Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K (m) and V (max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 A mu mol/min/mg, respectively. PG was found to have temperature optimum at 65 A degrees C and was totally stable at 45 A degrees C for 90 min. Half-life at 55 A degrees C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites

Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.

Construction and validation of a Bovine Innate Immune Microarray

Background: Microarray transcript profiling has the potential to illuminate the molecular processes that are involved in the responses of cattle to disease challenges. This knowledge may allow the development of strategies that exploit these genes to enhance resistance to disease in an individual or animal population. Results: The Bovine Innate Immune Microarray developed in this study consists of 1480 characterised genes identified by literature searches, 31 positive and negative control elements and 5376 cDNAs derived from subtracted and normalised libraries. The cDNA libraries were produced from 'challenged' bovine epithelial and leukocyte cells. The microarray was found to have a limit of detection of 1 pg/mu g of total RNA and a mean slide-to-slide correlation co-efficient of 0.88. The profiles of differentially expressed genes from Concanavalin A ( ConA) stimulated bovine peripheral blood lymphocytes were determined. Three distinct profiles highlighted 19 genes that were rapidly up-regulated within 30 minutes and returned to basal levels by 24 h; 76 genes that were upregulated between 2 - 8 hours and sustained high levels of expression until 24 h and 10 genes that were down-regulated. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray analysis. The results indicate that there is a dynamic process involving gene activation and regulatory mechanisms re-establishing homeostasis in the ConA activated lymphocytes. The Bovine Innate Immune Microarray was also used to determine the cross-species hybridisation capabilities of an ovine PBL sample. Conclusion: The Bovine Innate Immune Microarray has been developed which contains a set of well-characterised genes and anonymous cDNAs from a number of different bovine cell types. The microarray can be used to determine the gene expression profiles underlying innate immune responses in cattle and sheep.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

Prevalence of diabetic peripheral neuropathy and relation to glycemic control therapies at baseline in the BARI 2D cohort

We evaluated the associations between glycemic therapies and prevalence of diabetic peripheral neuropathy (DPN) at baseline among participants in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial on medical and revascularization therapies for coronary artery disease (CAD) and on insulin-sensitizing vs. insulin-providing treatments for diabetes. A total of 2,368 patients with type 2 diabetes and CAD was evaluated. DPN was defined as clinical examination score > 2 using the Michigan Neuropathy Screening Instrument (MNSI). DPN odds ratios across different groups of glycemic therapy were evaluated by multiple logistic regression adjusted for multiple covariates including age, sex, hemoglobin A1c (HbA1c), and diabetes duration. Fifty-one percent of BARI 2D subjects with valid baseline characteristics and MNSI scores had DPN. After adjusting for all variables, use of insulin was significantly associated with DPN (OR = 1.57, 95% CI: 1.15-2.13). Patients on sulfonylurea (SU) or combination of SU/metformin (Met)/thiazolidinediones (TZD) had marginally higher rates of DPN than the Met/TZD group. This cross-sectional study in a cohort of patients with type 2 diabetes and CAD showed association of insulin use with higher DPN prevalence, independent of disease duration, glycemic control, and other characteristics. The causality between a glycemic control strategy and DPN cannot be evaluated in this cross-sectional study, but continued assessment of DPN and randomized therapies in BARI 2D trial may provide further explanations on the development of DPN.

CR3 (CD11b/CD18) activation of nasal neutrophils: a measure of upper airway endotoxin exposure

Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control - 1 M nicotinamide; sham; positive control - 11 eta g of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548 +/- 15.65 RLU 100 mu l(-1); sham: 11469 +/- 2582 RLU 100 mu l(-1); positive control: 42026 +/- 16659 RLU 100 mu l (n = 7; p < 0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.