Impact of ethanol and ultrasound treatment on mesophilic aerobic bacteria, coliforms, and Salmonella Typhimurium on chicken skin.

The present study evaluated the efficacy of ethanol treatment (0, 30, 50, or 70%) alone or in combination with ultrasound (37 kHz, 380 W) for the reduction of natural indigenous mesophilic aerobic bacteria (MAB), coliforms, and inoculated Salmonella Typhimurium on chicken skin. Bacterial cells with loose, intermediate, or tight attachment to chicken skin were recovered by shaking in an incubator (200 rpm) for 5 min, stomaching for 1 min, or blending for 1 min, respectively. Chicken skins were inoculated with a suspension (7 log CFU/mL) of S. Typhimurium. Ethanol reduced the number of MAB, coliforms, and S. Typhimurium on the chicken skin in a concentration-dependent manner, whereas ultrasound treatment without ethanol was ineffective. A combination of 70% ethanol with ultrasound treatment was the most effective in reducing S. Typhimurium populations with loose, intermediate, and tight attachment (reduction by 2.86 log CFU/g, 2.49 log CFU/g, and 1.63 log CFU/g, respectively). However, chicken skin treated with 50% ethanol alone or with a combination of >50% ethanol and ultrasound showed significant changes in Hunter color values (a* and b*) and texture (shear force) (P > 0.05). On the other hand, a combination of 30% ethanol and ultrasound yielded the best results, leading to a reduction of S. Typhimurium by a >1.0 log CFU/g, but did not alter the color or texture of chicken skin. Thus, a combination of 30% ethanol and ultrasound appears to be the optimum treatment for reduction of microbial contamination in production and distribution of skin-on chicken products, and enhance poultry safety without decreasing food quality.

Therapeutic effects of kefir grain Lactobacillus-derived extracellular vesicles in mice with 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease.

Kefir is a fermented product from yeast and lactic acid bacteria, and has been associated with various health benefits including relieving inflammatory bowel disease. Recently, it has been shown that gram-positive bacteria produce extracellular vesicles (EV). The EV could be appearing as potentially important mediators of cell to cell interaction. In this study, we explored the role of kefir grain Lactobacillus-derived EV in modulating inflammation responses via alleviating the production of inflammatory cytokines in tumor necrosis factor-α (TNF-α)-induced inflammation in Caco-2 cells and the 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease mouse model. Kefir-derived Lactobacillus EV were isolated by ultracentrifugation of the culture medium of 3 different kefir-derived strains (i.e., Lactobacillus kefir, Lactobacillus kefiranofaciens, and Lactobacillus kefirgranum). Nanoparticle tracking analysis showed that the size of isolated kefir-derived Lactobacillus EV was within 80 to 400 nm, and kefir-derived Lactobacillus EV uptake into recipient Caco-2 cells was confirmed by fluorescence labeling. Treatment of each kefir-derived Lactobacillus EV onto TNF-α-stimulated Caco-2 cells significantly reduced the level of both mRNA expression and secretion of IL-8, and Western blot analysis revealed that such an effect was related to inhibition of TNF-α signaling mediated by reducing the phosphorylation of p65, a subunit of NF-kB. Subsequent administration of kefir-derived Lactobacillus EV into inflammatory bowel disease-induced mice significantly alleviated the body weight loss and rectal bleeding, and enhanced stool consistency. Histological examination showed that kefir-derived Lactobacillus EV substantially reduced the infiltration of transmural leukocytes and loss of goblet cells within the colon, and the serum level of myeloperoxidase was significantly lower in the EV-treated group than control group. Our study demonstrates that kefir-derived Lactobacillus EV can be potentially used for developing innovative strategies for alleviating inflammatory bowel disease.

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit.

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid-liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-microm Nova-Pak C18 column (150x3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2-10 microg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1-50 microg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 microg/l for plasma and vitreous humour using a 1-ml sample and was 1 microg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100-102% for plasma, 91-106% for aqueous humour and 96-111% for vitreous humour. The between-day recovery (n=9) was 90-114% for plasma, 83-115% for aqueous humour and 90-105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7-7.0% and 8.1-13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs.

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-microm Capcell Pak C18 column (150x4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine-HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1-100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2-93.9%, the between-day recovery (n=5) was 89.5-93.5%, and the absolute between-day recovery (n=5) was 77-81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59-5.82% and 3.17-4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.

Expression of the fibroblast growth factor receptor-1 in human normal tissues and tumors determined by a new monoclonal antibody.

OBJECTIVE: To characterize the distribution of the receptor for the fibroblast growth factor in human normal tissues and tumors using a new monoclonal antibody. DESIGN: Monoclonal antibodies for a kinase-insert region of fibroblast growth factor receptor-1 (FGFR-1) were generated. We conducted an immunohistological analysis of FGFR-1 using a highly specific antibody we generated, and we examined the distribution of this receptor in normal human tissues and in tumors. RESULTS: Intense positivity of FGFR-1 was observed in astrocytes in the brain, smooth muscles in the uterus, cardiac myocytes, respiratory epithelium in the lung, tubular epithelium in the kidney, acinar cells in the pancreas, follicular cells in the thyroid, and ductal and lobular epithelium in the breast. We also observed FGFR-1 expression in the fibroblasts and the tissue microvasculature. In addition to some nonepithelial tumors, some epithelial tumors expressed FGFR-1, including pancreatic adenocarcinomas, thyroid papillary carcinomas, invasive ductal carcinomas of the breast, lung adenocarcinomas, renal cell carcinomas, and colonic adenocarcinomas. Although FGFR-1 was expressed in colonic adenocarcinomas, which have invasive potential, tubular adenomas, which are noninvasive, did not express FGFR-1. CONCLUSION: We have been able to define the distribution of FGFR-1 in human normal tissues and tumors. Especially in colonic tumors, FGFR-1 expression may lead adenoma cells to invade and grow in the surrounding tissue.

The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA.

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H. Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.

Multiplicity of transforming growth factors in human malignant effusions.

Human malignant effusions were found to contain transforming growth factor (TGF) activity capable of stimulating anchorage independent growth of nontransformed rodent fibroblasts. Bio-Gel P-60 chromatography of acid-ethanol extracts demonstrated the presence of three populations of TGF activities in 57% of malignant effusions. Two activities were similar to those of TGF alpha and TGF beta as judged by their size (Mr approximately equal to 6,000 and approximately equal to 25,000, respectively), biological activity (ability to stimulate anchorage independent growth of NRK fibroblasts in the absence or presence of epidermal growth factor, respectively), and capacity to competitively inhibit binding of 125I-labeled epidermal growth factor to A-431 membranes and 125I-TGF beta to baby hamster kidney fibroblasts, respectively. In addition a third factor which stimulated anchorage independent growth of nontransformed rodent fibroblast and human colonic epithelial cells was also recovered following Bio-Gel P-60 chromatography of extracts from several cytology positive human malignant effusions of patients with colonic and breast carcinoma as well as other malignancies. The latter malignant effusion related transforming growth factor was not present in benign or cytology negative effusions. Malignant effusion related TGF factor was inactivated by sulfhydryl reducing agents, heat, and trypsin treatment but was stable in 1% acetic acid and ethanol. Partial purification was accomplished by chromatography of an acid-ethanol extract on Bio-Gel P-60 followed by high performance liquid chromatography with C18-mu Bondapak to yield a nearly pure protein with apparent molecular weights of 64,000 by sodium dodecyl sulfate-polacrylamide gel electrophoresis when run in nonreducing conditions and 32,000 when run in reducing conditions. Malignant effusion related TGF was able to stimulate anchorage independent growth of nontransformed fibroblasts in the absence of other growth factors. It did not competitively inhibit binding of 125I-labeled epidermal growth factor, 125I-TGF beta, or 125I-labeled platelet derived growth factor. Therefore, this factor isolated from human malignant effusions may be distinct from previously described transforming growth factors. Collectively these observations indicate that human malignant effusions contain a multiplicity of transforming growth factors. It is possible that the malignant effusion related transforming growth factors play a role or reflect the metastatic growth properties of various tumors.