Analysis of the immunoglobulin heavy chain variable region gene expression in Iranian patients with chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) results from clonal expansion of phenotypically mature but functionally immature B-lymphocytes. The incidence of this type of leukemia is low in Asian countries, whereas it is the most frequent type of leukemia in the West. Previous investigations mainly conducted in Western populations have demonstrated non-random rearrangement of certain immunoglobulin variable region heavy (VH) and/or light (VL) chain genes in different groups of B-CLL patients. Little is known about the profile of VH gene expression in Asian patients. In the present study, we determined the frequency of VH gene family usage in 59 Iranian patients with B-CLL. VH gene family of patients was determined by reverse transcriptase-polymerase chain reaction using VH1-VH7 family specific primers. The most frequently expressed VH gene family was found to be VH3 (45.8%) followed by VH4 (32.2%), VH1 (18.6%), VH5 (1.7%) and VH6 (1.7%), with no expression of VH2 and VH7 gene families. The results indicate a lower representation of the VH1 and VH2 gene families and a higher representation of the VH4 gene family in Iranian B-CLL patients compared to Western patients, suggesting involvement of ethnic and/or environmental factors in B-CLL disease initiation.

The effect of dexamethasone on defective nephrin transport caused by ER stress: a potential mechanism for the therapeutic action of glucocorticoids in the acquired glomerular diseases.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5' triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.

Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies.

Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.

Identification of cross-reactive and restricted epitopes localized on human chorionic gonadotropin beta-subunit by monoclonal antibodies.

Human chorionic gonadotropin (hCG) belongs to the family of glycoprotein hormones. All members of the family are composed of an identical alpha subunit and structurally related beta subunit which confers biological specificity. Specific quantification and functional analysis of hCG require the use of monoclonal antibodies recognizing different epitopes of hCGbeta. This study describes the production and characterization of monoclonal antibodies (MAbs) to hCGbeta with no cross-reactivity to other glycoprotein hormones. Spleen cells from Balb/c mice immunized with hCG were fused with mouse SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of highly purified and recombinant glycoprotein hormones, their subunits and peptides representing the C-terminal end of hCGbeta (hCGbeta-CTP) by ELISA and immunoblotting. The affinity constant (K(aff)) was also determined by ELISA. Three murine hybridomas designated G5M1, B12M2 and F4M3 were obtained that secrete MAbs specific for hCGbeta. The G5M1 MAb reacts only with hCGbeta, hCGbeta-CTP and intact hCG with no detectable cross-reaction with hCGalpha or any of the other glycoprotein hormones. The specificity of B12M2 MAb is very similar to G5M1, but it does not react with hCGbeta-CTP. The F4M3 MAb also has similar specificity to G5M1 and B12M2, but it strongly cross-reacts with hLH. The affinity constant (Kaff) of G5M1, B12M2 and F4M3 was found to be 4.28 x 10(9), 5.2 x 10(8), and 1.97 x 10(9) M(-1), respectively. Our results indicate that G5M1 and B12M2 MAbs are specific for hCG and recognize epitopes restricted to hCGbeta, but F4M3 recognizes a common epitope expressed both on hCGbeta and hLHbeta.

Generation and characterization of a mouse monoclonal antibody with specificity similar to staphylococcal protein A (SPA).

Human IgG is comprised of four subclasses (IgG(1), IgG(2), IgG(3), and IgG(4)). Each subclass possesses different biological properties. One of the differential specificities of human IgG subclasses is binding of Fc fragment of IgG(1), 2, and 4 but, not IgG(3) to staphylococcal protein A (SPA). This study was conducted to produce, select and characterize a monoclonal antibody (MAb) recognizing human IgG subclasses with specificity similar to SPA. Splenocytes from Balb/c mice immunized with Fc fraction of a human IgG(1) myeloma protein were fused with Sp2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAb was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. A murine hybridoma designated 6F11E1 was obtained that secretes an MAb specific for the Fc fragment of the immunizing protein. This MAb reacts with isotypic epitope common to IgG(1), 2 and 4 subclasses. An allelic epitope linked to IgG(3) molecules is also recognized by 6F11E1. This pattern of reactivity was found to be highly similar to that of SPA. Our findings imply that similar or overlapping epitopes are recognized by 6F11E1 and SPA.

Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.

Unraveling the molecular make-up of the glomerular podocyte slit diaphragm.

Recent discoveries in podocyte proteins involved in the renal filtration barrier have shed new light on the ultrastructure of the kidney filter and pathogenesis of proteinuria. The identification of nephrin, a component of the slit diaphragm, and the intracellular slit diaphragm associated proteins CD2AP and podocin has demonstrated the existence of proteins that directly contribute to a functional kidney filter. Mutations in the genes for these three proteins result in proteinuria and nephrotic syndrome, and these proteins are also likely to be involved more generally in the pathomechanisms of proteinuria. This new knowledge has been promoted particularly through the powerful methods of molecular genetics and molecular biology. In this minireview, we present the recent progress in research of the podocyte slit diaphragm.

Hepatitis C virus infection in patients with essential mixed cryoglobulinemia, multiple myeloma and chronic lymphocytic leukemia.

Increased prevalence of HCV infection in some lymphoproliferative diseases has been recently reported. In the present study, the frequency of anti-HCV antibody (Ab) together with hepatitis B surface (HBs) antigen (Ag) and anti-HBs Ab were determined in 42, 45 and 23 patients with essential mixed cryoglobulinemia (EMC), multiple myeloma (MM) and B-cell chronic lymphocytic leukemia (B-CLL), respectively. Thirty hospitalized patients with chronic rheumatoid arthritis (RA) were also included as a control. Specific antibodies to HCV antigens were detected by enzyme linked immunosorbent assay (ELISA) and positive results were confirmed by a recombinant immunoblot assay (RIBA). Our results demonstrated anti-HCV positivity in 69%, 11% and 4.3% of the EMC, MM and B-CLL samples tested, respectively. None of the RA patients were found to be anti-HCV positive. No significant differences were observed between the patients groups regarding the frequency of HBs Ag and anti-HBs Ab. Considering the low incidence of HCV infection in the control group and the normal population, these results confirm and extend previous reports on the possible role of HCV infection in the etiology of EMC and further suggest involvement of this virus in a subset of MM.

Congenital nephrotic syndromes.

Many acquired and familial renal diseases in man lead to kidney dysfunction and nephrotic syndrome. These diseases share a common pathological fate in the form of glomerular dysfunction and proteinuria. Classification of the disease is difficult because the onset of pathological appearance in congenital nephrotic syndrome (CNS) varies considerably. Recently, classification has been aided by applying molecular genetics to identify genes involved in the pathogenesis of proteinuria. Light has also been shed on the biology and mechanisms of glomerular filtration and the molecular pathogenesis of CNS.

Molecular cloning and characterization of starch-branching enzyme II from potato.

Full-length cDNA for starch branching enzyme (SBE) II of potato was isolated and sequenced. In potato, similarly to most other investigated plants, the SBE-II isoform differs from SBE-I by having an acidic amino-terminal extension and a shorter carboxyterminus. Two forms of SBE-II, migrating as 98 and 95 kDa proteins in 6% SDS-polyacrylamide gels, were associated to tuber starch. The latter form was 16 amino acids shorter in the amino terminus. Transcript of SBE-II was present in leaf tissue, whereas significant expression was not seen in tubers. On the other hand, a significant amount of SBE-I transcript was detected in tuber tissue but not in leaves.

The multiple forms of starch-branching enzyme I in Solanum tuberosum.

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeia and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.

Characterization of the 97 and 103 kDa forms of starch branching enzyme from potato tubers.

N-Terminal analysis, peptide mapping and partial peptide sequencing of the 97 and 103 kDa forms of starch branching enzyme from potato tubers showed that the two forms are highly related. A comparison with sequence data in the literature showed that these forms belong to the starch branching enzyme isoform I family. An internal cDNA fragment was obtained using PCR technology on potato tuber RNA with two oligonucleotide primers constructed from the peptide sequence data. Southern blot analysis using the PCR fragment as probe showed that there is only one gene locus encoding this isoform of the enzyme in Solanum tuberosum as well as in Solanum commersonii.