Analysis of human collagen sequences

The extracellular matrix is fast emerging as important component mediating cell-cell interactions, along with its established role as a scaffold for cell support. Collagen, being the principal component of extracellular matrix, has been implicated in a number of pathological conditions. However, collagens are complex protein structures belonging to a large family consisting of 28 members in humans; hence, there exists a lack of in depth information about their structural features. Annotating and appreciating the functions of these proteins is possible with the help of the numerous biocomputational tools that are currently available. This study reports a comparative analysis and characterization of the alpha-1 chain of human collagen sequences. Physico-chemical, secondary structural, functional and phylogenetic classification was carried out, based on which, collagens 12, 14 and 20, which belong to the FACIT collagen family, have been identified as potential players in diseased conditions, owing to certain atypical properties such as very high aliphatic index, low percentage of glycine and proline residues and their proximity in evolutionary history. These collagen molecules might be important candidates to be investigated further for their role in skeletal disorders

Lactobacillus GG for treatment of acute childhood diarrhoea: An open labelled, randomized controlled trial

Background & objectives : 0 Randomized controlled trials in developed countries have reported benefits of Lactobacillus GG (LGG) in the treatment of acute watery diarrhoea, but there is paucity of such data from India. The study was aimed to evaluate the efficacy and safety of Lactobacillus GG in the treatment of acute diarrhoea in children from a semi-urban city in north India. Methods: In this open labelled, randomized controlled trial 2000 children with acute watery diarrhoea, aged between 6 months to 5 years visiting 0 outpatient department and emergency room of a teaching hospital in north India were enrolled. The children were randomized into receiving either Lactobacillus GG in dose of 10 billion cfu/day for five days or no probiotic medication in addition to standard WHO management of diarrhoea. 0 Primary outcomes were duration of diarrhoea and time to change in consistency of stools. Results : 0 Median (inter quartile range) duration of diarrhoea was significantly shorter in children in LGG group [60 (54-72) h vs. 78 (72-90) h; P<0.001]. Also, there was faster improvement in stool consistency in children receiving Lactobacillus GG than control group [36 (30-36) h vs. 42 (36-48) h; P<0.001]. There was significant reduction in average number of stools per day in LGG group (P<0.001) compared to the control group. These benefits were seen irrespective of rotavirus positivity in stool tests. Interpretation & conclusions : 0 Our results showed that the use of Lactobacillus GG in children with acute diarrhoea resulted in shorter duration and faster improvement in stool consistency as compared to the control group

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

<div><p>Background</p><p>Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart.</p> <p>Objectives</p><p>The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant.</p> <p>Methods</p><p>H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-κB, known to regulate gelatinase B, was also examined.</p> <p>Results</p><p>Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and <i>in-situ</i> gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B.</p> <p>Conclusions</p><p>The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling.</p> </div

Effect of curumin on gelatinolytic activity.

<p><b>A</b>) <b>Gel-diffusion </b><b>assay</b>: Upper gel: Various concentrations of trypsin (1-Blank; 2-5 µg/µl; 3-10 µg/µl; 4-15 µg/µl; 5-20 µg/µl; 6-25 µg/µl; 7-30 µg/µl; 8-35 µg/µl) were added to different wells and protease activity was observed as digested zones around it. A standard curve of the enzyme activity in units as a function of diameter of zone was prepared. The enzyme activity for different samples shown in the lower gel (Control; NE-treated; NE+Curcumin-treated; Curcumin-treated alone) was calculated from the standard graph and represented as a histogram (*P<0.01). The difference of NE-treated was significant to control as well as NE+Curcumin-treated group. <b>B</b>) <b>Gelatin </b><b>Zymography</b>: Samples in different lanes of zymograms starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. The fold change in the activity for bands corresponding to MMP-9 with respect to control was quantified using ImageJ and plotted as histogram (*P<0.01, **P<0.05). <b>C</b>) <b><i>in-situ</i></b><b>Gelatin </b><b>Zymography</b>: The experiment was carried out under different experimental conditions above and images captured by fluoresencent microscope at 20X magnifications are represented.</p

Expression levels of MMP-9 on curcumin treatment.

<p><b>A</b>) <b>RT-PCR </b><b>for </b><b>MMP-9</b>: mRNA expression seen through semiqunatitative RT-PCR Samples in different lanes starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. qPCR results obtained were normalized against beta actin and plotted as a histogram (*P<0.05). <b>B</b>) <b>Western </b><b>Blotting </b><b>for </b><b>MMP-9</b>: Samples in different lanes starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. Protein expression observed through Western blotting was quantitated by NIH ImageJ software. Results obtained were normalized against beta actin and plotted as a histogram (*P<0.01). <b>C</b>) <b>Immunocytochemistry </b><b>for </b><b>MMP-9</b>: Images captured by fluoresencent microscope at 20X magnifications are represented. NE-treated group showed a significant difference compared to control and NE+Curcumin-treated group in all experiments shown in the figure.</p

Nuclear localization of NF-κB.

<p>(<b>A</b>) <b>Immunofluorescence </b><b>for </b><b>NF-κB</b>: Nuclear localization of NF-κB was compared between H9c2 Control cells, NE-treated cells NE+Curcumin-treated and Curcumin-treated alone cells. Blue color represents DAPI staining of nucleus. NF- κB was stained green. (<b>B</b>) <b>Western </b><b>Blotting</b>: Nuclear and cytosolic proteins from different Samples in different lanes for both nuclear and cytosolic extract panels starting from the left represented as Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated alone. Protein expression observed through Western blotting was quantitated by NIH ImageJ software. Results obtained were normalized against beta actin for cytoplasmic extracts and Lamin A/C for nuclear extracts and plotted as a histogram (*P<0.01, **P<0.05). NE-treated group showed a significant difference compared to control and NE+Curcumin-treated group.</p

Proposed Model for the study.

<p>An illustration of the proposed mechanism of prevention of NE-induced cardiotoxicity by curcumin through downregulation of gelatinase B. It shows that curcumin targets different signaling players in the pathway.</p

Curcumin prevents Norepinephrine induced cardiac stress in H9c2 cells.

<p><b>A</b>) <b>Analysis </b><b>of </b><b>FACS </b><b>Forward </b><b>Scatter</b>: Histogram represents comparison of cell size in Control (Uninduced cells), NE-treated (Hypertrophic), NE+Curcumin-treated and CurcuminNE-treated alone experimental groups (*P<0.01). <b>B</b>) <b>Analysis </b><b>of </b><b>protein </b><b>content</b>: The statistical representation of total protein concentration (mg/ml) for all four groups (*P<0.05). <b>C</b>) <b>RT-PCR </b><b>analysis </b><b>for </b><b>ANF</b>: Starting from left, samples in different lanes represent, Lane-1: Control; Lane-2: NE-treated; Lane-3: NE+Curcumin-treated; Lane-4: Curcumin-treated. The bands were quantitated by NIH ImageJ software and fold intensity with respect to control after normalization was plotted as a histogram (*P<0.05). The difference in NE-treated group was statistically significant in comparison to control and NE+Curcumin-treated groups in all experiments shown in the figure.</p