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I Blaha

Publications and source records attributed to I Blaha.

16 recordsLinked to original sources
PubMed1995

Synthesis and evaluation of a non-radioactive gene probe for the detection of C.perfringens alpha toxin.

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

Bacillus cereus↗
PubMed1994

Immunohistochemical detection of bcl-2 protein in liver lesions: bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia.

A proto-oncogene, bcl-2, encodes a protein that inhibits programmed cell death (apoptosis) and may play a role in cell and tissue differentiation. As bcl-2 appears to be involved in the turn-over of stem or precursor cells, it is thought to be operational in carcinogenesis pathways. However, apart from certain lymphomas, only limited data are available on the frequency of its expression in solid tumors. Immunohistochemical analysis with an antibody specific for bcl-2 protein was used to detect the protein in hepatocellular carcinomas and in one of the putative precursor lesions, liver cell dysplasia. We detected bcl-2 protein in 5 of 37 hepatocellular carcinomas. Immunoreactivity was not related to type, grade, or extent of PCNA staining of the tumours. No bcl-2 protein staining was observed in three types of liver cell dysplasia. Thus, bcl-2 is abnormally expressed in some hepatocellular carcinomas but not in potential tumour precursor cells.

Carcinoma, Hepatocellular↗
PubMed1993

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame.

Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

Amino Acid Sequence↗
PubMed1992

Detection of hepatitis B virus DNA in the liver of children with chronic hepatitis B by in situ hybridization and its relation to other viral markers.

The aim of the study was to detect hepatitis B virus (HBV) DNA by in situ hybridization (ISH) with a 35S-labeled radioactive probe in frozen liver biopsy tissue sections of 63 hepatitis B virus surface antigen (HBsAg)-positive children. The results were compared to other markers of viral replication. HBV DNA was detected in 48 children. Of the 15 negative cases, four had hepatitis B envelope antigen (HBeAg), 10 anti-HBe, and one neither HBeAg nor anti-HBe. Free HBV DNA in serum and liver was positive in one patient. Forty of the positive children were HBeAg- and six anti-HBe-positive; two were negative for both. Of 45 36 had HBV DNA in serum. In 38 of 47 HBV DNA and in 31 of 42 HBcAg could be detected in the liver. The HBV DNA signals were located mainly over the cytoplasm of hepatocytes. The distribution of HBV DNA in the tissue was classified as homogeneous, inhomogeneous with focal patches, and focal. It is concluded that in situ hybridization is a reliable method for detection of HBV DNA in liver tissue of children with chronic hepatitis B. The technique, which can be applied to small amounts of liver tissue, provides informations about the distribution of replicative viral sequences, complementing laboratory data, liver histochemistry, and histology.

Adolescent↗
PubMed1991

Subsite specificity of the proteinase from myeloblastosis associated virus.

The subsite requirements of the aspartic proteinase from the myeloblastosis-associated virus (MAV) for the cleavage of peptide substrates were studied with a series of synthetic peptides of general structure Ala-Thr-P4-P3-P2-P1*Nph-Val-Arg-Lys-Ala. The residues in positions P4, P3, P2 and P1 were varied and the kinetic parameters for the cleavage of substrates in 2.0 M NaCl were spectrophotometrically determined at pH 6.0 and 37 degrees C. The acceptance of amino acid residues in particular subsites is similar to that observed with the human immunodeficiency virus type 1 (HIV-1) proteinase in our earlier studies on the same substrate series: hydrophobic or aromatic residues are preferable in P1 position, a broad variety of residues are acceptable in P3 whereas the residues occupying P2 plays the decisive role in the substrate cleavage as evidenced by its dramatic influence on both kcat and Km values. The most remarkable difference between the two enzymes was found in P3 and P4 subsites. In P3, the introduction of negatively charged glutamate increases the substrate binding by the MAV proteinase 12-fold and decreases binding by the HIV-1 proteinase. In P4, Pro in this series is a favourable residue for the MAV proteinase and is strongly inacceptable for HIV-1 the proteinase. The pH profile of the cleavage was studied with a chromogenic substrate and differences between HIV-1 and MAV proteinases are discussed.

Amino Acid Sequence↗
PubMed1991

Specificity studies on retroviral proteinase from myeloblastosis-associated virus.

The specificity of the p15 proteinase of myeloblastosis-associated virus (MAV) was tested with nonviral high molecular weight substrates and with synthetic peptides. Peptides with sequences spanning known cleavage sites in viral polyproteins of Rous sarcoma virus (RSV) and avian leukemia viruses, as well as in BSA and HSA, were synthesized, and the rate of their cleavage by the MAV proteinase was compared. Synthetic peptides require for successful cleavage at least 4 residues at the N-terminal side and 3 residues at the C-terminal side. The proteinase shows a preference for hydrophobic residues with bulky side chains (Met, Tyr, Phe) in P3, although Arg and Gln can also be accepted. Small hydrophobic residues are required in P2 and P2', and large hydrophobic residues (Tyr, Met, Phe/p-nitro-Phe) are preferred in both P1 and P1'. The difference between the specificity of the p15 proteinase and that of the HIV-1 proteinase mostly pertains to position P2' of the substrate, where bulkier side chains are accepted by the HIV-1 proteinase (Richards et al., 1990). A good chromogenic substrate for the MAV and RSV proteinases was developed and used to further characterize the MAV proteinase activity with respect to ionic strength and pH. The activity of the proteinase is strongly dependent on ionic strength and pH. Both the kcat and Km values contribute to a higher cleavage efficiency at higher salt concentrations and show a bell-shaped pH dependence curve with a sharp maximum at pH 5.5 (kcat) and 6.5 (Km).

Amino Acid Sequence↗
PubMed1991

Studies on the role of the S4 substrate binding site of HIV proteinases.

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.

Amino Acid Sequence↗
PubMed1990

Engineering enzyme subsite specificity: preparation, kinetic characterization, and X-ray analysis at 2.0-A resolution of Val111Phe site-mutated calf chymosin.

Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.

Amino Acid Sequence↗
PubMed1989

[A highly sialylated, embryonic form of the neural cell adhesion molecule in Wilms tumor: identification of a cell adhesion molecule as a onco-developmental antigen].

The neural cell adhesion molecule NCAM is a general Ca2+ -independent cell adhesion molecule which exerts important functions during the development of the nervous system. NCAM polypeptides exist in various isoforms all of which have a similar extracellular domain structure containing a homophilic binding site for establishment of cell-cell contacts. A common structural feature of NCAM is the presence of homopolymers of alpha 2,8-linked sialic acid residues, the so-called polysialic acid, which is developmentally regulated. It undergoes a change from a highly less sialylated short chain form from embryonic to adult life. The polysialic acid regulates the homophilic adhesive properties of NCAM. The highly sialylated form of NCAM typically found in developing tissues decreases homophilic NCAM-NCAM interactions and, therefore, cell-cell contacts due to its large excluded volume and electric repulsive forces. We have used a monoclonal antibody against polysialic acid, polyclonal antibodies against NCAM, polysialic acid specific bacteriophage-encoded endoneuraminidases and a NCAM cDNA to investigate this molecule by light and electron microscopic immunolabeling, in situ hybridization and immunochemistry. The highly sialylated form of NCAM was found to be expressed in a developmentally-regulated fashion in embryonic kidney, undetectable in the normal adult kidney and re-expressed in Wilms tumor. In Wilms tumor the blastemal cell masses as well as epithelial differentiations such as tubules and glomeruloid bodies were immunolabeled whereas the stroma did not label for polysialic acid. Combination of immunoprecipitation and immunoblotting using antibodies against polysialic acid and NCAM, respectively, directly demonstrated structural relationship of renal polysialic acid with NCAM polypeptide. By immunoblot analysis two NCAM isoforms of approximately 120 and 140 kD were found in Wilms tumor. Immuno-electron microscopy provided direct morphological evidence for prevention of cell-cell contacts due to the presence of a thick cell surface coat composed of polysialic acid. In nephroblastomatosis complexes only blastemal cells exhibited immunocytochemically detectable polysialic acid. All other investigated kidney tumors, i.e. clear cell sarcoma, malignant rhabdoid tumor, cystic nephroma and renal cell carcinoma, were negative. These data allow the conclusion that the highly sialyated, embryonic form of NCAM is an onco-developmental antigen in human kidney. At the same time this is the first observation that a molecule with a well defined role for controlled cell migration and differentiation during embryonic organ development represents an onco-developmental antigen.

Cell Adhesion Molecules, Neuronal↗
PubMed1988

Evaluation of polysialic acid in the diagnosis of Wilms' tumor. A comparative study on urinary tract tumors and non-neuroendocrine tumors.

The polysialic acid moiety of the neural cell adhesion molecule has been shown to represent an onco-developmental antigen which can be detected in both embryonic human kidney and Wilms' tumor but not in normal adult human kidney. In the present comparative study, Wilms' tumors, clear cell (bone-metastasizing) sarcomas of kidney, cystic nephromas, renal cell carcinomas, transitional cell carcinomas and papillomas of the renal pelvis, ureter and urinary bladder (as well normal transitional epithelium from these regions). Ewing sarcomas, hepatoblastomas, rhabdomyosarcomas, and carcinomas of the stomach, colon, exocrine pancreas, lung, and esophagus, were investigated immunohistochemically for the presence of polysialic acid. In addition, immunoblot analysis was performed in selected tumors. With the exception of Wilms' tumor, none of the tumors investigated was positive for polysialic acid. In Wilms' tumor, blastemal cells and all epithelial components were positive but no immunostaining was observed in the stroma. These observations emphasize the potential value of a monoclonal anti-polysialic acid antibody in identifying blastemal metanephric cells and their epithelial differentiatives in Wilms' tumor.

Animals↗
PubMed1988

Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.

The long chain form of polysialic acid characteristic of the low adhesive embryonic form of the neural cell adhesion molecule NCAM is temporally and spatially expressed in developing kidney but undetectable in normal adult kidney. Therefore, this molecule represents a developmentally regulated antigen in kidney contrasted with neural tissue, where it is also detectable in the adult brain. This investigation of 25 Wilms' tumors comprising all different histologic types demonstrates expression of this molecule under conditions of malignant growth. Immunostaining was observed in Wilms' tumors with both a monoclonal anti-polysialic acid antibody and a polyclonal anti-NCAM polypeptide antiserum. Intense cell surface staining sensitive to endosialidases specifically hydrolyzing alpha 2,8 linked (poly)sialic acid was detectable in blastemal regions, and weaker, variable labeling was seen over tubules and glomeruloid bodies. The stroma was not stained. This is evidence indicating that Wilms' tumor originates from the embryonic equivalent of induced metanephrogenic mesenchyme. It seems unlikely however, that the stroma is derived from the blastema. The same high molecular mass broad band typical of the embryonic form of NCAM was revealed by immunoblot analysis of homogenates from Wilms' tumor as well as from embryonic kidney and brain. In situ hybridization demonstrated the presence of mRNA for NCAM in all but stromal elements of Wilm's tumors. Thus, polysialic acid is present on NCAM and represents a new oncodevelopmental antigen in human kidney. Polysialic acid was greatly reduced or absent by immunohistochemistry and immunoblotting in necrotic tumor areas.

Adult↗
PubMed1988

Blastemal cells of nephroblastomatosis complex share an onco-developmental antigen with embryonic kidney and Wilms' tumor. An immunohistochemical study on polysialic acid distribution.

Previous investigations on polysialic acid of the neural cell adhesion molecule NCAM in human kidney have demonstrated its presence during nephrogenesis in embryonic kidney, absence in normal adult kidney, and reexpression in Wilms' tumor. These data showed that polysialic acid of NCAM is an onco-developmental antigen in human kidney and provided more direct evidence for the metanephric origin of Wilms' tumor. In the present study, five cases of Wilms' tumor associated with nephroblastomatosis complexes were immunohistochemically investigated with a monoclonal antibody for the presence of polysialic acid. Regardless of the type of nephroblastomatosis complex, ie, renal nodular blastema, simple tubular metanephric hamartoma, sclerosing metanephric hamartoma with adenoma, or incipient Wilms' tumor, immunoreactivity for polysialic acid was found in the blastemal cells, but was undetectable in all other structural elements. Because only blastemal cells exhibited a characteristic feature of embryonal differentiating metanephric derivatives, it appears that Wilms' tumor has its origin not exclusively in nodular renal blastema but rather in blastemal cells present in the various forms of nephroblastomatosis complex. The presence of polysialic acid of NCAM in blastemal cells in such lesions indicates that further events in addition to the expression of the embryonic form of this cell adhesion molecule may be involved in the pathogenesis of Wilms' tumor.

Antigens, Neoplasm↗
PubMed1987

Ectopic thymus in the neck; a case report and review of the literature.

A soft, poorly defined mass in the right upper neck of a 7-week-old boy was shown on histology to be ectopic thymus. As aberrant thymic tissue often does change into cysts or neoplasms removal is the treatment of choice. Its persistence in the upper neck seems to be very rare. Embryology, incidence, differential diagnosis and treatment are discussed with a review of the literature.

Choristoma↗