Altered expression of ezrin, E-Cadherin and ß-Catenin in cervical neoplasia.

High-grade cervical squamous intraepithelial lesions (CIN) as well as squamous cell carcinoma and adenocarcinoma of the cervix are associated with persistent high-risk human papillomavirus (HPV) infection. A number of cellular events play a role in HPV pathogenesis and in the development of cervical lesions, including alterations in cell adhesion and motility. The crucial plasma membrane - cytoskeleton linker protein ezrin of the Ezrin-Radixin-Moesin (ERM) protein family is involved in the regulation of cell morphology, cell adhesion and invasion. Based on our previous work on ERM proteins we sought out to study the expression of ezrin in cervical premalignant lesions. We also studied the expression of E-cadherin and ß-catenin, which play an important role in epithelial cell adhesion. We observed intensifying expression of ezrin along with progressing grade of neoplasia. Ezrin staining was found to colocalize with p16 staining in high-risk HPV associated lesions. Expression of E-cadherin and ß-catenin was found to be altered along with the severity of the lesion, similar to ezrin. Enhanced expression of ezrin in cervical HPV associated lesions suggests a role in the development of cervical neoplasia and cancer. Further clinical evaluation should reveal the feasibility of ezrin as a biomarker for the progression of cervical lesions.

[Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood].

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.

The role of histidine residues in the catalytic act of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme. Based on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis. The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques. The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum. It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions. Mutants H140D and H233D produced only minor amounts of alpha-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics. It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially.

Phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus: purification, gene cloning and sequencing.

Two peaks of aspartate aminotransferase (AspAT) catalytic activity were observed during DEAE chromatography of a protein extract from alkalophilic B. circulans. The enzyme purified from the major peak appeared to be not aspartate but phosphoserine aminotransferase (PSAT) with a considerably high AspAT side activity. The sequence of the enzyme N-terminus was determined, and the PSAT gene was cloned as two separate fragments. DNA sequencing revealed the open reading frame for the PSAT starting from TTG, putative ribosomal binding site and terminator of transcription. The PSAT gene encodes a protein of 361 amino acids (M(r) 39793) which shows moderate homology to other known phosphoserine aminotransferases (36-46% of identity, 60-64% of similarity). The PSAT from the alkalophile shares with all of them the consensus sequence pattern around the pyridoxal 5'-phosphate attachment site.

Thiol/disulfide exchange between small heat shock protein 25 and glutathione.

Murine small heat shock protein 25 (Hsp25) carries a single Cys-residue at position 141 of its amino acid sequence. In glutathione redox buffers, Hsp25 equilibrates between reduced protein (PSH), mixed disulfide (PSSG) and protein dimer (PSSP) forms. At highly oxidative conditions, native Hsp25 predominantly forms PSSP while denatured Hsp25 forms PSSG. Conversion of PSSP to PSSG correlates with urea and temperature denaturation of tertiary and/or quaternary structure of Hsp25. At pH 7.5, 25 degreesC, the second-order rate constant for the formation of PSSP in the reaction of native PSH with GSSG is 20.1+/-1.4 M-1 min-1. This is approximately 3-fold lower than the reaction velocity of GSSG with a typical, unhindered thiol of pKa 8.6. At redox equilibrium, the fractions of PSSP, PSSG, and PSH depend on the concentration of GSH and less on the ratio [GSH]/[GSSG] (R). At a constant R, the fractions of PSSG and PSH species depend similarly on GSH concentration, being approximately equal in glutathione redox buffers with low R. It is concluded that in oligomeric complexes, Hsp25 subunits in vitro form stable dimers, in which the reacting -SH groups are in a proximity to form intersubunit disulfide bonds. Within a reaction of one of these -SH groups with GSSG, steric hindrances and electrostatic repulsion complicate penetration of another reduced or oxidized glutathione molecule to the reaction site.

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol.

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.

Dimer structure as a minimum cooperative subunit of small heat-shock proteins.

Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.

Two "unrelated" families of ATP-dependent enzymes share extensive structural similarities about their cofactor binding sites.

Two proteins, D-alanine:D-alanine ligase and cAMP-dependent protein kinase, share a remarkable degree of structural convergence despite having different three-dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super-secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures.

Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.

Expression and biochemical analyses of the recombinant potato virus X 25K movement protein.

The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5'-proximal gene of three overlapping MP genes forming a 'triple gene block'. The PVX 25K MP (putative NTPase-helicase) has been synthesized in Escherichia coli as a recombinant containing a six-histidine tag at the amino terminus. The His-tagged 25K protein was purified in a one-column Ni-chelate affinity chromatography procedure. In the absence of any other viral factors, this protein had obvious Mg2+-dependent ATPase activity, which was stimulated slightly (1.7-1.9-fold) by various polynucleotides. Like other viral proteins possessing ATPase-helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro. The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.

Immobilization of aspartate aminotransferase on agarose.

Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.

Inorganic pyrophosphatase-labelled enzyme immunoassay for beta 2-microglobulin.

beta 2-Microglobulin was purified from human peritoneal dialysate by ultrafiltration, gel chromatography and DEAE-high performance chromatography. Anti-beta 2-monoclonal antibodies were developed in mice and a pair of the antibodies was utilized to develop an enzyme-linked immunosorbent assay (ELISA) kit for the analyte with utilizing a new enzyme label, inorganic pyrophosphatase (EC 3.6.1.1). The sensitivity of the assay was 0.6 microgram/l (3 x SD) and the assay was linear up to absorbance values of around 2.0. No hook effect occurred in any putative concentrations of beta 2-microglobulin in serum. The precision of the assay of one run varied within 5-7% CV and the interassay precision was 2.8-8.6% CV, while the recovery was 99.2 +/- 6.0%. Excellent correlation occurred against an established radioimmunoassay method. All the used reagents were storable for a minimum of 1 year at +4 degrees C. It was decided that inorganic pyrophosphatase is a label of choice for ELISA.

Molecular modeling of the steric structure of the envelope F1 antigen of Yersinia pestis.

Steric structure of the envelope F1 protein of Yersinia pestis was reconstructed by computer modeling taking into account structural similarities between F1 and interleukins (IL)-1 alpha, -beta, -ra and by using the known atomic coordinates for huIL-1 beta obtained by the X-ray crystallography. Of 18 hydrophobic positions forming a hydrophobic core in all the proteins studied with the IL-1-like conformation, 15 positions are occupied by hydrophobic residues in F1 protein as well. Of 8 homologous positions occupied by the amino acid residues of similar charge in all huIL-1 alpha, -beta, -ra, 5 positions are conserved in F1 antigen. The B-cell epitope accessible to antibodies in polymeric F1 is exposed as an hydrophilic loop at the surface opposite to the C-terminal sequence, forming a conserved binding site with periplasmic molecular chaperones.

Cyclophilin A and an antibody against cyclosporin A resemble each other in their binding sites.

The three-dimensional structures of cyclosporin A complexed with cyclophilin A or Fab fragment of a monoclonal antibody were compared. In these two complexes conformations of the fragment D-alanine8-N-methylvaline11 in cyclosporin A were in a good agreement. In addition, cyclophilin A and the Fab fragment had related arrangements of the aromatic amino acids in their binding sites, implying that antibody independently utilizes similar structural themes for binding cyclosporin A as cyclophilin A.

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2.

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.

Molecular model of an alternative splice variant of human IL-4, IL-4 delta 2, a naturally occurring inhibitor of IL-4-stimulated T cell proliferation.

The molecular model of IL-4 delta 2, a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame, is described. The second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4 delta 2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relative to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural pattern is transformed to the IL-4 delta 2 structure with a down-up-down-down structural pattern.

Conservative hydrophobic interdomain contacts of IFN-gamma remain in P17 matrix protein of HIV-1.

A constructed scheme of the surface layers containing helices C, D, and E' of various polypeptide chains which participate in the interdomain contacts in IFN-gamma demonstrated two sites of localization of the conservative hydrophobic amino acids. An analogous scheme of the interaction of helices B, C, and D in the p17 matrix protein of HIV-1 showed that the majority of the hydrophobic positions are similar. These data confirm the structural similarity between p17 and IFN-gamma.

Some new aspects of molecular mechanisms of cyclosporin A effect on immune response.

A few protein targets were found to display a specific high-affinity interaction with the immunosuppressant cyclosporin A (CsA): cytosolic cyclophilins (CyP)A, B, C, D, E containing from 122 to 174 amino acid residues in a polypeptide chain, and secreted forms of CyP; CyP-40, 40-kDa CsA-binding polypeptide complexed with steroid receptor (SR); CyP-related 150-kDa receptor of natural killer (NK) cells; interleukin 8 (IL-8); actin; a family of molecular chaperones hsp70 and P-glycoprotein (P-GP). All CyPs possess peptidyl-prolyl cis-trans isomerase activity (PPIase) and may serve as ATP-independent molecular chaperone proteins. The CsA-CyP complexes are specific inhibitors of Ca(2+)-and calmodulin-dependent protein phosphatase calcineurin (CaN). The inhibition of CaN blocks the activation of genes of IL-2, IL-2R, IL-4, etc. in T cells. In addition, immunosuppressive and/or antiinflammatory activity of CsA can be executed via CyP-40 and hsp 70 complexed with SR, and following the interaction with CyP-related receptor of NK and with IL-8. CsA binding to CyPC, P-GP and actin may throw light on the biochemical events leading to nephrotoxicity and graft vessel disease, two major side effects produced by CsA. The discovery of the interaction of human immunodeficiency virus type 1 (HIV-1) Gag protein with CyP and effective disruption of this interaction by CsA may be important for our understanding of the pathology caused by this immunosuppressive virus and will inspire therapeutic strategies to nip HIV in the bud. Bacterial immunophilins (ImPs) contribute to the virulence of pathogenic microorganisms. Elucidation of molecular mechanisms of microbial ImPs' action in the pathogenesis of bacterial infections may lead to new strategies for designing antibacterial drugs.

Aspartate aminotransferase from an alkalophilic Bacillus contains an additional 20-amino acid extension at its functionally important N-terminus.

Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (M(r) 47,439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.