Biomedical subjects

Paweł Mak

Publications and source records attributed to Paweł Mak.

8 recordsLinked to original sources

PubMed2006

Purification and characterization of eight peptides from Galleria mellonella immune hemolymph.

Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against gram-negative and gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 microM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.

Amino Acid Sequence↗

PubMed2006

The length of menstrual bleeding and the risk of urogenital infections in the context of the activity of hemoglobin-derived microbicidal peptides.

OBJECTIVE: Well-known natural unspecific antimicrobial factors acting in the vagina are additionally reinforced during menstrual bleeding by hemocidins--a recently discovered novel class of microbicidal peptides generated proteolytically from hemoglobin. The aim of the presented research was to investigate the relation between the average length of menstrual bleeding and the frequency of urogenital infections. We expected that the shorter menstrual bleeding might increase the risk of urogenital infections because is synonymous with the shorter period of exposition on bactericidal action of hemocidins. STUDY DESIGN: The study contains statistical analysis of an average declared length of menstrual bleeding in the group of 267 young, sexually active women with the symptoms of urogenital infections. The control group consisted of 300 young healthy women. RESULTS: The length of menstrual bleeding in the group of patients with urogenital infections (average 4.35 days) was statistically significantly shorter than in the control group (average 4.95 days). The average length of the menstrual cycle was equal and counted ca. 28 days in both groups. CONCLUSION: The length of menstrual bleeding seems to be a significant factor in the vaginal ecology maintenance.

Adolescent↗

PubMed2005

Effects of elastase and cathepsin G on the levels of membrane and soluble TNFalpha.

Neutrophil elastase (NE) and cathepsin G (CG), the proteolytic enzymes localized in azurophil granules of neutrophils (PMN), are involved in PMN responses to various stimuli. When released at sites of inflammation, they participate in the degradation of numerous proteins involved in the regulation of the immune response. In this study, we employed ADAM17(-/-) fibroblasts stably transfected with cDNA of human pro-tumor necrosis factor alpha (proTNFalpha) (ADAM17(-/-)TNF(+)) to investigate the effects of NE and CG on shedding and degradation of TNFalpha. Both NE and CG were found to diminish the level of membrane TNFalpha (mTNFalpha) as measured by flow cytometry. This process was accompanied by the accumulation of biologically active soluble TNFalpha (sTNFalpha) in the culture medium, as determined by an increase in both the cytotoxic activity of TNFalpha and its ability to serve as a co-stimulator in the induction of inducible nitric oxide synthase (iNOS). However, in contrast to CG, NE at high concentrations was able to degrade sTNFalpha released from the cell surface. Using soluble recombinant human TNFalpha, we identified Val(93)-Ala(94) and Val(117)-Glu(118) as the NE cleavage sites within the sTNFalpha molecule. Taken together, the ability of NE and CG to modulate levels of membrane and soluble forms of TNFalpha may contribute to the proinflammatory activity of neutrophils.

ADAM Proteins↗

PubMed2005

New generation of peptide antibiotics.

The increasing antibiotic resistance of pathogenic bacteria calls for the development of alternative antimicrobial strategies. Possible approaches include the development of novel, broad-spectrum antibiotics as well as specific targeting of individual bacterial virulence factors. It is impossible to decide currently which strategy will prove more successful in the future since they both promise different advantages, but also introduce diverse problems. Considering both approaches, our laboratory's research focuses on the evaluation of hemocidins, broad-spectrum antibacterial peptides derived from hemoglobin and myoglobin, and staphostatins, specific inhibitors of staphopains -- Staphylococcus aureus secreted proteases that are virulence factors regarded as possible targets for therapy. The article summarizes recent advances in both fields of study and presents perspectives for further development and possible applications.

Anti-Bacterial Agents↗

PubMed2004

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma.

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.

Amino Acid Sequence↗

PubMed2004

Antibacterial hemoglobin peptides in human menstrual blood.

This work documents that normal menstrual vaginal blood of healthy females is exceptionally rich in hemocidins--hemoglobin (Hb) fragments having bactericidal properties. The peptide fractions were isolated from the plasma of vaginal discharge of three healthy nulliparous women and subjected to identification by automatic sequencing as well as by mass spectrometry. All 44 identified peptides originate from Hb (mainly from the N-terminal part of alpha-globin) and all demonstrated differential killing activity toward Escherichia coli. The screening of antimicrobial activity was performed using two synthetic peptides identical to those found in menstrual blood. These peptides were active mainly toward Gram-negative bacteria and to a less degree toward Gram-positive bacteria. Our results confirm recent observations that Hb-derived fragments manifest pronounced antibacterial activity and suggest that these peptides help in maintaining human vaginal homeostasis during physiologic menstrual bleeding.

Adolescent↗

PubMed2003

Collagen type II modification by hypochlorite.

Oxidation of proteins is a common phenomenon in the inflammatory process mediated by highly reactive agents such as hypochlorite (HOCl/OCl(-)) produced by activated neutrophils. For instance, in rheumatoid arthritis hypochlorite plays an important role in joint destruction. One of the major targets for HOCl/OCl(-) is collagen type II (CII) - the primary cartilage protein. In our study, HOCl/OCl(-) mediated collagen II modifications were tested using various methods: circular dichroism (CD), HPLC, ELISA, dynamic light scattering (DLS), fluorimetry and spectrophotometry. It was shown that hypochlorite action causes deamination with consecutive carbonyl group formation and transformation of tyrosine residues to dichlorotyrosine. Moreover, it was shown that ammonium chloramine (NH(2)Cl) formed in the reaction mixture reacts with CII. However, in this case the yield of carbonyl groups and dichlorotyrosine is lower than that observed for HOCl/OCl(-) by 50%. CD data revealed that collagen II exists as a random coil in the samples and that chlorination is followed by CII fragmentation. In the range of low HOCl/OCl(-) concentrations (up to 1 mM) 10-90 kDa peptides are predominant whereas massive production of shorter peptides was observed for high (5 mM) hypochlorite concentration. DLS measurements showed that chlorination with HOCl/OCl(-) decreases the radius of collagen II aggregates from 30 to 6.8 nm. Taking into account the fact that chlorinated collagen is partially degraded, the DLS results suggest that smaller micelles are formed of the 10-90 kDa peptide fraction. Moreover, collagen chlorination results in epitope modification which affects CII recognition by anti-CII antibodies. Finally, since in the synovial fluid the plausible hypochlorite concentration is smaller than that used in the model the change of size of molecular aggregates seems to be the best marker of hypochlorite-mediated collagen oxidation.

Amino Acids↗

PubMed2003

Properties of chemically oxidized kininogens.

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.

Amino Acids↗