Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.     

Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.     

Glutathione <Emphasis Type="BoldItalic">S</Emphasis>-transferase P1 gene polymorphisms and susceptibility to coronary artery disease in a subgroup of north Indian population

The present study aimed to investigate the association of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) polymorphisms of GSTP1 with coronary artery disease (CAD) in a subgroup of north Indian population. In the present case–control study, CAD patients (\(n = 200\)) and age-matched, sex-matched and ethnicity-matched healthy controls (\(n = 200\)) were genotyped for polymorphisms in GSTP1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype distribution of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) polymorphisms of GSTP1 gene was significantly different between cases and controls (\(P = 0.005\) and 0.024, respectively). Binary logistic regression analysis showed significant association of A/G (odds ratio (OR): 1.6, 95% CI: 1.08–2.49, \(P = 0.020\)) and G/G (OR: 3.1, 95% CI: 1.41–6.71, P \(=\) 0.005) genotypes of GSTP1 \(\hbox {g}.313\hbox {A}{\!>\!}\hbox {G}\), and C/T (OR: 5.8, 95% CI: 1.26–26.34, \(P = 0.024\)) genotype of GSTP1 \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) with CAD. The A/G and G/G genotypes of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and C/T genotype of \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) conferred 6.5-fold increased risk for CAD (OR: 6.5, 95% CI: 1.37–31.27, \(P = 0.018\)). Moreover, the recessive model of GSTP1 \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) is the best fit inheritance model to predict the susceptible gene effect (OR: 2.3, 95% CI: 1.11–4.92, \(P = 0.020\)). In conclusion, statistically significant associations of GSTP1 \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) (A/G, G/G) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) (C/T) genotypes with CAD were observed.     

Heterogeneous nanomechanical properties of type I collagen in longitudinal direction

Biomechanics and Modeling in Mechanobiology - Collagen is an abundant structural biopolymer in mammal vertebrates, providing structural support as well as mechanical integrity for connective...     

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

We present results of Raman spectroscopic studies carried out on optically trapped red blood cells with Raman excitation wavelength in Q‐band region of the hemoglobin (Hb) absorption spectrum. The results obtained suggest that when exposed to the Raman excitation laser the RBCs get deoxygenated due to photo‐dissociation of oxygen from hemoglobin. For smaller exposure durations (5 s) the level of deoxygenation increases with an increase in power. However, for longer exposure durations the deoxygenated hemoglobin in the cells gets irreversibly oxidized to form a low spin ferric derivative of hemoglobin. The rate of oxidation depends upon the initial level of deoxygenation; higher the initial level of deoxygenation, higher is the rate of oxidation. However, the RBCs deoxygenated via oxygen deprivation (i.e. N₂ purging) were found to be very stable against any laser induced effect. These observations suggests that in case of laser induced deoxygenation of RBCs the free oxygen generated by photo‐dissociation acts as the oxidizing agent and leads to oxidative damage of the RBCs.

     

A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.     

In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.     

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO₂ group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.