PubMed Health⌕ Search

Biomedical subjects

M Karas

Publications and source records attributed to M Karas.

At least 19 recordsLinked to original sources
PubMed2006

Influence of the matrix on analyte fragmentation in atmospheric pressure MALDI.

In this paper, we report the measurement of the degree of analyte fragmentation in AP-MALDI as a function of the matrix and of the laser fluence. The analytes include p-OCH3-benzylpyridinium, three peptides containing the sequence EEPP (which cleave very efficiently at the E-P site), and three deoxynucleosides (dA, dG, and dC), which lose the neutral sugar to give the protonated base. We found that the matrix hardness/softness was consistent when comparing the analytes, with a consensus ranking from hardest to softest: CHCA >> DHB > SA approximately THAP > ATT > HPA. However, the exact ranking can be fluence-dependent, for example between ATT and HPA. Our goal here was to provide the scientific community with a detailed dataset that can be used to compare with theoretical predictions. We tried to correlate the consensus ranking with different matrix properties: sublimation or decomposition temperature (determined using thermogravimetry), analyte initial velocity, and matrix proton affinity. The best correlation was found with the matrix proton affinity.

Atmospheric Pressure↗
PubMed2006

[Frequency view on genome changes testing].

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab_a_vysetreni/nr lab_dna_diag/dna_lab_db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

Gene Frequency↗
PubMed2005

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry.

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Bacterial Proteins↗
PubMed2005

Quality control of tablets by Near Infrared (NIR)-Spectroscopy.

Today, NIR-spectroscopy is an established analytical technique not only in the identification of raw materials but also in the quantification of active ingredients in tablets. In this work calibration models were set up with tablets of the same active ingredient but of miscellaneous origin and manufacturess. Consequently the tablets had different excipients and appearance. The pharmaceutical preparations used included atenolol 100 mg tablets, enalapril 20 mg tablets and acetylsalicylic acid (ASS) tablets of different dosage units. In order to proof if the calibration models set up are generally feasible the assay declared by the manufacturer was used to calculate the partial least square (PLS) calibration. With respect to enalapril tablets simultaneous analysis by HPLC, according to USP 26 was carried out. It was investigated if such methods allow a determination of active ingredients in tablets within limits of +/- 10% of declaration. It was shown that it is possible to set up calibration models to quantify active ingredients in tablets independent of adjuvants or optical appearance. Additionally it could be shown that NIR-spectroscopy is also applicable to determine the concentration of active ingredients in blister-packed tablets.

Atenolol↗
PubMed2004

Nanospray 'taxation' and how to avoid it.

In mass spectrometric analysis with nanospray ionization, some analytes were found to appear in spectra with a delay of tens of minutes, while a few others could not be detected at all. The effect was found to be related to cation-exchange chromatography with negative charge on the glass surface, and with the most affected peptide or protein ions having strong localization of positive charge in blocks of two or more adjacent basic amino acid residues (e.g. melittin). The 'affinity' to the glass surface was studied with a peptide mixture and bovine serum albumin (BSA) tryptic digest solutions at sub-micromolar concentration. About 20% fewer tryptic peptides could be identified from spectra recorded with a glass nanospray capillary compared to those acquired with either conventional 1 microL/min electrospray or a quartz nanospray capillary. Protein identification studies are not likely to be seriously affected by this loss, but other protein applications, such as investigations of mutations or post-translational modifications, may suffer due to reduced sequence coverage. Ways to avoid losses of useful ions are discussed.

Aerosols↗
PubMed2003

Are human platelet alloantigens (HPA) minor transplantation antigens in clinical bone marrow transplantation?

The role of human platelet alloantigens (HPA) in clinical bone marrow allotransplantation was investigated. The leading hypothesis was that HPA alloepitopes act as minor histocompatibility antigens and aggravate graft-versus-host disease (GVHD). To exclude the effect of MHC disparity, only HLA identical donor-recipient pairs were entered into the study. The influence of HPA compatibility on overall survival, occurrence of relapses and haematopoietic recovery was also investigated. A total of 223 patients who received a graft from an HLA-identical sibling, genotyped for HPA -1, -2, -3, -4 and -5, were observed over a post-transplant period of 24 months following the protocol recommended by EBMT. The data from patients having received grafts from HPA compatible donors were compared to data from patients having received grafts that were mismatched in HPA allotypes in the GVH direction. Analysis of the incidence of acute and chronic (GVHD), overall survival, relapse incidence, haematopoietic recovery and some other clinical parameters did not reveal any significant difference between the HPA-matched and -mismatched groups of patients, regardless of their age. Our results give no evidence that HPA-1, -2, -3 and -5 alloantigens should be considered minor transplantation antigens in clinical bone marrow transplantation.

Adult↗
PubMed2001

Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS.

In ESI MS, some classes of biomolecules are detected only with low signal intensities due to difficulties in achieving efficient analyte desolvation, either because an analyte tends to fragment already at gentle desolvation conditions (i.e., noncovalent protein complexes or nucleotides) or because an analyte requires very strong activation in order to remove solvent molecules (i.e., carbohydrates). Even though the pressure in the first pumping stage of the ESI instrument is known to have an influence on the desolvation conditions, it has never been the focus of a detailed investigation. The role of the pressure in the first pumping stage is systematically interrogated in this study for several model substances. Ion signal intensities and signal-to-noise ratios are significiantly enhanced if the pressure in the first pumping stage is increased and adjusted, and analyte fragmentation can be substantially reduced. Thus, besides thermal heating and the acceleration in the nozzle-skimmer region, which are usually optimized, the pressure in the first pumping stage is an additional important desolvation parameter.

Concanavalin A↗
PubMed2001

Analyte incorporation and ionization in matrix-assisted laser desorption/ionization visualized by pH indicator molecular probes.

Despite the spreading applications of matrix-assisted laser desorption/ionization (MALDI), its fundamental understanding is still limited and under constant debate. This report focuses on the initial state of the analyte in the host matrix. pH indicator dyes serve as molecular probes since their color directly indicates their (de)protonation state. For a set of matrixes at their intrinsic pH, solution color was maintained, delivering clear proof for analyte incorporation in the solution charge state. Moreover, substantial solvent inclusion is determined by 1H NMR spectroscopy. MALDI mass spectra show a clear correlation to the dye charge state. However, the dominant solution species are not observed exclusively in the mass spectra, pointing to a proton transfer or proton neutralization activity of the matrix.

Journal Article↗
PubMed2001

Development of a high-performance liquid chromatographic method for the determination of 11-keto-beta-boswellic acid in human plasma.

A validated HPLC method for the determination of 11-keto-beta-boswellic acid (KBA) in human plasma was developed. The method involves the solid-phase extraction of KBA from plasma followed by a separation with reversed-phase HPLC. Calibration was based on external standardisation and ranged between 0.1 and 2.0 microg KBA per ml plasma. Linearity was established over the entire calibration range and in each case the coefficient of correlation (r2) was above 0.99. The recovery of KBA from plasma was 85.7%. It was further demonstrated that the method can be applied successfully to monitor the level of KBA in plasma.

Calibration↗
PubMed2001

Insulin-like growth factor-I (IGF-I) receptor activation rescues UV-damaged cells through a p38 signaling pathway. Potential role of the IGF-I receptor in DNA repair.

The activated insulin-like growth factor-I receptor (IGF-IR) is implicated in mitogenesis, transformation, and anti-apoptosis. To investigate the role of the IGF-IR in protection from UV-mimetic-induced DNA damage, 4-nitroquinoline N-oxide (4-NQO) was used. In this study we show that the activation of the IGF-IR is capable of rescuing NWTb3 cells overexpressing normal IGF-IRs from 4-NQO-induced DNA damage as demonstrated by cellular proliferation assays. This action was specific for the IGF-IR since cells expressing dominant negative IGF-IRs were not rescued from 4-NQO UV-mimetic treatment. DNA damage induced by 4-NQO in NWTb3 cells was significantly decreased after IGF-IR activation as measured by comet assay. IGF-I was also able to overcome the cell cycle arrest, observed after 4-NQO treatment, thereby enhancing the ability of NWTb3 cells to enter S phase. Interestingly, the p38 mitogen-activated protein kinase pathway was shown to represent the main signaling pathway involved in the IGF-IR-mediated rescue of UV-like damaged cells. The ability of the IGF-IR to induce DNA repair was also demonstrated by infecting NWTb3 cells with UV-irradiated adenovirus. Activation of the IGF-IR resulted in enhanced beta-galactosidase reporter gene activity demonstrating repair of the damaged DNA. This study indicates a direct role of the IGF system in the rescue of damaged cells via DNA repair.

3T3 Cells↗
PubMed2001

The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells.

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439-452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2(+) and Thy1.2(-) splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4(+) and CD8(+) cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Alleles↗
PubMed2001

Mass spectrometric investigations of human milk oligosaccharides.

Human milk oligosaccharides (OS) have been fractionated by gel permeation chromatography (GPC) and analyzed by mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS) and electrospray ionization (ESI) ion trap/MS were used. Using a large human milk pool, the MALDI-TOF/MS analysis of high-molecular-mass GPC fractions showed that complex, multiply fucosylated and/or sialylated OS are present over a larger mass range than described previously Acidic oligosaccharides could be detected from low-retention-time GPC fractions with masses up to 4000 Da, which has not been reported before.

Chemical Fractionation↗
PubMed2001

TCR stimulation protects CD8+ T cells from CD95 mediated apoptosis.

Activation of T cells through the T-cell receptor (TCR) induces the expression of Fas Ligand (CD95L). In turn, CD95L binds to the Fas receptor (CD95) and rapidly induces apoptosis in cycling cells. This interaction is involved in the elimination of reactive lymphocytes during an immune response. However, TCR activation cannot always trigger apoptosis because an effective immune response would then be compromised. Here we show that a short (2 to 3 h) activation of T cells through the TCR simultaneously induces an increase in CD95L mRNA and a dramatic decrease in caspase-8 mRNA levels and proteolytic activity in human CD8(+) T cells. In addition, there is a small reduction in CD95 mRNA and CD95 levels on the cell surface. We found that preactivation of T cells protected them from apoptosis induced by either religation of the TCR or direct exposure to CD95L. These results suggest a mechanism by which cycling CD95-sensitive peripheral T cells, become protected from CD95 mediated deletion when actively engaged in the specific recognition of target cells.

Apoptosis↗
PubMed2001

The influence of electrostatic interactions on the detection of heme-globin complexes in ESI-MS.

The heme-globin complexes of hemoglobin and myoglobin are investigated in positive-ion mode and negative-ion mode using a nano-ESI source coupled to a quadrupole ion trap MS and an orthogonal time-of-flight MS. The extent of dissociation of these noncovalent complexes upon collisional activation and thus their gas-phase stability is strongly dependent on the polarity of the ESI-MS experiment as well as on the charge of the prosthetic group (ferri-heme [Fe3+-heme]+ vs. ferro-heme [Fe2+-heme]+/-0). The results clearly point to the important role of electrostatic interactions on the gas phase stability of noncovalent complexes and therefore the ion signals observed in ESI-MS experiments.

Animals↗
PubMed2001

Allogeneic BMT in patients above 45 years of age: a single center experience.

Increasing age has been reported to be associated with worse outcome and higher occurrence of complication after allogeneic bone marrow transplantation. We analysed a cohort of 39 patients between the ages of 45 and 57 (median 49 years) with different hematologic malignancies who had undergone BMT in our institution over the preceding 4 years. Pretransplant conditioning consisted of Bu/CY2, GVHD prophylaxis of a combination of cyclosporine and "short" methotrexate. At present 54% of patients remain alive (with a median follow-up 44 months), the probability of survival at 5 years is 53% (5-year DFS 78%). The 5-year survival probability in the control group of younger patients is 53% (P = 0.8003). Main causes of death were GVHD (4 patients, 10%), relapse (5 patients, 13%) and infection (6 patients, 15%). The incidence of acute GVHD grade II-IV was 51% (grade III-IV 0% patients), the incidence of chronic GVHD 49% (limited 18% and extensive 31% patients). Our results suggest that allogeneic BMT can be performed in patients above the age of 45 years with acceptable morbidity and mortality, especially if a family HLA matched donor is available.

Actuarial Analysis↗
PubMed2001

The insulin-like growth factor I receptor-induced interaction of insulin receptor substrate-4 and Crk-II.

Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y(700), Y(717), Y(743), and Y(779)). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.

3T3 Cells↗
PubMed2001

Influence of caffeine on allyl alcohol-induced hepatotoxicity in rats. I. In vivo study.

Cotreatment of rats with a low hepatotoxic dose (30.7 mg/kg, i.p.) of allyl alcohol (AA) and a higher, but nontoxic, dose (150 mg/kg, oral) of caffeine (CF) potentiated the hepatotoxicity of AA. This was verified by significantly higher levels of plasma alanine aminotransferase (ALT) activity and histopathologically greater severity of lesions in the periportal hepatocytes than those due to AA alone. Treatment of rats with 4-methylpyrazole (4-MP) (0.5 mmol/kg, i.p.) (an inhibitor liver alcohol dehydrogenase) for 30 minutes, followed by similar cotreatment with AA and CF, completely prevented the elevation of plasma levels of ALT and histological damage induced by cotreatment with CF and AA 24 hours following their administration. Severe liver damage induced by cotreatment with CF and AA was further, markedly enhanced by phenobarbital pretreatment (80 mg/kg, i.p., 3 days). Thus, extensive necrosis of periportal hepatocytes was noted, as well as edema and accumulation of inflammatory cells in the necrotic foci caused by such pretreatment. The depression of hepatic nonprotein sulfhydryls resulting from CF plus AA was much more severe than that caused by AA or CF alone and appeared as early as 30 minutes after administration. However, much less marked depletion of protein thiols was observed following similar treatments. Significant increase in lipid peroxidation (as measured by melondialdehyde [MDA] formation) was also observed in rat liver but only 24 hours after administration. The production ofMDA in the rat liver was significantly higher after administration of AA plus CF than after administration of AA alone. Pretreatment of rats with phenobarbital further significantly enhanced the formation of 2,4-dinitrophenylhydrazine (DNP)-reactive metabolite(s) (measured as DNP-acrolein adduct equivalents) in rat liver induced by AA (30.7 mg/kg) plus CF (150 mg/kg) within 1 hour following such treatment. Cotreatment with AA and a higher dose of CF resulted in significantly higher excretion of urinary thioethers or mercapturic acids than in rats treated with AA alone. Thus, these data suggest that an increased bioactivation pathway of acrolein involving a P450 mixed-function oxidase system caused by CF may be involved in such potentiating effects of CF on AA-induced hepatotoxicity in rats.

Acrolein↗