Knockdown of Cytosolic Glutaredoxin 1 Leads to Loss of Mitochondrial Membrane Potential: Implication in Neurodegenerative Diseases

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, α-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of β-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP

Prospective cohort study of body mass index and the risk of hospitalisation: findings from 246361 participants in the 45 and Up Study

Objective To quantify the risk of hospital admission in relation to fine increments in body mass index (BMI). Design, setting and participants Population-based prospective cohort study of 246 361 individuals aged ≥45 years, from New South Wales, Australia, recruited from 2006-2009. Self-reported data on BMI and potential confounding/mediating factors were linked to hospital admission and death data. Main outcomes Cox-models were used to estimate the relative risk (RR) of incident all-cause and diagnosis-specific hospital admission (excluding same day) in relation to BMI. Results There were 61 583 incident hospitalisations over 479 769 person-years (py) of observation. In men, hospitalisation rates were lowest for BMI 20–-2 (age-standardised rate:120/1000 py) and in women for BMI 18.5– -2 (102/1000 py); above these levels, rates increased steadily with increasing BMI; rates were 203 and 183/1000 py, for men and women with BMI 35-50 kg m -2, respectively. This pattern was observed regardless of baseline health status, smoking status and physical activity levels. After adjustment, the RRs (95% confidence interval) per 1 kg m -2 increase in BMI from ≥20 kg m -2 were 1.04(1.03-1.04) for men and 1.04(1.04-1.05) for women aged 45-64; corresponding RRs for ages 65-79 were 1.03(1.02-1.03) and 1.03(1.03-1.04); and for ages ≥80 years, 1.01(1.00-1.01) and 1.01(1.01-1.02). Hospitalisation risks were elevated for a large range of diagnoses, including a number of circulatory, digestive, musculoskeletal and respiratory diseases, while being protective for just two-fracture and hernia. Conclusions Above normal BMI, the RR of hospitalisation increases with even small increases in BMI, less so in the elderly. Even a small downward shift in BMI, among those who are overweight not just those who are obese, could result in a substantial reduction in the risk of hospitalisation

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação para o Desenvolvimento Tecnológico da Engenharia (FDTE)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, BrasilUniversidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, BrasilFAPESP: 2010/52521-6CNPq: 483009/2010-