Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.     

Optimizing Expression of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Surface Protein a,PspA: Serocross-Reactivity within Families of Antisera Induced Against Clades 1 and 3

Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family.     

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.     

Translational control of the ascorbic acid transporter SVCT2 in human platelets

Reactive oxygen species (ROS) and redox state have emerged as physiological mediators, controlling blood coagulation and thrombosis. The redox balance is obviously linked to the presence of antioxidants; in particular, vitamin C appears to be a key modulator of platelet oxidative state, since these cells physiologically accumulate ascorbic acid and, moreover, platelet ascorbate plays a role during aggregation. Here, we showed that platelets could compensate for fluctuations in ascorbate levels by modulating the expression of the Na+-dependent transporter SVCT2. Furthermore, the use of anucleated cells demonstrated, for the first time, that SVCT2 expression could be regulated at the translational level. The control of ascorbic acid uptake, through regulation of its carrier, was not only related to substrate availability, but it also occurred during platelet activation, which was accompanied by vitamin C deprivation and alteration in the redox state. Finally, we showed that changes in intracellular ascorbic acid content had physiological relevance, since they modulate the surface sulfhydryl content and the thrombus viscoelastic properties. Beside its role during aggregation, vitamin C may also have important effects during postaggregatory events.     

Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group

Pathogenesis of chronic inflammatory diseases is associated with excessive elastase release through neutrophil degranulation. In the present study, inhibition of human neutrophil degranulation by four flavonoids (myricetin, quercetin, kaempferol, galangin) was evaluated by using released elastase as a biomarker. Inhibitory potency was observed in the following order: quercetin > myricetin > kaempferol = galangin. Quercetin, the most potent inhibitor of elastase release also had a weak inhibitory effect on the enzyme catalytic activity. Furthermore, the observed effects were highly dependent on the presence of a catechol group at the flavonoid B-ring. The results of the present study suggest that quercetin may be a promising therapeutic agent in the treatment of neutrophil-dependent inflammatory diseases.     

Development of a ligand blot assay using biotinylated live cells

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.     

Natural infectivity of Xylella fastidiosa Wells et al. in sharpshooters (Hemiptera: Cicadellidae) from coffee plantations of Parana, Brazil

Xylella fastidiosa Wells et al., a gram-negative and xylem limited bacterium, causes significative economic on several crops, such as the leaf scorch in coffee. It is transmitted by xylem feeding insects and four sharpshooters species have been reported as vectors of X. fastidiosa in coffee. The objective of this study was to determine the natural infectivity of X. fastidiosa in five species of sharpshooters from coffee trees: Acrogonia citrina Marucci & Cavichioli, Bucephalogonia xanthophis (Berg), Dilobopterus costalimai Young, Oncometopia facialis (Signoret) and Sonesimia grossa (Signoret). Samples were collected from coffee plantations in five counties of the North and Northwest regions of the State of Parana, Brazil, from October 1998 through November 2001. A total of 806 samples containing three to five insects were examined for the presence of X. fastidiosa by using PCR and nested PCR tests. X. fastidiosa was present in samples of all five species of sharpshooters collected in the two coffee regions. The average level of natural infectivity potential was 30.4%. However, this natural infectivity ranged from 2.2% for O. facialis to 68.8% for A. citrina. Sharpshooters collected in the spring tended to have lower natural infectivity of X. fastidiosa as compared to those collected in other seasons. The results obtained showed the high potential of dissemination of X. fastidiosa by different insect vectors in coffee trees in Parana.     

Time dependent modifications of Hep G2 cells during exposure to static magnetic fields

Morphological modifications, i.e., cell shape, cell surface sugar residues, cytoskeleton, and apoptosis of Hep G2 cells during 24 h exposure to 6 mT static magnetic field (static MF) were studied by means of light and electron microscopy and cytochemistry. Progressive modifications of cell shape and surface were observed during the entire period of exposure to static MF. Control cells were polyhedric with short microvilli covering the cell surface, while those exposed to static MF, were elongated with many irregular microvilli randomly distributed on the cell surface. At the end of the exposure period, the cells had a less flat shape due to partial detachment from the culture dishes. However, throughout the period of exposure under investigation, the morphology of the organelles remained unmodified and cell proliferation was only partially affected. In parallel with cell shape changes, the microfilaments and microtubules, as well as the quantity and distribution of surface ConA-FITC and Ricinus communnis-FITC labeling sites, were modified in a time dependent manner. Apoptosis, which was almost negligible at the beginning of experiment, increased to about 20% after 24 h of continuous exposure. The induction of apoptosis was likely due to the increment of [Ca2+]i during exposure. In conclusion, the data reported in the present work indicates that 6 mT static MF exposure exerts time dependent biological effects on Hep G2 cells.     

Whole-cell Bordetella pertussis vaccine component modulates the mouse immune response to an unrelated soluble antigen

Several factors are involved in the selective activation of Th1 or Th2 cells, such as different physical characteristics of antigens and the type of antigen-presenting cells involved in the immune response, among others. To study the influence of a particulate antigen on Th1/Th2 cell differentiation during the immune response to another antigen, we analysed the immune response to tetanus toxoid (soluble antigen) in BALB/c mice immunized with one of the three following vaccines: tetanus and diphtheria toxoids (DT), or DT associated with whole-cell Bordetella pertussis or its soluble antigens (DTPw and DTPa, respectively). Similar total antibody levels were observed for all vaccines. DT vaccine showed a higher IgG1/IgG2a ratio than the similar values observed for DTPw and DTPa vaccines. DT- and DTPa-primed spleen cells showed a Th2 (IL-5) profile while a Th1/Th2 (IFN gamma, IL-5) profile was observed for DTPw. IL-6 was only produced by DTPw-primed cells. Besides, IL-12 levels induced by DTPw were three times higher than the ones induced by both DT and DTPa. Our findings indicate that whole-cell B. pertussis priming modifies the tetanus immune response from Th2 to Th1/Th2 type probably via inflammatory mechanisms. In addition, in the light of conflicting reports regarding the mechanisms of protection induced by DTP vaccines, we studied the pertussis immune response. Only DTPw immunization generated memory T cells capable of proliferating with B. pertussis as an in vitro stimulus. Results might indicate that these cells may not play a key role in protecting against B. pertussis when the host is vaccinated with DTPa.