatic mutation of NRF2 has been analysed in a wide variety of human cancers, whereas somatic mutation of KEAP1 has been reported only in lung and gall bladder cancers. The aim of our study was to investigate whether KEAP1 mutations are widespread in human cancers. Methods and results: We analysed 499 cancer tissues from lung, breast, colon, stomach, liver, larynx and prostate, and leukaemias, by single‐strand conformation polymorphism analysis. We detected somatic mutations of KEAP1 in gastric (11.1%), hepatocellular (8.9%), colorectal (7.8%), lung (4.6%), breast (2.0%) and prostate (1.3%) carcinomas. Allelic losses of the KEAP1 locus were identified in 42.9% of cancers with KEAP1 mutations, but no NRF2 mutations were detected in these cancers. The NRF2‐activated cytoprotective proteins (NAD(P)H dehydrogenase quinone 1 and glutamine‐cysteine ligase catalytic subunit) were expressed in all of the cancers with KEAP1 mutations. Conclusions: Our data show that KEAP1 mutations occur widely in solid cancers, irrespective of histological type. Biallelic inactivation of KEAP1 and increased levels of cytoprotective proteins in the cancers suggest that KEAP1 mutations might protect cancer cells from oxidative insults and play a role in the development of solid cancers.     

Analysis of the Three Dimensional Relationships Among Components of Human Vascular Subendothelium by Stereo-Tilt Immunoelectron Microscopy

Abstract

Stereo-tilt immunoelectron microscopy offers a method for analyzing the inter-relationships among components of the subendothelium at the 3 dimensional level. It has been demonstrated that von Willebrand factor (vWF) is important in the process of platelet adhesion following endothelial injury by serving as a bridge between constituents of the vascular subendothelium and platelet membrane receptors. We had previously shown that type VI collagen microfibrils serve as a binding site for vWF in human vascular subendothelium, and immunolabeling studies of vascular tissues by routine transmission electron microscopy showed apparent co-localization of the 2 proteins.

We investigated the relationships among vWF, fibrillin and type VI collagen in human vascular subendothelium with double labeling immunogold localization techniques in conjunction with stereo-tilt electron microscopy to examine the 3 dimensional ultrastructure of vWF-microfibril complexes.

Our studies confirmed that vWF co-localizes with the type VI collagen microfibrils in subendothelium and not with fibrillin as suggested by others.

The vWF is present in subendothelium in the form of electron-dense aggregates having diameters varying between 65–80 nm that decorate the type VI collagen microfibrils and having structural similarities to intracellular Weibel- PaIade bodies. The 3 dimensional representations show that the labeling of the 2 molecular species are not necessarily directly adjacent to one another and can be some distance from each other. These results confirm our previous observations on the association between type VI collagen and vWF. Importantly, they also show that colocalization studies where immunolabeling is performed in situ and observed by electron microscopy must be evaluated 3 dimensionally before one can determine conclusively that the 2 components are associated with one another. (The J Histotechnol 21:205–212, 1998)     

Exercise-induced shear stress is associated with changes in plasma von Willebrand factor in older humans

Shear stress is the frictional force of blood against the endothelium, a stimulus for endothelial activation and the release of von Willebrand factor (vWF). This study tested the hypothesis that the increase in shear stress associated with exercise correlates with plasma vWF. Young (n = 14, 25.7 ± 5.4 years) and older (n = 13, 65.6 ± 10.7 years) individuals participated in 30 min of dynamic handgrip exercise at a moderate intensity. Brachial artery diameter and blood flow were measured using ultrasound Doppler and blood samples were collected before, immediately after, and following 30 min of recovery from exercise with plasma levels of vWF. Plasma levels of vWF increased (P < 0.05) by 6 ± 2% in young individuals and 4 ± 1% in older individuals immediately after exercise. The change in plasma vWF was linearly correlated with the increase in shear stress during exercise in older individuals (post-exercise: r = 0.78, 30 min recovery: r = 0.77, P < 0.01), but no association was found in the young individuals. These changes in plasma levels of vWF in humans suggest that aging influences endothelial activation and hemostasis.     

Liposomes or traditional adjuvants: induction of bactericidal activity by the macrophage infectivity potentiator protein (Mip) of Neisseria meningitidis

Currently, one of the main approaches to achieve a vaccine for serogroup B Neisseria meningitidis is based on outer membrane proteins with low antigenic variability among strains. Since these proteins tend to be minor components of the outer membrane, recombinant production is required to obtain them in sufficient amounts for evaluation and development of vaccines. In this study, we analysed the ability of recombinant macrophage infectivity potentiator (rMip) protein to induce protective bactericidal activity in mice. The rMip protein was cloned from N. meningitidis strain H44/76 and was used to immunise mice, and the sera obtained were tested against the homologous and several heterologous N. meningitidis strains. The sera were obtained using the rMip alone, with adjuvant Al(OH)₃, or after inclusion into liposomes. Bactericidal activity was variable depending on the strain, although high titres were seen against strains H44/76 and NmP27. Liposomes enhanced fourfold the reactivity against the homologous strain. The results presented suggest that the rMip protein should be considered a promising candidate for the improvement of future protein‐based vaccines.     

Different anaesthesia methods affect the development of hepatoblastoma after platelet activation

This study aims to compare the influence of different anaesthesia methods on the mechanisms involved in the development of hepatoblastoma (HB). HB rabbit models were constructed and divided into three groups: disoprofol, pentobarbital sodium and HB groups. After anaesthesia, rabbit blood was collected from the tail vein. Haematological analysis (platelets) and an ELISA was used to measure the thrombopoietin (TPO) and 5‐hydroxytryptamine (5‐HT). Flow cytometry was used to determine expression of P‐selectin and PAF. The expression of 5‐HTR2B, PCNA, vWF, P70s6k, 4E‐BP1, mTOR and FRAP was determined in the tumour itself or in vascular tissues obtained from the rabbits. The platelet content in the disoprofol group. The content or expression of TPO, 5‐HT, P‐selectin, PAF, 5‐HTR2B, PCNA, vWF, P70s6k, 4E‐BP1, mTOR and FRAP was significantly higher in the disoprofol group compared to pentobarbital sodium and HB groups. Expression of these molecules was much higher in the pentobarbital sodium group compared with the HB group. These findings suggest that disoprofol anaesthesia can promote HB development via the mTOR/p70S6K1 and FRAP signalling pathway.     

Assessment of immunogenic characteristics of Hemiscorpius lepturus venom and its cross-reactivity with venoms from Androctonus crassicauda and Mesobuthus eupeus

Abstract

Hemiscorpius lepturus (H. lepturus), one of the most venomous scorpions in tropical and sub-tropical areas, belongs to the Hemiscorpiidae family. Studies of antibodies in sera against the protein component of the venom from this organism can be of great use for the development of engineered variants of proteins for eventual use in the diagnosis/treatment of, and prevention of reactions to, stings. In the present in vitro study, the proteins of H. lepturus venom, which could specifically activate the production of immunoglobulin G (IgG) in victims accidently exposed to the venom from this scorpion, were evaluated and their cross-reactivity with venoms from two other important scorpion species including Androctonus crassicauda and Mesobuthus eupeus assessed. H. lepturus venom was analyzed with respect to its protein composition and its antigenic properties against antibodies found in sera collected from victims exposed to the venom of this scorpion within a previous 2-month period. The cross-reactivity of the H. lepturus venom with those from A. crassicauda and M. eupeus was assessed using ELISA and immunoblotting. Electrophoretic analysis of the venom of H. lepturus revealed several protein bands with weights of 8–116 KDa. The most frequent IgG-reactive bands in the test sera had weights of 34, 50, and 116?kDa. A weak cross-reactivity H. lepturus of venom with venoms from A. crassicauda and M. eupeus was detected. The results of immunoblotting and ELISA experiments revealed that H. lepturus venom activated the host immune response, leading to the production of a high titer of antibodies. Clearly, a determination of the major immunogenic components of H. lepturus venom could be valuable for future studies and ultimately of great importance for the potential production of recombinant or hypo-venom variants of these proteins.     

Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants

The P3 protein encoded by Shallot yellow stripe virus onion isolate (SYSV-O) interacted in the Yeast Two-hybrid (Y2H) system and in co-immunoprecipitation (Co-IP) assays with the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein that is encoded by the rbcL gene of its onion host. Dissection analysis by Y2H showed that the main part of SYSV P3 (amino acids 1–390) and onion RbcL (amino acids 1–137) were responsible for the interaction. The P3 proteins encoded by Onion yellow dwarf virus (OYDV), Soybean mosaic virus Pinellia isolate (SMV-P), and Turnip mosaic virus (TuMV) also interacted with RbcL, suggesting that a P3/RbcL interaction might exist generally for potyviruses. An interaction between P3 of these potyviruses and the small subunit of RubisCO (RbcS) was also demonstrated. Moreover, the P3N-PIPO protein encoded by a newly identified open reading frame embedded within the P3 cistron also interacted with both RbcL and RbcS. It is possible that the potyvirus P3 protein affects the normal functions of RubisCO which thus contributes to symptom development.     

Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia

 Nine axenic isolates of Giardia originating from four different host species in Switzerland were subjected to genetic analysis using the polymerase chain reaction (PCR) to amplify segments of genes encoding different trophozoite variant surface proteins (VSPs). Three genotypes were identified on the basis of product yield, size, and restriction-fragment-length polymorphisms (RFLPs). Five G. duodenalis isolates (O1, B1, B2, B3-1A1 and C1 – from a sheep, three calves and a dog, respectively) were classified as belonging to genetic group I of Andrews et al. (1989). DNA amplified from the VSP genes tsp11, tsa417 and vsp1267 of these isolates was indistinguishable in size and restriction characteristics from that amplified from group-I Giardia isolated from humans in Australia. One human-derived Swiss isolate (H2-17A1), typed as belonging to genetic group II, yielded a vsp1267-specific PCR product that was indistinguishable by size or restriction sites from the equivalent 1.6-kb product amplified from human-derived Australian group-II organisms. This isolate also yielded 1.8-kb tsp11 and 0.52-kb tsa417/tsp11-like PCR products possessing RFLPs typical of group-II organisms. Three isolates (O2-4A1, O3 and H3-15K2 – originating from two sheep and a human, respectively) represent a novel genotype that is closely related to genetic groups I and II. These three isolates exhibited identical RFLPs in their tsp11 PCR products and failed to yield a vsp1267 PCR product. An antiserum specific for the 90-kDa VSP of the sheep-derived clone O2-4A1 reacted strongly by immunofluorescence and on Western blots with surface proteins from the O2, O3 and H3 isolates only – consistent with the genetic classification determined above. The data provide no evidence for the occurrence of host-specific genotypes. Received: 13 January 1995 / Accepted: 20 May 1995     

Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species

Centromeres play an important role in chromosome transmission in eukaryotes and comprise specific DNA and proteins that form complexes called kinetochores. In tobacco, although a centromere-specific histone H3 (NtCENH3) and centromeric DNA sequence (Nt2-7) have been identified, no other kinetochore components have been determined. In this study, we isolated and characterized cDNAs encoding two centromeric proteins CENP-C and MIS12 from Nicotiana tabaccum. Two CENP-C homologues, NtCENP-C-1 and -2, isolated from N. tabaccum were similar to CENP-C from N. sylvestris and N. tomentosiformis, respectively. Similarly, two Mis12 homologues, NtMIS12-1 and -2, in N. tabaccum were shown to originate from N. sylvestris and N. tomentosiformis, respectively. Both respective homologues for CENP-C and Mis12 were expressed at the same level. This indicates that in a tetraploid species, N. tabaccum, two ancestral genes encoding the centromeric proteins participate equally in the functioning of centromeres.     

Potential cross‐reactivity of monoclonal antibodies against clinically relevant mycobacteria

Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette–Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non‐tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation‐inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody‐producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme‐linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest cross‐reactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in‐silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones.     

Synthesis and Characterization of Constitutional Isomeric Poly(amide‐ester)s from Adipoyl Chloride and 4‐(2‐Aminoethyl)phenol

Ordered [head‐to‐head (H‐H) or tail‐to‐tail (T‐T)] poly(amide‐ester) ( 6 a ) was prepared by polycondensation from adipoyl chloride ( 1 ) and N,N ′‐bis(4‐hydroxy phenethyl)adipamide ( 5 ), which was prepared from the selective acylation of 4‐(2‐aminoethyl)phenol ( 2 ) with N,N ′‐adipoylbis[benzoxazoline‐2‐thione] ( 4 ). And H‐T ordered poly(amide‐ester) ( 6 b ) was prepared by self‐polycondensation of 4‐carboxy‐N‐(4‐hydroxyphenethyl)‐adipamide ( 7 ) in the presence of DBOP. Random poly(amide‐ester) ( 6 c ) was synthesized by polymerization of 1 with 2 . The microstructure of the polymers obtained was investigated by ¹H and ¹³C NMR spectroscopy, and it was found that the polymers obtained had the expected ordered structure. Furthermore, DSC and WAXD results demonstrated that the constitutional regularity of polymers influenced their thermal properties and crystallinity.     

Novel function of histamine signaling via histamine receptors in cholesterol and bile acid metabolism: Histamine H2 receptor protects against nonalcoholic fatty liver disease

We have reported that the function of histamine and its receptors (HRs) has a close relationship with the development of nonalcoholic fatty liver disease (NAFLD). However, much less is known regarding its pathogenic and molecular mechanism(s), including the early stage of hepatic and intestinal function for lipid and bile acid (BA) metabolism. We used H1R and H2R knockout mice (H1/2R‐KO) to clarify those pivotal roles in cholesterol/BA metabolism, in which H1/2R‐KO mice were separately fed a short‐term 1% cholesterol or cholic acid (CA) diet. [³H]Cholesterol absorption study revealed that significantly enhanced accumulation occurred in the jejunum, blood and liver, but not in the feces, of H2R‐KO mice, compared to wild‐type and H1R‐KO mice. Furthermore, four weeks after the high‐cholesterol diet, the H2R‐KO jejunum but not liver exhibited increased expressions of cholesterol transporters, consistent with higher plasma lipoprotein levels. Five days after CA diet, the H2R‐KO mice showed significantly higher expressions of ileal BA‐reabsorption and hepatic BA‐efflux factors, corresponding to higher serum but lower fecal BA levels. The following long‐term CA diets resulted in severe injury to the H2R‐KO liver. Histamine/H2R signaling might have a protective role in the initial phase during NAFLD progression, correlated with cholesterol and BA metabolism in the liver/intestine.     

Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis

In this study we show that sera from dogs naturally infected with Leishmania infantum contain antibodies that specifically react against the parasite H2B and H4 histones. The Leishmania H2B and the amino-terminal region of the histone H4, expressed as fusion proteins, when confronted with sera from canine viscerocutaneous leishmaniasis (VCL) dogs, were recognized by 63% and 47%, respectively. No reactivity was detected when sera from dogs naturally infected with pathogens other than Leishmania were used. Using a collection of synthetic peptides covering the complete sequence of both proteins, we have determined that the main linear antigenic determinants are located in the amino-terminal domains of these histones. The humoral response against histones H2B and H4 induced during canine leishmaniasis was found to be specific for Leishmania histones, since no cross-reactivity of the VCL sera with mammal histones was observed. Also, a comparative study of the prevalence of antibodies among VCL sera against the four core histones of L. infantum was performed. Although a large heterogeneity of the humoral responses against these proteins was found, histones H2A and H3 seem to be more prevalent immunogens than histones H2B and H4 during canine natural leishmaniasis. The origin of the anti-histone humoral response and its possible implications in the pathogenesis of Leishmania infection are discussed.     

Genetic variation within the NR1H2 gene encoding liver X receptor β associates with insulin secretion in subjects at increased risk for type 2 diabetes

The liver X receptors (LXRs)-α and -β play a crucial role in control of insulin production and secretion in pancreatic β-cells. We hypothesized that common variants in the NR1H2 and NR1H3 genes, encoding LXR-β and -α, respectively, may alter pancreatic β-cell function. One thousand five hundred seventy-four subjects of European ancestry with elevated risk for type 2 diabetes were genotyped for the two NR1H2 single nucleotide polymorphisms (SNPs) rs2248949 and rs1405655 and for the four NR1H3 SNPs rs11039149, rs3758673, rs12221497 and rs2279238, and association studies with metabolic traits were performed. Metabolic characterization comprised an oral glucose tolerance test (OGTT) in all participants and, in addition, a hyperinsulinemic–euglycemic clamp and an intravenous glucose tolerance test (IVGTT) in subsets. One hundred per cent of common genetic variation (minor allele frequency ≥1%) within the NR1H2 and NR1H3 loci (D′ = 1.0; r2 ≥ 0.8) were covered by the six chosen tagging SNPs. NR1H2 rs2248949 was nominally associated with OGTT-derived first-phase insulin secretion and proinsulin conversion to insulin and significantly associated with the AUC of insulin levels during the IVGTT (p = 0.007) after adjustment for age, gender, BMI and insulin sensitivity in the dominant model, with the minor allele conferring reduced pancreatic β-cell function to the carriers. In subjects of European ancestry at increased risk for type 2 diabetes, common variation within the NR1H2 gene impaired insulin secretion, which may facilitate the development of type 2 diabetes.     

Comparative proteomic analysis of passaged Helicobacter pylori

In order to identify the proteins associated with Helicobacter pylori colonization in mice, we used 2‐dimensional gel electrophoresis (2‐DE) to analyze the membrane‐ and soluble‐cellular proteins extracted from H. pylori strain 26695 and the mouse‐passaged homolog 88‐3887. We defined 2‐ and 3‐fold changes in protein expression as the threshold values for differential expression in the membrane‐protein and whole‐cell‐protein fractions, respectively. The differentially expressed proteins were identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF/TOF). A total of 29 proteins, including 16 membrane‐ or membrane‐associated proteins (13 upregulated, 3 downregulated) and 13 cellular proteins (10 upregulated, 3 downregulated) were differentially expressed between the strains 26695 and 88‐3887. Among the upregulated proteins, 10 proteins had been previously shown to be associated with the mouse colonization, and 13 upregulated proteins were shown to be associated with the adaptation of H. pylori in murine hosts for the first time in this study. The identified proteins were classified as proteins related to metabolism, stress response, virulence, or adhesion. The data presented in this report indicated that there were subsets of upregulated proteins in mouse‐adapted H. pylori. In particular, the adhesins, virulence factors, and stress‐response proteins are likely to contribute to colonization in mice. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)