Characteristics of Aerococcus viridans isolated from bovine subclinical mastitis and its effect on milk SCC, yield, and composition.

Aerococcus viridians (A. viridans), an environmental Gram-positive bacterium, has been documented to be associated with bovine mastitis. However, its exact role in bovine mastitis and the changes it brings about in milk characteristics are not yet known. The objectives of the current study were to describe the antibiotic resistance of A. viridans from bovine mastitis as well as the correlation between existence of this pathogen in udders and the somatic cell counts (SCC), daily milk yield, and composition of individual cow. One-year sampling for subclinical mastitis composite milk was conducted based on monthly DHI data from September 2013 to August 2014, in a commercial herd located in Beijing, China. All samples were cultured and pathogens were identified using microbiology method. A. viridians isolates were further identified by API identification system and 16S ribosomal RNA (rRNA) sequencing method. Kirby-Bauer disk diffusion method was used to test the antibiotic resistance of A. viridians against kinds of antimicrobial substance. SCC, milk yield, and milk composition data were from monthly Dairy Herd Improvement (DHI) results. Results showed that a total of 279 (16.67%) A. viridans isolates were identified from among 1674 bacterial isolates cultured from milk samples with high SCC. The incidence of mastitis caused by A. viridans was the highest (48-53%) during the summer season. Majority of the isolates were susceptible to most of antimicrobial compounds tested, especially to ß-lactams, but were found to be resistant (50-90%) to aminoglycosides, sulfonamides, and tetracycline. The average SCC of the A. viridans infected cows was significantly higher (1000.0 × 103 cells/mL) (P < 0.01) as compared to healthy cows (72.4 × 103 cells/mL) and daily milk yield decreased (P > 0.05) by 1.86 kg/day. Reductions were also observed in fat content (P > 0.05), lactose (P < 0.01), and total solids (P > 0.05), whereas protein content increased significantly (P < 0.01) in milk samples of cows infected with A. viridans. The results of this study suggest that A. viridans could be considered as an emerging aetiological agent of bovine subclinical mastitis wherein it exerts an effect on SCC, milk yield, and composition.

Engineering of chaperone systems and of the unfolded protein response.

Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR.

Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.

P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca(V)2.1 alpha1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca(V)2 channels and suggest that the ability of individual Ca2+ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.