Identification of tetracycline- and erythromycin-resistant Gram-positive cocci within the fermenting microflora of an Italian dairy food product.

AIMS: Microbiological and molecular analysis of antibiotic resistance in Gram-positive cocci derived from the Italian PDO (Protected Designation of Origin) dairy food product Mozzarella di Bufala Campana. METHODS AND RESULTS: One hundred and seven coccal colonies were assigned to Enterococcus faecalis, Lactococcus lactis and Streptococcus bovis genera by ARDRA analysis (amplified ribosomal DNA restriction analysis). Among them, 16 Ent. faecalis, 26 L. lactis and 39 Strep. bovis displayed high minimum inhibitory concentration (MIC) values for tetracycline, while 17 L. lactis showed high MIC values for both tetracycline and erythromycin. Strain typing and molecular analysis of the phenotypically resistant isolates demonstrated the presence of the tet(M) gene in the tetracycline-resistant strains and of tet(S) and erm(B) in the double-resistant strains. Southern blot analysis revealed plasmid localization of L. lactis tet(M), as well as of the erm(B) and tet(S) genes. Genetic linkage of erm(B) and tet(S) was also demonstrated by PCR amplification. Conjugation experiments demonstrated horizontal transfer to Ent. faecalis strain JH2-2 only for the plasmid-borne L. lactis tet(M) gene. CONCLUSIONS: We characterized tetracycline-and erythromycin-resistance genes in coccal species, representing the fermenting microflora of a typical Italian dairy product. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are of particular relevance from the food safety viewpoint, especially in the light of the potential risk of horizontal transfer of antibiotic-resistance genes among foodborne commensal bacteria.

Diabetes-linked zinc transporter ZnT8 is a homodimeric protein expressed by distinct rodent endocrine cell types in the pancreas and other glands.

BACKGROUND AND AIMS: Zinc is abundant in pancreas, being required by endocrine islet cells for hormone secretion and by exocrine acinar cells as pancreatic juice component. ZnT8 is a member of the SLC30A family of zinc transporters whose overexpression in cultured pancreatic beta cells leads to increased insulin secretion in response to glucose, suggesting a possible role in regulating glycemia. ZnT8 was therefore proposed as a therapeutic target for diabetes, and recent genome-wide association studies identified polymorphisms in the ZNT8 gene conferring increased type 2 diabetes risk. METHODS AND RESULTS: As limited information was available on the biochemical properties of ZnT8 and on its endogenous expression, we have raised a specific polyclonal antibody and immunostained protein extracts, cell lines and tissue sections. We show that ZnT8 forms a very stable dimer that requires biological membranes to properly assemble. We demonstrate localization of murine ZnT8 to the secretory granules in pancreatic beta and alpha islet cells. Moreover, we show that ZnT8 is also expressed in other secretory cell types, namely the cubical epithelium that lines thyroid follicles and the cortex of the adrenal gland, suggesting a more widespread role in endocrine secretion. CONCLUSION: We provide novel insights into the features of the ZnT8 transporter, of special relevance in light of its proposed role as therapeutical target for diabetes treatment.

Immune response in relation to zinc status, sex and antioxidant defence in Italian elderly population: the ZENITH study.

OBJECTIVE: Evaluation of some immune markers in Italian elderly population in relation to zinc status, gender and antioxidant defence. DESIGN: Observational study. SETTING: Italian population. SUBJECTS: Apparently healthy, free-living subjects, 56 men and 52 women, aged 70-85 y, enrolled in Italy. METHODS: Lymphocytes were unstimulated or stimulated with the mitogen phytohemoagglutinin (PHA). The proliferative capacity was measured as incorporation of [3H]-thymidine and reported as stimulation index (SI). Cytokine secretion by lymphocytes was determined by ELISA. The antioxidant enzyme activities were measured using commercial kits. RESULTS: Dietary zinc intake, as well as zinc in serum, red blood cells and urine were on the normal range of values and did not show any difference between men and women. The proliferative response showed a high variability without significant differences between men and women. The amount of secreted pro- and anti-inflammatory cytokines was similar in men and women. No differences were found in the activity of antioxidant enzymes in lymphocytes, namely superoxide dismutase, glutathione peroxidase and catalase, between men and women. An association between SI and serum zinc level in men was found. SI resulted negatively correlated with interleukin (IL)-1beta (R2 = 0.036 and P = 0.012) and IL-10 (R2 = 0.34 and P = 0.040) only in men. IL-10 of PHA-stimulated lymphocytes was negatively correlated with red blood zinc in men (R2 = 0.41 and P = 0.008), while IL-10 of unstimulated and PHA-stimulated lymphocytes were negatively correlated with serum zinc in women (R2 = 0.38 and P = 0.020; R2 = 0.31 and P = 0.040, respectively). No correlation was observed between immune markers and antioxidant enzyme activities. CONCLUSIONS: Only weak differences on immune response between men and women were observed. However, zinc status appears to have more influence on the ability of lymphocytes to proliferate in men than in women.

Isolation, expression and characterization of carp retinol-binding protein.

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.

Identification and sequencing of beta-myrcene catabolism genes from Pseudomonas sp. strain M1.

The M1 strain, able to grow on beta-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One beta-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique beta-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on beta-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for beta-myrcene catabolism in Pseudomonas sp. strain M1.