Biomedical subjects

M Kania

Publications and source records attributed to M Kania.

At least 19 recordsLinked to original sources

PubMed2006

A phase II trial of pemetrexed plus gemcitabine in locally advanced and/or metastatic transitional cell carcinoma of the urothelium.

BACKGROUND: Both pemetrexed and gemcitabine have single-agent activity in bladder cancer, but the combination of these two drugs has not been previously evaluated for safety and efficacy in this disease. Thus, the objectives in the current study were to determine overall response rate (ORR), progression-free survival, overall survival and safety and toxicity in chemonaive patients with locally advanced and/or metastatic transitional cell carcinoma of the urothelium. PATIENTS AND METHODS: Gemcitabine 1250 mg/m2 was administered over 30 min i.v. on days 1 and 8, and pemetrexed 500 mg/m2 over 10 min i.v. on day 8 after gemcitabine, every 21 days. RESULTS: Sixty-four patients were enrolled, 11 female and 53 male, median age 65 years (range 38-81), median WHO performance status of 1. Visceral metastases were present in 55% of patients. ORR among 47 patients evaluable for response was 28% (95% CI 16% to 43%) and ORR for the intention-to-treat population was 20% (95% CI 11% to 32%) with three CR and 10 PR. Median response duration was 11.2 months and median overall survival 10.3 months (95% CI 8.1-14.6 months). CTC grade 3/4 hematologic toxicities included anemia (19%), thrombocytopenia (9%), neutropenia (38%), febrile neutropenia (17%) and neutropenic sepsis (3%). Grade 3/4 non-hematologic toxicities included elevated transaminases (12%), dyspnea (8%), fatigue (8%) and stomatitis (5%). There was one toxic death due to neutropenic sepsis. CONCLUSIONS: The combination of pemetrexed and gemcitabine had a manageable safety profile. However, efficacy was apparently not superior to that of single-agent gemcitabine.

Adult↗

PubMed2004

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.

Biosensing Techniques↗

PubMed2001

[Levels of carnitine and homocysteine in plasma of long-term hemodialysis patients with chronic renal failure].

Homocysteine and carnitine are the metabolic products of exogenous amino acids. Increased plasma concentration of homocysteine but decreased or normal carnitine concentration are usual findings in haemodialysis patients with chronic renal failure. This study aimed to evaluate the interrelationship (if any) between the above mentioned metabolites in haemodialysis uraemic patients. 44 haemodialyzed patients with chronic renal failure--HD (25 female, 19 male, mean age 47 +/- 12 years) were enrolled into this study. Blood samples for estimation of plasma glucose, cholesterol, triglycerides, albumin, folic acid, vitamin B12, homocysteine (HC), total (TC) and free (FC) carnitine were withdrawn after overnight fasting before subsequent haemodialysis session. In all subjects whole body total fat mass (TFM) and lean mass (TLM) were assessed by dual X-ray absorptiometry (DEXA). Hyperhomocysteinaemia was found in 90.9% patients, while carnitine deficiency in 22.7% of all analysed subjects. Both hyperhomocysteinaemia and carnitine deficiency was found in 18.2% of haemodialysis patients. Folic acid deficiency regardless of prescribed supplementation was observed in 9.5% patients. A significant positive correlation was found between plasma concentration of TC or FC and TLM (tau = 0.332, p < 0.001; tau = 0.298, p < 0.01 respectively). A negative correlation was observed between plasma concentration of folic acid and homocysteine (tau = -0.201, p < 0.05). No significant relationship was noted between homocysteinaemia, total and free plasma carnitine levels and anthropometrical parameters. In conclusion, plasma concentration of homocysteine and carnitine are independent indicators of abnormal amino acid metabolism in uraemic patients.

Adult↗

PubMed1999

Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study.

Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations. A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant. The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.

Bacterial Proteins↗

PubMed1998

Structure-based design of beta-lactamase inhibitors. 1. Synthesis and evaluation of bridged monobactams.

Bridged monobactams are novel, potent, mechanism-based inhibitors of class C beta-lactamases, designed using X-ray crystal structures of the enzymes. They stabilize the acyl-enzyme intermediate by blocking access of water to the enzyme-inhibitor ester bond. Bridged monobactams are selective class C beta-lactamase inhibitors, with half-inhibition constants as low as 10 nM, and are less effective against class A and class B enzymes (half-inhibition constants > 100 microM) because of the different hydrolysis mechanisms in these classes of beta-lactamases. The stability of the acyl-enzyme complexes formed with class C beta-lactamases (half-lives up to 2 days were observed) enabled determination of their crystal structures. The conformation of the inhibitor moiety was close to that predicted by molecular modeling, confirming a simple reaction mechanism, unlike those of known beta-lactamase inhibitors such as clavulanic acid and penam sulfones, which involve secondary rearrangements. Synergy between the bridged monobactams and beta-lactamase-labile antibiotics could be observed when such combinations were tested against strains of Enterobacteriaceae that produce large amounts of class C beta-lactamases. The minimal inhibitory concentration of the antibiotic of more than 64 mg/L could be decreased to 0.25 mg/L in a 1:4 combination with the inhibitor.

Acylation↗

PubMed1998

26S proteasome structure revealed by three-dimensional electron microscopy.

In 26S proteasomes, "19S cap complexes" associate with either one or both ends of the barrel-shaped 20S core complex. These regulatory complexes which comprise about 20 different subunits, including 6 ATPases of the AAA family, are thought to recognize ubiquitinated substrate proteins, to dissociate and unfold them before threading them into the 20S core where they are degraded. Here, we examine the structure of 26S proteasomes from Drosophila embryos and Xenopus oocytes by electron microscopy. Image analysis reveals a rather flexible linkage between the 19S caps and the 20S core, with a peculiar wagging-type movement of the caps relative to the core. At this stage of the analysis, it is not clear whether this movement is relevant in terms of function. Three-dimensional reconstructions, taking this into account, provide first insights into the remarkably complex structure of the 19S caps and allows us to put forward a composite model of the entire 26S complex.

Animals↗

PubMed1997

Import of human and Thermoplasma 20S proteasomes into nuclei of HeLa cells requires functional NLS sequences.

Proteasomes are present both in the nucleus and cytoplasm of eukaryotic cells. Their localization is regulated and changes during the cell cycle. Nuclear localization signal (NLS) type sequences were identified in proteasomes from various organisms. In addition, acidic complementary sequences were identified (cNLS) which could interact with the positively charged NLS, masking or unmasking them and thereby modulating nuclear import. In this paper we show that fluorescently labeled human erythrocyte 20S proteasomes accumulate in the nucleus of digitonin-permeabilized cells. This translocation is ATP-dependent and occurs through the nuclear pore complex as is shown by blocking of the nuclear pores with wheat germ agglutinin. In addition, we used 20S proteasomes from Thermoplasma acidophilum as a model system. Recombinant 20S proteasomes from the archaebacterium Thermoplasma acidophilum are imported into nuclei of HeLa and 3T3 cells similar to their eukaryotic counterpart. We constructed mutants in the putative NLS and cNLS region to study their effect on import. The NLS mutant was not imported into nuclei and showed cytoplasmic staining only. This indicates that this sequence is indeed responsible for nuclear targeting. Mutational studies of the cNLS do not support the involvement of this sequence in regulation of nuclear transport.

3T3 Cells↗

PubMed1997

[Reconstructive surgery in iatrogenic bile duct injuries].

The paper presents the results of surgical treatment performed in 54 patients with iatrogenic injury of biliary tract. All cases of injury were made during open and laparoscopic cholecystectomy. We concluded that Roux-en-Y hepatico-jejunostomy should be recommended. There were no deaths after surgery. This technic was successful therapeutic management more than 95% of cases.

Anastomosis, Roux-en-Y↗

PubMed1996

Design, synthesis, and evaluation of 2 beta-alkenyl penam sulfone acids as inhibitors of beta-lactamases.

A general method for synthesis of 2 beta-alkenyl penam sulfones has been developed. The new compounds inhibited most of the common types of beta-lactamase. The level of activity depended very strongly on the nature of the substituent in the 2 beta-alkenyl group. The inhibited species formed with the beta-lactamase from Citrobacter freundii 1205 was sufficiently stable for X-ray crystallographic studies. These, together with UV absorption spectroscopy and studies of chemical degradation, suggested a novel reaction mechanism for the new inhibitors that might account for their broad spectrum of action. The (Z)-2 beta-acrylonitrile penam sulfone Ro 48-1220 was the most active inhibitor from this class of compound. The inhibitor enhanced the action of, for example, ceftriaxone against a broad selection of organisms producing beta-lactamases. The organisms included strains of Enterobacteriaceae that produce cephalosporinases, which is an exceptional activity for penam sulfones.

Ceftriaxone↗

PubMed1995

Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis.

The role of asparagine 152 in the catalytic mechanism of Escherichia coli AmpC beta-lactamase has been investigated by site-directed mutagenesis. The residue has been replaced by aspartic acid, glutamic acid, histidine, and leucine. All the substitutions had similar effects on the activity toward substrates and inhibitors. The rate of substrate hydrolysis decreased by factors of 500-5000. The rates of both acylation (2-50-fold decrease) and deacylation (50-500-fold decrease) were affected, indicating a role for Asn152 in both processes. The wild-type AmpC beta-lactamase appears to exist as an equilibrium mixture of two forms, identified by their different kinetic properties. The Asn152 mutations affected the activity of the slow-reacting form much more than that of the fast-reacting form, but they did not appear to affect the interconversion of these two kinetic forms. Comparison of these observations with results obtained with mutation of the equivalent residues in other classes of penicillin-sensitive enzyme indicates that there are quite profound differences between the catalytic mechanisms of these enzymes despite a high degree of conservation of amino acids in the active center, and of the overall three-dimensional structure.

Anti-Bacterial Agents↗

PubMed1995

TLD postal dose intercomparison for megavoltage units in Poland.

The aim of the TLD pilot study was to investigate and to reduce the uncertainties involved in the measurements of absorbed dose and to improve the consistency in dose determination in the regional radiotherapy centres in Poland. The intercomparison was organized by the SSDL. It covered absorbed dose measurements under reference conditions for Co-60, high energy X-rays and electron beams. LiF powder type MT-N was used for the irradiations and read with the Harshaw TLD reader model 2000B/2000C. The TLD system was set up and an analysis of the factors influencing the accuracy of absorbed dose measurements with TL-detectors was performed to evaluate and minimize the measurement uncertainty. A fading not exceeding 2% in 12 weeks was found. The relative energy correction factor did not exceed 3% for X-rays in the range 4-15 MV, and 4% for electron beams between 6 and 20 MeV. A total of 34 beams was checked. Deviation of +/- 3.5% stated and evaluated dose was considered acceptable for photons and +/- 5% for electron beams. The results for Co-60, high energy X-rays and electron beams showed that there were two, three and no centres, respectively, beyond acceptance levels. The sources of errors for all deviations out of this range were thoroughly investigated, discussed and corrected, however two deviations remained unexplained. The pilot study resulted in an improvement of the accuracy and consistency of dosimetry in Poland.

Cobalt Radioisotopes↗

PubMed1994

The role of tyrosine 150 in catalysis of beta-lactam hydrolysis by AmpC beta-lactamase from Escherichia coli investigated by site-directed mutagenesis.

The kinetics of beta-lactam hydrolysis by wild-type AmpC beta-lactamase from Escherichia coli and three mutant proteins created by substitution of tyrosine 150 have been examined. The catalytic efficiency was decreased 10- to 1000-fold according to the substrate and mutant being studied. The effect of the mutation was much stronger with rapidly hydrolyzed substrates (e.g., cephalothin) than it was with slowly hydrolyzed substrates (e.g., ceftriaxone). With the latter substrates, the mutagenesis had a much stronger effect on apparent affinity than it did on rates of catalysis. Indeed, the enzyme appeared to be more reactive toward certain of the slowly hydrolyzed substances (e.g., methicillin, aztreonam, and ceftriaxone). These observations were not compatible with an obligatory role of tyrosine 150 in catalysis. The analysis of the effects of the mutation on activity was complicated by the observation of at least two, kinetically distinct, forms of the enzymes. It appeared that mutation of tyrosine 150 influenced the kinetic properties of one state and that this residue is involved in the partitioning of the enzyme between the different reactive states.

Acylation↗

PubMed1994

[Clinical protocols concerned with disorders of fatty acid beta oxidation].

Inborn defects in fatty acid oxidation are a newly described group of diseases affecting infants and child. This disorder frequently masquerades as Reye's syndrome or sudden infant death syndrome. The known defects, now totaling 11, share many clinical similarities. Clinical manifestations include episodes of coma and hypoglycemia that are induced by fasting, or chronic, progressive muscle weakness and cardiomyopathy. Affected patients are often free of symptoms between episodes. A characteristic organic aciduria or enzyme activity in cultured skin fibroblasts permit diagnosis. Because of high mortality and morbidity rates, early diagnosis is desirable. treatment for survivors siblings is simple and is associated with avoidance of prolonged fasting. Systemic screening of the general population should be initiated to identify those affected and start treatment before they become ill.

Child↗

PubMed1992

Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor beta.

Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20,000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-R beta. These clones cover about 200 kb and have 170 kb in common. TNF-R beta is 9.3 kb long and contains two introns within the protein-coding sequence.

Base Sequence↗

PubMed1991

High-affinity receptor for interferon-gamma (IFN-gamma), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-gamma receptor protein.

High-affinity receptors for human IFN-gamma were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high-affinity binding site for each cell. One cross-linked complex between IFN-gamma and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N-linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN-gamma and antibody epitopes characteristic of the extracellular domain of the IFN-gamma receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high-affinity IFN-gamma receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high-affinity IFN-gamma receptor protein. N-linked sugars may give structural stability to the IFN-gamma receptor and are unlikely to be directly involved in IFN-gamma binding.

Antibodies, Monoclonal↗

PubMed1989

Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins.

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.

Animals↗

PubMed1987

Quaternary structure of ornithine aminotransferase in solution and preliminary crystallographic data.

Ornithine aminotransferase was purified from rat liver and crystallized in the presence of ammonium sulphate and poly(ethylene glycol) (PEG 4000). The crystallographic threefold symmetry observed for the resulting two crystal forms stimulated a re-examination of the enzyme's quaternary structure in solution by analytical ultracentrifugation and chemical cross-linking. The results indicate that the oligomeric state or ornithine aminotransferase, under conditions similar to those used in crystallization experiments, is a hexamer (Mr = 256,000) rather than a tetramer or higher oligomers as reported previously. The subunits of the enzyme are identical (Mr = 45,000). Only the hexagonal prismatic crystals obtained with PEG 4000 were suitable for crystallographic studies and diffracted X-rays to a resolution of at least 0.16 nm. However, these crystals contained an unusual element of disorder which was persistent under a variety of conditions and was only noticeably diminished in the presence of the non-ionic detergent octyl beta-glucoside. The crystals apparently belong to the trigonal space group P3(1)12 (or enantiomorph) with axial lengths of a = 19.5 nm, c = 5.9 nm and contain three monomers per asymmetric unit.

Cross-Linking Reagents↗

PubMed1987

Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.

N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.

Aldose-Ketose Isomerases↗