Kinetic Properties of a Novel Fusarium solani (phospho)lipase: A Monolayer Study

Using the monomolecular film technique, we studied interfacial properties of Fusarium solani lipase (FSL). This lipolytic enzyme was found to be unique among the fungal lipases possessing not only a lipase activity but also a high phospholipase one.The FSL was able to hydrolyze dicaprin films at various surface pressures. The surface pressure dependency, the stereospecificity, and the regioselectivity of FSL were performed using optically pure stereoisomers of diglyceride (1,2‐sn‐ dicaprin and 2,3‐sn‐dicaprin) and a prochiral isomer (1,3‐sn‐dicaprin) spread as monomolecular films at the air–water interface. The FSL prefers adjacent ester groups of the diglyceride isomers (1,2‐sn‐dicaprin and 2,3‐sn‐dicaprin) at low and high surface pressures. Furthermore, FSL was found to be markedly stereospecific for the sn‐1 position of the 1,2‐sn‐enantiomer of dicaprin at both low and high surface pressures.Moreover, FSL shows high activities on phospholipids monolayers. However, this enzyme displays high preference to zwitterionic phospholipids compared to the negatively charged ones. Chirality, 25:35‐38, 2012. © 2012 Wiley Periodicals, Inc.     

Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109

PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer.     

Considerations when using discriminant function analysis of antimicrobial resistance profiles to identify sources of fecal contamination of surface water in Michigan

The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.     

Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination

The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Relapsed follicular lymphoma sequentially treated with rituximab and 90Y-ibritumomab tiuxetan. Case report

BACKGROUND: Monoclonal antibodies have dramatically changed the treatment possibilities for follicular lymphoma. (90)Y-ibritumomab tiuxetan (Zevalin) is the first radioimmunotherapy agent approved for the treatment of relapsed and resistant follicular lymphoma patients. Long-term benefit was observed especially for patients achieving CR after radioimmunotherapy. METHODS AND RESULTS: A 65-year-old female patient with the second relapse of CD20 positive follicular lymphoma and multiple concomitant diseases was treated with four weekly doses of rituximab (375 mg/m(2)). (18)F-fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET-CT) demonstrated only partial response to therapy with persistent PET scan positivity in enlarged abdominal lymph nodes. Therefore, it was decided to treat her with a 1200-MBq (32-mCi) dose of (90)Y-ibritumomab tiuxetan radioimmunotherapy. No acute complications were noted afterwards. Hematological nadirs were reached 4 weeks later, with a platelet count of 24 x 10(9)/l that normalized within the next 2 weeks. The patient had neither infection nor bleeding complications. Eight weeks after radioimmunotherapy, the PET-CT scans documented only 3 lymph nodes around the abdominal aorta, maximum size 2 x 1 cm. The PET scan analysis proved no accumulation of (18)F-fluoro-deoxy-glucose in any lymph nodes or other organs and tissues. CONCLUSIONS: Sequential treatment with rituximab and (90)Y-ibritumomab tiuxetan may be an interesting alternative in cases of relapsed follicular or other indolent lymphomas in pretreated or older patients with other concomitant diseases.     

Association between plasma antibody response and protection in rainbow trout Oncorhynchus mykiss immersion vaccinated against Yersinia ruckeri

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge.Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1x10⁹ CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.     

Sources and pathways of spread of vancomycin-resistant enterococci in hemato-oncological patients

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.     

Evidence of DNA rearrangements in the 128-kilobase pBtoxis plasmid of Bacillus thuringiensis israelensis

BUPM97 is a novel Tunisian isolate of Bacillus thuringiensis israelensis presenting insecticidal activity against Culex pipiens larvae. The δ-endotoxins pattern of this strain was different from that of the reference strain B. thuringiensis israelensis H14. Therefore, the study of its cry genes content was carried out by restriction-fragment-length-polymorphism (RFLP) using specific cry genes probes and by DNA sequencing. It was clearly demonstrated that in the strain BUPM97 the cry4A and cry10A genes were deleted from the B. thuringiensis israelensis 128-kb pBtoxis plasmid. In addition, a strong DNA sequence polymorphism was evidenced in the same plasmid downstream from the cry4B gene. This very particular DNA dynamic evidenced in this new strain of B. thuringiensis israelensis should be taken into consideration, regarding the strain stability during the industrial production of B. thuringiensis bioinsecticides.     

Microbial communities in large-scale wood piles and their effects on wood quality and the environment

The demand of renewable energy sources, i.e. biomass, is steadily increasing worldwide to reduce the need of fossil energy sources. Biomass such as energy crops, woody species, forestry and agricultural residues are the most common renewable energy sources. Due to uneven demand for wood fuel, the material is mostly stored outdoors in chip piles or as logs until utilisation. Storage of biomass is accompanied by chemical, physical and biological processes which can significantly reduce the fuel quality. However, heating plants require high-quality biomass to ensure efficient operation, thereby minimising maintenance costs. Therefore, optimised storage conditions and duration times for chipped wood and tree logs have to be found. This paper aims at reviewing available knowledge on the pathways of microbial effects on stored woody biomass and on investigations of the fungal and bacterial community structure and identity. Moreover, potential functions of microorganisms present in wood chip piles and logs are discussed in terms of (1) reduction of fuel quality, (2) catalysing self-ignition processes, and (3) constituting health risk and unfriendly work environment.