Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.     

Dynamical correlation of non-stationary signals in time domain—A comparative study

In a thorough study, the multitaper (MTM) and the extended continuous wavelet-transform (CWT) coherence-analysis methods were compared in terms of there application in determining the dynamics from the electroencephalogram (EEG) and electromyogram (EMG) signals of patients with Parkinsonian tremor. The main aim of the study in a biological point of view is to analyze whether the basic tremor frequency and its “first harmonic” frequency of Parkinsonian tremor are really harmonically related or are in fact distinct processes.The extension of the CWT is achieved by using a Morlet wavelet as the analysis window with an adjustable relative bandwidth which gives the flexibility in setting a desired frequency resolution. In order to obtain a perspective view of the two methods, they were applied to two different model signals to determine their actual threshold in detecting short-lived changes in the analysis of non-stationary signals and to determine their noise thresholds by adding external noise to the signals to test the reduction in coherence to be not merely due to the random fluctuations in stochastic signals. Beyond applying an autoregressive 2nd-order and a coupled van der Pol model system, however, also true EEG and EMG data from five Parkinson patients were used. The results were compared in terms of the time and frequency resolutions of these two methods, and it was determined that the multitaper method was able to detect reduction in power and coherence as short as 1 s. The extended CWT analysis only revealed gaps that were longer than 3 s.The time gaps in the coherence indicate the loss of connection between the cortex and muscle during the respective time intervals. This more accurate analysis of the MTM was also seen in the dynamical EEG–EMG coherence at the tremor frequency and its “first harmonic” of Parkinsonian patients.In terms of our “biological” aim, this shows distinct prevalence of the corticomuscular coupling at those frequencies over time. Applying this method to biological data reveals important aspects about their dynamics, e.g., the distinct dynamics between basic frequency and “first harmonic” frequency over time in Parkinsonian tremor.     

ZnO nanoparticles augment ALT,AST, ALP and LDH expressions in C2C12 cells

The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions in C2C12 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles (1–5 mg/ml) on C2C12 cell viability at 48 and 72 h. ZnO nanoparticles increased ALT, AST, ALP and LDH enzyme mRNA expression and their activities in C2C12 cells. In conclusion, the present study showed that ZnO nanoparticles increased these enzyme activities and its mRNA expression in C2C12 cells in a dose-dependent manner.     

Co-culture of C2C12 and 3T3-L1 preadipocyte cells alters the gene expression of calpains, caspases and heat shock proteins

The present study was carried out to understand the co-culture effect of C2C12 and 3T3-L1 preadipocyte cells on calpain, caspase, and heat shock protein (Hsp) systems. Calpains, caspases, and heat shock proteins play critical roles in the growth and development of mammalian cells. Cells were co-cultured using transwell inserts with a 0.4-??m porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 transferred to 3T3-L1 plates. Following co-culture for 24 and 48?h, the cells in the lower well were harvested for analysis. Calpains include ??-calpain, m-calpain, and their specific inhibitor calpastatin. The expression pattern of ??-calpain did not change in the co-cultured C2C12 and 3T3-L1 cells, whereas m-capain mRNA expression significantly reduced in the 48-h co-cultured 3T3-L1 cells. Calpastatin mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Caspase-7 mRNA expression did not change in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells. Caspase-3 mRNA expression significantly reduced in the 24- and 48-h co-cultured 3T3-L1 cells; caspase-9 mRNA had a significant reduction only at 48?h, whereas caspase-9 mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Hsp27 and Hsp90 mRNA expressions are significantly reduced in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells, whereas Hsp70 mRNA expression significantly increased in the 48-h co-cultured 3T3-L1 cells. The co-culture reflects three-dimensional views of C2C12 and 3T3-L1 cell types as in vivo, which is quite distinct from the one-dimensional monocultured C2C12 and 3T3-L1 cells.     

Homobrassinolide induced conformational changes in hexokinase: a possible mechanism for its antidiabetic potential

Hormonal regulation of cell growth and development, tissue morphology, metabolism and physiological function in animals and man is a well‐established knowledge domain in modern biological science. The present study was carried out to investigate the structural stability of hexokinase when exposed to diabetic levels of glucose and its binding efficiency. The fluorescence study indicated that 28‐homobrassinolide was able to protect or restore the native structure of hexokinase. Proteins are synthesized and fold into the native form to become active. The inability of a protein molecule to remain in its native form is called as protein misfolding and this is because of several factors. Protein aggregation and misfolding are known to play a critical role in several human diseases including diabetes. Homobrassinolide interaction with hexokinase was studied by UV–Vis spectrophotometer and fluorescence spectrophotometer. Results were suggested that the denatured hexokinase was renatured upon binding with homobrassinolide. In silico, docking study was performed to recognize the binding activity of homobrassinolide against a subunit of the glucokinase, and homobrassinolide was able to bind to the drug binding pocket of glucokinase. The glide energy is ?7.1 kcal/mol, suggesting the high binding affinity of homobrassinolide to glucokinase. Overall, these studies predict that the phytohormone 28‐homobrassinolide would function as an anti‐diabetic when present in human and animal diet by augmenting the hexokinase enzyme activity in the animal cell. Copyright © 2015 John Wiley & Sons, Ltd.     

Genome-scale model-driven strain design for dicarboxylic acid production in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Background

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application.

Results

In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica.

Conclusion

In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.