Depression, anxiety in ovarian cancer patient.

OBJECTIVE: A summary article, which is concluding available data about the psychological alterations, especially depression and anxiety, in ovarian cancer patients. DESIGN: Revue article. SETTING: Department of Obstetrics and Gynaecology, University Hospital in Hradec Králove. DISCUSSION: Depressive symptoms could arise as a consequence of the stress, which is the response to oncological diagnosis, treatment or relapse of the oncological disease. This depressive condition is raising concerns in patients, family and health care professionals because it is significantly contributing to morbidity and at the same time is leading to the increase of the health care costs. In general the alteration of the physical and mental functions is reducing the average life expectancy. The patients with serious gynecological cancer diagnosis are requiring psychological support, which is not always satisfactory from their family. It would therefore be desirable to establish professional centers or clinics providing counseling and psychotherapy. CONCLUSION: The summary of the available research data about depression in a women with gynaecological cancer has pointed out not only connection between depression and ovarian cancer, but has also underlined the importance of this problematic for daily praxis and further intensive research.

Occurrence of serum antibodies against wheat alpha-amylase inhibitor 0.19 in celiac disease.

The alcohol-soluble fraction of wheat gluten (gliadins) induces in genetically susceptible individuals immunologically mediated celiac disease (CLD). However, gliadins and related cereal proteins are not unique foodstuff targets of CLD patients´ immune system. Non-gluten wheat alpha-amylase inhibitor 0.19 (AAI 0.19) has been found to be capable of activating human monocyte-derived dendritic cells and inducing pro-inflammatory status in intestinal mucosa of patients with celiac disease (CLD). The possible contribution of this reactivity in incomplete remission of CLD patients on a gluten-free diet (GFD) is matter of contention. In an attempt to characterize the antigenicity of AAI 0.19 in patients with active CLD, patients on a GFD and healthy controls we developed ELISA employing wheat recombinant AAI 0.19. Using this test we revealed a significant (P<0.001) elevation of IgA anti-AAI 0.19 antibodies (Ab) in patients with active CLD (12 out of 30 patients were seropositive) but also in CLD patients on a GFD (15/46), in contrast to healthy controls (2/59). Anti-AAI 0.19 IgG Ab levels were increased (P<0.001) only in patients with active CLD (14/30) in contrast to the controls. Interestingly, the levels of anti-AAI 0.19 IgG Ab were decreased in CLD patients on a GFD (P<0.001, 1/46) compared to the controls (1/59). Notably, 20 out of 30 patients with active CLD were positive either for IgA or for IgG anti-AAI 0.19 Ab. Thus, the majority of CLD patients developed a robust IgA and IgG Ab response against AAI 0.19. These findings may contribute to the broadening of the knowledge about CLD pathogenesis.

Effect of trapping method on species identification of phlebotomine sandflies by MALDI-TOF MS protein profiling.

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood-feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI-TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in-house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI-TOF MS protein profiling for species identification.

Prevalence of Mycoplasma hominis and Ureaplasma urealyticum in women undergoing an initial infertility evaluation.

AIMS: Mycoplasma hominis and Ureaplasma urealyticum are potentially pathogenic bacterial species that are frequently isolated from the urogenital tract of women. These pathogens could be responsible for various genitourinary diseases and have been associated with adverse pregnancy outcomes and female fertility problems. The aim of this study was to analyse the presence of M. hominis and U. urealyticum in the cervical canal of uterus of women with and without fertility problems. METHODS: Endocervical swabs obtained from women with reproductive problems and fertile women were tested by both cultivation and polymerase chain reaction. The antimicrobial susceptibility to the azithromycin, ciprofloxacin, doxycycline and erythromycine of the isolated strains of M. hominis and U. urealyticum was also tested by the microdilution broth method. RESULTS: A total of 111 women with fertile problems were examined. U. urealyticum was detected in samples from 44 (39.6%) women. M. hominis was detected in significantly fewer samples, i.e. only from 9 (8.1%) samples. From these, 6 (5.4%) women were positive for both microorganisms. The fertile group consisted from 23 women. The presence of U. urealyticum was detected in 8 (34.7%) of them. M. hominis was detected only in the mixture with U. urealyticum in 3 (13.0%) cases. The most effective antibiotic against both species in our study was doxycycline. CONCLUSION: The results show slightly higher incidence of M. hominis and U. urealyticum in the genitourinary tract of women with fertility problems compare with control group. The potential negative effect of these species on the reproduction ability of women was not observed.

C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-ß in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment.

Proteins implicated in the increase of adhesivity induced by suberoylanilide hydroxamic acid in leukemic cells.

We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases.

DnaK and GroEL chaperones are recruited to the Bacillus subtilis membrane after short-term ethanol stress.

AIMS: To find out membrane tolerance strategy to ethanol in Bacillus subtilis that possesses a powerful system of protection against environmental stresses. METHODS AND RESULTS: Cytoplasmic membranes of B. subtilis were severely affected by even short-term exposure to 3% (v/v) ethanol: the growth rate and membrane protein synthesis were markedly reduced, and no adaptive alterations in phospholipids were detected. Simultaneously, steady-state DPH fluorescence anisotropy (r(ss)) showed that the membrane rigidity increased substantially. Analysis of the membrane phosphoproteome using in vitro labelling with [γ-(32) P]ATP revealed the association of DnaK and GroEL chaperones with membrane, indicating a stress induction process. Upon a long-term 3% (v/v) ethanol stress, the cell growth accelerated slightly and the composition of polar head groups and fatty acids of membrane phospholipids underwent an extensive reconstruction. Correspondingly, membrane fluidity turned back to the original r(ss) values of the control cells. CONCLUSIONS: In B. subtilis, the adaptive response to short-term ethanol stress comprises the recruitment of molecular chaperones on the impaired membrane structure; consequently, the phospholipid synthesis is restored and membrane fluidity adapts properly to the continuing ethanol stress. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings underline the role of membrane lipids in establishing tolerance towards ethanol and also suggest the contribution of molecular chaperones to the membrane and cell recovery.

Effect of starter unit availability on the spectrum of manumycin-type metabolites produced by Streptomyces nodosus ssp. asukaensis.

AIMS: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. METHODS AND RESULTS: Structural variations of manumycins lie only in the diverse initiation of the 'upper' polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A. CONCLUSIONS: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.

A new approach to the isolation and characterization of wheat flour allergens.

BACKGROUND: The incidence of food allergy to wheat is increasing. Its diagnosis depends on the purity of major allergens and their inclusion in tests. Isolation and characterization of wheat allergens are therefore of utmost importance. OBJECTIVE: To purify and identify wheat flour allergens most frequently recognized by patients' IgE antibodies and to study their allergenicity. METHODS: Water/salt-soluble extracts from wheat flour were prepared and separated using a combination of ultrafiltration, isoelectric focusing and liquid chromatography. Purified proteins were analysed by immunoblotting using pooled sera from patients with atopic dermatitis who possessed IgE specific to wheat. Wheat proteins found to bind IgE were subsequently identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The frequency and intensity of IgE binding of isolated proteins were tested using individual sera from patients and controls. RESULTS: We developed a procedure that allows isolation of wheat allergens from natural sources. Twenty-seven potential wheat allergens have been successfully identified; of these, the following seven are newly reported in food allergy: endogenous α-amylase/subtilisin inhibitor, trypsin/α-amylase inhibitor (AAI) CMX1/CMX3, thaumatin-like protein (TLP), xylanase inhibitor protein-1, ß-glucosidase, class II chitinase and 26 kDa endochitinase. TLP and wheatwin were shown to activate patients' basophils to a similar extent as two well-known allergens, lipid transfer protein (Tri a 14) and AAI 0.19 (Tri a 28.0101). CONCLUSION AND CLINICAL RELEVANCE: Our new approach enables the isolation of water/salt-soluble wheat allergens in their native form in amounts sufficient both for biological testing (in vivo and in vitro) and for physicochemical characterization. Such studies will lead to a more detailed knowledge of allergenicity of wheat proteins and to improved accuracy of diagnostic tests.

Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis.

Stress proteomes of the cytoplasmic membrane fraction of Bacillus subtilis trp (C2)-exposed to acid pH and ethanol were characterized. Although these stress factors impair the cell function in a specific manner, they share the ability to denature proteins. Therefore, specific and general stress proteins in the membranes were investigated. Both ethanol (3 %) and pH 5.0 increase the doubling time from 17 to 25 min. Isolated cytoplasmic membranes were subjected to an optimized 2D PAGE analysis which permitted the separation and analysis of ≈450 distinct protein spots. Two alternative methods of protein detection were compared, i.e. silver staining and (35)S-L-methionine pulse labeling; the stress induced proteins were identified by MALDI-TOF MS. After ethanol stress, five proteins were increased, viz. YdaP, Ctc, YfhM, YjcH and YwaC. Acid stress proteins were AcoB, YkwC, SodA, YjcH and YwaC. Proteins YjcH and YwaC were increased after ethanol as well as acid pH treatment.

Changes in cell adhesivity and cytoskeleton-related proteins during imatinib-induced apoptosis of leukemic JURL-MK1 cells.

The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin ß1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors.

Glucosylglycerate is an osmotic solute and an extracellular metabolite produced by Streptomyces caelestis.

Streptomyces caelestis DSM 40084 produces two osmolytes, viz. 2-O-(alpha-D-glucopyranosyl)-zeta-glyceric acid (GG) and trehalose. Both compounds were isolated and identified by nuclear magnetic resonance spectroscopy and mass spectrometry. A very sensitive regulation of the cell osmolytes was demonstrated in exponentially growing cultures. The intracellular levels of GG and trehalose increased 2x in response to a step change of medium osmolarity caused by 0.3% NaCl. 1H NMR analysis of the cell extracts did not confirm the presence of additional osmolytes. GG is a S. caelestis metabolite commonly released from the cells; its concentration reached 3 g/L during the cultivation in a yeast extract--(NH4)2SO4-glycerol medium. This is the first report on the occurrence of the ionic osmolyte GG in the genus Streptomyces and on its free excretion to the medium.

Oligosaccharides produced by submerged cultures of Claviceps africana and Claviceps sorghi.

Oligosaccharides produced by submerged cultures of C. africana and C. sorghi were isolated by semipreparative HPLC. Structure of 6-O-beta-D-fructofuranosyl-D-glucopyranose (blastose), 1,6-bis-O-(beta-D-fructofuranosyl)-alpha-D-glucopyranoside (neokestose) and two sugar alcohols, 1-O-beta-D-fructofuranosyl-D-mannitol (fructosylmannitol) and 1,6-bis-O-(beta-D-fructofuranosyl)-D-mannitol (bisfructosylmannitol) was determined by NMR spectrometry. MALDI TOF MS analysis revealed molecular ions [M+Na]+ that indicate the presence of other tetra- and pentasaccharides (m/z = 689.4 and 851.5, respectively) and corresponding sugar alcohol (m/z = 691.4). Rapid conversion of sucrose into series of oligosaccharides and corresponding sugar alcohols was observed in all tested strains.

Physiological dendrogram of Claviceps spp. based on sucrose metabolism in submerged cultures and its comparison with phylogenetic tree.

Sixteen isolates of Claviceps spp. were analyzed for the production of polysaccharides, oligosaccharides, and sucrose metabolism under conditions of submerged fermentation. Physiological markers calculated by the Verhulst-Pearl law were used for hierarchical cluster analysis. Low correlation was found between physiologically based dendrogram and phylogenetic analysis constructed from an alignment of rDNA sequences. To confirm the intraspecific uniformity of physiological markers three isolates of C. africana from different hosts and locations were included. The influence of genotype, physiological variability, environmental location and habitat on metabolite production is discussed.

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Characterization of the rplB gene from Streptomyces collinus and its protein product by mass spectrometry.

Ribosomal protein L2 is the largest protein components of 50S subunits. The protein is implicated in peptidyl transferase activity and binds to functionally important domains of 23S rRNA. The rplB gene, which codes for ribosomal protein L2 was cloned from Streptomyces collinus. The gene rplB was isolated from BamHI fragment (3.0 kb) of chromosomal DNA possessing two partial and four complete ORF's in the order from 5' to 3': rplC, rplD, rplW, rplB, rpsS, and rplV. The gene organization corresponds to the S10 operon. Gene rplB (834 bp) encodes a polypeptide chain of 278 amino acids. The molecular mass calculated from genomic structure is 30.5 kDa and pI 11.87. Protein L2 is rich in positively charged amino acids (Arg 36, Lys 20, and His 11). N-terminal domain possesses topology similar to the oligonucleotide/oligosaccharide binding OB folds. The availability of genome sequence makes it possible to identify L2 protein by mass spectrometry, moreover it facilitates the characterization of its potential posttranslational modifications. To confirm the protein sequence derived from the rplB gene the tryptic peptides of L2 were analyzed by mass spectrometric techniques. The obtained data matched exactly with the results of DNA sequencing.

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Oxidation of lincomycin by hydrogen peroxide restricts its potential biotransformation with haloperoxidases.

Lincomycin biotransformation was conducted by using Streptomyces venezuelae and Streptomyces phaeochromogenes cell-free extracts. Reaction products were isolated and identified by MS and NMR spectroscopy as lincomycin sulfoxide and lincomycin sulfone. Both compounds arise also by chemical oxidation with hydrogen peroxide; this reaction represents a new efficient way for the preparation of lincomycin sulfoxide and lincomycin sulfone and simultaneously excludes the biotransformation of lincomycin using haloperoxidases.

Chemoenzymatic preparation of silybin beta-glucuronides and their biological evaluation.

Chemoenzymatic glucuronidation of the optically pure silybin A (1) using ovine liver glucuronyl transferase afforded three beta-glucuronides of silybin, substituted at phenolic OH groups at the positions C-20 (2), C-7 (3), and C-5 (4) formed in the yields 27, 62.5, and 2.5%, respectively. Using these standards, it was shown that the main silybin conjugate in humans is its 20-beta-D-glucuronate (2), while the C-7 regioisomer (3) was formed in lower proportion. The rate of conjugation of (natural) silybin diastereomers 10S, 11S and 10R, 11R, and therefore also their metabolism in humans is rather different. The radical scavenging activity of 2 is considerably lower than that of its aglycone (1); however, the activity of 3 is higher than in the silybin. These findings corroborate the hypothesis that, at physiological pH, the exclusive target for one-electron oxidation of the silybin molecule is the o-methoxy-phenolic structure at C-19, C-20. This is first pharmacological study using optically pure silybin.