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M Ptak

Publications and source records attributed to M Ptak.

At least 19 recordsLinked to original sources
PubMed2005

The influence of collagenase treatment on the production of TNF-alpha, IL-6 and IL-10 by testicular macrophages.

Testicular macrophages (TMf) are located in the interstitial tissue of the male gonad. Highly purified TMf populations can be prepared either by the mechanical shaking of dispersed testicular tissues or by enzymatic digestion with collagenase followed by cell adherence, rosetting and gradient centrifugation. TMf obtained by the enzymatic procedure produced significantly more cytokines (IL-6, IL-10 and TNF-alpha) than TMf isolated by the mechanical method and this effect is long-lasting. Our results indicate that isolation of tissue macrophages by enzymatic digestion may influence their functional activity, and suggest that critical evaluation of the method used to obtain these cells should be the regular practice.

Animals↗
PubMed2005

Association between CYP2C9 slow metabolizer genotypes and severe hypoglycaemia on medication with sulphonylurea hypoglycaemic agents.

AIMS: The genetically polymorphic cytochrome P450 (CYP) enzyme CYP2C9 metabolizes most sulphonylurea oral hypoglycaemic agents. The aim of this study was to test the hypothesis that individuals with genotypes predicting low CYP2C9 activity may be at a higher risk of severe drug-associated hypoglycaemia. METHODS: In a case-control study, 20 diabetic patients admitted to the emergency department with severe hypoglycaemia during sulphonylurea drug treatment were compared with a control group of 337 patients with type 2 diabetes but without a history of severe hypoglycaemia. A large sample of 1988 healthy Caucasian subjects served as a second control group. RESULTS: The CYP2C9 genotypes *3/*3 and *2/*3 that are predictive of low enzyme activity were more common in the hypoglycaemic group than in the comparison groups (10%vs <2%, respectively: odds ratio 5.2; 95% confidence interval 1.01, 27). Furthermore, the diabetic patient group with severe hypoglycaemia exhibited lower body mass indexes, higher rates of renal failure, were older compared with the diabetic group without severe hypoglycaemia, and were being treated with higher doses of glibenclamide. CONCLUSIONS: These findings suggest that among other factors, individuals with genetically determined low CYP2C9 activity are at an increased risk of sulphonylurea-associated severe hypoglycaemia. Thus, genotyping might be a tool for the better prediction of adverse effects caused by oral hypoglycaemic agents.

Aged↗
PubMed2004

Hormonal counterregulation during severe hypoglycaemia under everyday conditions in patients with type 1 and insulin-treated type 2 diabetes mellitus.

AIM: To determine the counterregulatory hormonal responses to severe hypoglycaemia (SH) in type 1 versus insulin-treated type 2 diabetic patients under everyday conditions. METHODS: Counterregulatory hormones were determined in 28 consecutive type 1 and thirteen insulin-treated type 2 diabetic patients (age 54 +/- 18 vs. 75 +/- 13 yrs; diabetes duration 27 +/- 16 vs. 21 +/- 6 yrs) with SH requiring emergency treatment. Blood samples were taken prior to and after effective treatment of SH. SH was defined as an event with neuroglycopenic presentation requiring external intervention by administration of intravenous glucose or oral carbohydrates. 68 % (19/28) of type 1 diabetic patients but none of those with type 2 diabetes had reduced awareness of hypoglycaemia. RESULTS: Plasma glucose levels were 30 +/- 14 prior to and 179 +/- 82 mg/dl after treatment of SH; the time between the two measurements was 54 +/- 26 minutes. With the exception of higher levels of human growth hormone in type 1 patients - which were attributed to younger age - the other counterregulatory responses to SH showed no significant differences in type 1 vs. type 2 diabetic patients. In both groups glucagon responses were virtually absent while moderate catecholamine responses could be demonstrated. Treatment with beta-blockers did not affect hormonal counterregulation in type 1 diabetic patients. CONCLUSIONS: In patients approaching the insulin-deficient end of the spectrum of type 2 diabetes the hormonal responses to SH are comparable to those in patients with longstanding type 1 diabetes. Thus, in advanced type 2 diabetes the risk of developing SH may be similar to that in individuals with type 1 diabetes.

Adolescent↗
PubMed2003

Hormonal counterregulation and consecutive glimepiride serum concentrations during severe hypoglycaemia associated with glimepiride therapy.

OBJECTIVE: To examine the release of counterregulatory hormones and consecutive glimepiride serum concentrations during severe hypoglycaemia (SH) associated with glimepiride therapy. METHODS: In nine type-2 diabetic patients [age 81+/-9 (65-93) years; diabetes duration 9+/-4 (3-15) years; initial blood glucose 33+/-16 (10-54) mg/dl (1.8+/-0.9 mmol/l); HbA1c 7.2+/-1.1 (5.6-8.7)%; creatinine clearance 49+/-33 (15-107) ml/min] who experienced SH associated with glimepiride therapy with neuroglucopenic presentation, insulin, C-peptide, glucagon, epinephrine, norepinephrine, cortisol, adenocorticotrophic hormone (ACTH), human growth hormone (HGH) and pancreatic polypeptide (PP) were determined in blood samples taken at 4-h intervals prior to and during treatment with glucose i.v. Serum from the same samples was screened for sulphonylurea-type oral antidiabetics. Glimepiride concentrations were determined by a validated atmospheric pressure chemical ionization liquid chromatographic-mass spectrometry (APCI-LC-MS) assay. RESULTS: Once treatment had begun, normoglycaemia was maintained; most glimepiride levels were below the limit of detection (LOD <0.01 mg/l) and further sulphonylureas could be excluded. The secretion of glucagon and epinephrine as counterregulatory hormonal responses was unaffected. In addition, protracted marked increases of cortisol and norepinephrine levels were demonstrated. Protracted stimulation of insulin and C-peptide occurred in a period of up to 24 h after SH. No significant protracted responses were observed for ACTH, HGH or PP. CONCLUSION: In SH associated with glimepiride therapy, no correlation between glimepiride serum concentrations and the protracted stimulation of insulin and C-peptide was observed. The secretion of glucagon and epinephrine as counterregulatory hormonal responses was unaffected. Protracted increased release of cortisol might be a medium-term indicator of glimepiride-associated SH.

Aged↗
PubMed2003

Modulation of macrophage activity by proteolytic enzymes. Differential regulation of IL-6 and reactive oxygen intermediates (ROIs) synthesis as a possible homeostatic mechanism in the control of inflammation.

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-alpha production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.

Animals↗
PubMed2000

Heat-aggregated immunoglobulins increase in vivo immunogenicity of mouse hapten (TNP)-derivatized macrophages by upregulation of interleukin-12 secretion and expression of B7-1 and B7-2 costimulatory molecules.

Antigen-antibody complexes (IC) can up or down regulate immune responses by induction of immunoregulatory cells. We have studied the effect of mouse heat-aggregated immunoglobulin (Ig) (HA) which have many biological activities similar to IC on immunogenicity of TNP-substituted macrophages (TNP-Mphi). Our results show that: (1) mouse oil-induced peritoneal macrophages treated with HA produce in vitro significantly higher levels of interleukin (IL-1beta), tumor necrosis factor (TNF)-alpha, IL-6, IL-10 and particularly IL-12 and express more B7-1 and B7-2 and ICAM-1 cell surface costimulatory molecules than Mphi treated with monomeric Ig (MM); (2) Mphi derivatized with TNP, treated or not with MM, induce in vivo antigen-specific unresponsiveness. In contrast TNP-Mphi treated with HA induce significant contact sensitivity reaction even when injected into previously tolerized recipient animals. Treatment of recipients with anti-IL-12 Ab prevents immunization by TNP-Mphi-HA. These results indicate that bypass of tolerance by treatment of TNP-Mphi with HA is a result of an increased production of IL-12 by these cells and an enhanced expression of costimulatory molecules important in T cell-Mphi interactions. We suggest that a similar overcoming of tolerance through the action of IC may be responsible for the generation of autoantibodies of heterologous specificity in pathological conditions in which such complexes are formed.

Animals↗
PubMed2000

Cross-reactivity of TNP immune effector T cells that mediate contact hypersensitivity and inflammatory bowel disease in the mouse.

BACKGROUND: Experiments were aimed to test the cross-reactivity of immune Th1 cells that mediate contact hypersensitivity (CHS) or inflammatory bowel disease (IBD) to TNP in the mouse. METHODS: CBA/J mice were immunized either epicutaneously or intrarectally with TNP and after appropriate time intervals were challenged with antigen in a crossed manner. The CHS reaction was measured by the ear swelling test. IBD was quantified by increase of colon weight and myeloperoxidase level. Both reactions were confirmed histologically. In passive-transfer experiments, mesenteric lymph node cells of animals sensitized intrarectally and peripheral lymph node and spleen cells of mice immunized epicutaneously were used. In some experiments, before being immunized mice were made either unresponsive to the TNP hapten by induction of suppressor T cells, or resistant to suppression after induction of upregulatory T cells. RESULTS: Irrespective of the mode of sensitization upon appropriate challenge with antigen all mice developed a good CHS reaction as well as significant IBD. This cross-reactivity could be passively transferred by immune cells. In mice in which antigen-specific down- or upregulatory cells were induced before sensitization both CHS and IBD to TNP were modulated accordingly. CONCLUSION: TNP hapten deposited on skin or on mucosal surfaces induces effector cells that recognize antigen independent of its tissue localization, and produce a local inflammatory reaction. TNP-specific up- and downregulatory cells, shown before to regulate the CHS reaction, similarly modulate the generation and development of hapten-induced IBD.

Animals↗
PubMed1999

Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.

Amino Acid Sequence↗
PubMed1998

Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data.

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221-1225], our structural study evidences the importance of the beta-sheet structure and also suggests that anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.

Amino Acid Sequence↗
PubMed1998

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: a model for a potential in vivo lipid carrier protein.

The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 A3 without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by alpha-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture.

Amino Acid Sequence↗
PubMed1998

Solution structure of Ace-AMP1, a potent antimicrobial protein extracted from onion seeds. Structural analogies with plant nonspecific lipid transfer proteins.

The three-dimensional solution structure of Ace-AMP1, an antifungal protein extracted from onion seeds, was determined using 1H NMR spectroscopy and molecular modeling. This cationic protein contains 93 amino acid residues and four disulfide bridges. Its structure was determined from 1260 NOE-derived distance restraints and 173 dihedral restraints derived from NOEs and 3JCaHNH coupling constants. The global fold involves four helical segments connected by three loops and a C-terminal tail without regular secondary structures, except for a 3(10)-helix turn and a beta-turn. The most striking feature is the absence of any continuous cavity running through the whole molecule as found in recently determined structures of nonspecific transfer proteins extracted from wheat and maize seeds, although their global folds are very similar. Consistent with the absence of a cavity in the core of Ace-AMP1, it was found that this protein, in contrast to ns-LTPs, does not bind fluorescently labeled phospholipids in solution. On the other hand, Ace-AMP1 is able to interact with phospholipid membranes as shown by the release of carboxyfluorescein from the lumen of artificial liposomes and by the induction of alterations in fluorescence polarization of fluorescently labeled phospholipids embedded in artificial liposomes.

Amino Acid Sequence↗
PubMed1998

A novel composite 90 degrees pulse sequence which provides distortionless NMR spectra and suppresses without destroying the water magnetization.

A novel 90 degrees composite pulse sequence which allows one to record 1D and 2D NMR spectra without disturbing the water magnetization is described. A home-written program was used to optimize the pulse angles for which the pulse sequence response fitted best the desired excitation profile, producing a neat and distortionless spectrum with a broad null excitation at the carrier frequency. The resulting pulse sequence was first evaluated using the simulation program "PENCIL" and then tested on two protein samples. A 3.5 degrees phase shift of the last pulse was required to cancel correctly the water signal. The pulse scheme was appended to a NOESY pulse sequence. Inspection of the water cross section revealed interactions between water and some protons of drosomycine, a small insect antifungal protein.

Amino Acids↗
PubMed1998

Macrophage function in alloxan diabetic mice: expression of adhesion molecules, generation of monokines and oxygen and NO radicals.

The increased incidence of bacterial and mycotic infections in poorly controlled diabetic patients or animals is frequently attributed to impaired activities of professional phagocytes (granulocytes, macrophages) in hypoinsulinaemic milieu. We measured production of monokines (IL-6 and tumour necrosis factor-alpha (TNF-alpha)), active NO and reactive oxygen intermediates (ROIs), as well as expression of several cell surface adhesion molecules (Mac-1, -2 and -3, intercellular adhesion molecule-1 (ICAM-1) and Fc gammaRII), by thioglycollate medium-induced peritoneal macrophages of normoglycaemic and alloxan diabetic CBA/J mice (blood glucose level in the range 300 or 500 mg/dl). Macrophages of animals with moderate diabetes (300 mg/dl) produced significantly more IL-6 and TNF-alpha and ROIs than cells of control mice and showed an increased expression of all cell surface molecules, except Mac-3. NO/NO2 production was not affected. Administration of insulin restored enhanced values to normal levels, except for the production of ROIs which remained unusually high. We conclude that two separate mechanisms influence macrophage physiology in diabetes--lack of saturation of insulin receptors on macrophages and an indirect effect due to formation of advanced glycosylation endproducts (AGE) on their surfaces. The latter is possibly responsible for increased generation of ROIs, since it cannot be down-regulated by prolonged insulin treatment. How the increased activity of macrophages of moderately diabetic mice (enhanced production of proinflammatory monokines and oxygen radicals as well as expression of molecules) is related to their ability to kill bacteria is now under investigation.

Alloxan↗
PubMed1998

Aggregated immunoglobulin protects immune T cells from suppression: dependence on isotype, Fc portion, and macrophage FcgammaR.

We determined the regulatory properties of heat-aggregated immunoglobulins (HA-Ig) that possess many activities of immune complexes (IC), such as binding and activation of cells via immunoglobulin Fc gamma receptors (FcgammaR). HA-Ig protected contact sensitivity (CS) effector T cells from antigen-specific immunosuppression, while monomeric IgG were inactive. This anti-suppressive activity of HA-Ig was antigen non-specific, and depended on the species from which Ig was derived, i.e. mouse and rat HA-Ig were protective in mice, and of other species were inactive. The protecting activity of HA-Ig was confined to IgG2a and IgG3, and to a lesser degree to IgG1 isotypes, and resided in the Fc domain. Removal of phagocytic cells from the CS-immune target cells, or blocking with anti-FcgammaR mAb, abolished HA-Ig protection of CS-effector T cells from suppression. We suggest that HA-Ig multimers acted via Fc domains, in one of two ways: by binding to FcgammaR of macrophages to produce positive-acting cytokines, or by blocking FcgammaR on macrophages, to compete with suppressive factors that can also bind to FcgammaR. If HA-Ig protection of T cells is generalized, it is likely that IC in vivo may non-specifically overcome suppression of responses to antigen that normally are under the control of T suppressive cells, and thus may contribute to the development of autoimmunity.

Animals↗
PubMed1997

1H NMR and fluorescence studies of the complexation of DMPG by wheat non-specific lipid transfer protein. Global fold of the complex.

Plant non-specific lipid transfer proteins (LTPs) are proteins which transfer lipids between membranes in vitro and are believed to be involved in the transport of cutin monomers to the cuticle layer in vivo or in the plant defence against phytopathogens. The complexation of DMPG, a diacyl phospholipid, by wheat ns-LTP, a protein extracted from wheat seeds, was followed by 1H NMR and fluorescence spectroscopy. The global fold of the protein was calculated using the DIANA software package from a list of 968 distance constraints. The internal cavity volume, a feature common to all known ns-LTP structures, was estimated to be 750 A3 using the 'CAVITE' program. This model of the complex was obtained by inserting a lipid molecule in the cavity and was energy minimized. The study showed that the protein fold described for the free form was only weakly affected by the insertion of the bulky lipid. Observation of some intermolecular NOEs between the protein and the lipid glycerol moiety revealed that the cavity entrance was located between residues His35 and Arg44. The resulting solution structure was compared to the crystal structure of the maize ns-LTP/palmitate complex.

Arginine↗
PubMed1997

Refined solution structure of the anti-mammal and anti-insect LqqIII scorpion toxin: comparison with other scorpion toxins.

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatus was refined and compared with other long-chain scorpion toxins. This structure, determined by 1H-NMR and molecular modeling, involves an alpha-helix (18-29) linked to a three-stranded beta-sheet (2-6, 33-39, and 43-51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 A for C alpha atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CS alpha beta (Cysteine Stabilized alpha-helix beta-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na+ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins.

Amino Acid Sequence↗
PubMed1997

Solution structure of drosomycin, the first inducible antifungal protein from insects.

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.

Amino Acid Sequence↗