A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases. 相似文献
Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.
Insulin resistance (IR) triggers endothelial dysfunction, which contributes to erectile dysfunction (ED) and cardiovascular disease.
Aim
To evaluate whether IR was related to ED in young adult patients.
Methods
A total of 283 consecutive men complaining of ED at least six months were enrolled, with a full medical history, physical examination, and laboratory tests collected. Quantitative Insulin Sensitivity Check Index (QUICKI) was used to determine IR. The severity of ED was assessed by IIEF-5 questionnaire. Endothelial function was assessed by ultrasonographic examination of brachial artery flow mediated dilation (FMD).
Results
IR was detected in 52% patients. Subjects with IR had significant higher total cholesterol, triglycerides, low density lipoprotein-cholesterol (LDL-c), glycated haemoglobin (HBA1c), high sensitivity C-reactive protein (hs-CRP) and body mass index (BMI), but showed significant lower IIEF-5 score, FMD%, high density lipoprotein -cholesterol (HDL-c), testosterone, sex hormone binding globulin (SHBG) levels than patients without IR. Multiple regression analysis showed QUICKI and testosterone were independent predictors of IIEF-5 score. Furthermore, the incidence of IR was correlated with the severity of ED.
Conclusions
Compared with other CVFs, IR was found as the most prevalent in our subjects. Besides, IR was independently associated with ED and its severity, suggesting an adverse effect of insulin resistance on erectile function. 相似文献
Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI+] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI+]. 相似文献