Tumour suppressor protein sMEK1 links to IRE1 signalling pathway to modulate its activity during ER stress.

The Endoplasmic Reticulum is a pervasive, dynamic cellular organelle that performs a wide range of functions in the eukaryotic cell, including protein folding and maturation. Upon stress, ER activates an adaptive cellular pathway, namely Unfolded Protein Response, that transduces information from ER to nucleus, restoring homeostasis in the ER milieu. UPR consists of three membrane-tethered sensors; IRE1, PERK and ATF6. Among all the UPR sensors, the IRE1 branch acts as a central pathway that orchestrates several pathways to determine cell fate. However, the detailed knowledge underlying the whole process is not understood yet. Previously, we determined the sMEK1 as one of the interacting partners of IRE1. sMEK1 is a protein phosphatase, which has been indicated in a number of critical cellular functions like apoptosis, cell proliferation, and tumour suppression. In this study, we evaluated the role of sMEK1 on the IRE1 signalling pathway. Our data indicate that sMEK1 can inhibit IRE1 phosphorylation under ER stress. This inhibitory effect of sMEK1 could be reflected in its downstream effectors, Xbp1 and RIDD, which are downregulated in the presence of sMEK1. We also found that the repressing effect of sMEK1 was specific to the IRE1 signalling pathway and could be preserved even under prolonged ER stress. Our findings also indicate that sMEK1 can inhibit IRE1 and its downstream molecules under ER stress irrespective of other UPR sensors. These results help to draw the mechanistic details giving insights into different molecular connections of UPR with other pathways.

Unravelling effects of phytochemicals from buckwheat on cholesterol metabolism and lipid accumulation in HepG2 cells and its validation through gene expression analysis.

BACKGROUND: The objective of this research was to elucidate the hypocholesterolemic effects of a bioactive compound extracted from buckwheat, and to delineate its influence on the regulatory mechanisms of cholesterol metabolism. The compound under investigation was identified as quercetin. MATERIAL AND RESULTS: In vitro experiments conducted on HepG2 cells treated with quercetin revealed a significant reduction in intracellular cholesterol accumulation. This phenomenon was rigorously quantified by assessing the transcriptional activity of key genes involved in the biosynthesis and metabolism of cholesterol. A statistically significant reduction in the expression of HMG-CoA reductase (HMGCR) was observed, indicating a decrease in endogenous cholesterol synthesis. Conversely, an upregulation in the expression of cholesterol 7 alpha-hydroxylase (CYP7A1) was also observed, suggesting an enhanced catabolism of cholesterol to bile acids. Furthermore, the study explored the combinatory effects of quercetin and simvastatin, a clinically utilized statin, revealing a synergistic action in modulating cholesterol levels at various dosages. CONCLUSIONS: The findings from this research provide a comprehensive insight into the mechanistic pathways through which quercetin, a phytochemical derived from buckwheat, exerts its hypocholesterolemic effects. Additionally, the observed synergistic interaction between quercetin and simvastatin opens up new avenues for the development of combined therapeutic strategies to manage hyperlipidemia.

Elucidating the EXOSC3-IRE1α interaction: a convergent study incorporating computational, in vitro and in vivo studies.

The Unfolded protein response (UPR) is an adaptive signalling pathway which is triggered by accumulation of unfolded/misfolded protein in ER lumen. The UPR consist of three transmembrane proteins-IRE1α, PERK and ATF6 that sense ER stress which leads to activation and downstream signaling from ER lumen to cytosol to restore homeostasis. IRE1α is an evolutionary conserved arm of UPR and acts as an interaction platform for many potential proteins that become activated under ER stress conditions. We investigated potential partners of IRE1 α through MS studies and found EXOSC3 as one of the binding partner of IRE1α. Exosomal complex proteins have 3'5' exonuclease properties (EXOSC3) that play an important role in mRNA surveillance. This property of exosomal proteins coincides with IRE1α ribonuclease activities and its mechanism of action is similar to that of IRE1α-RIDD pathway which degrades any unstable mRNA that disrupts cellular homeostasis. At the same time, studies have shown that knockdown of EXOSC3 causes ER stress in human cells, so we speculated that there might be a functional crosstalk between IRE1α and EXOSC3 under ER stress conditions. Therefore, we employed computational tools to predict and explore the stability and dynamics of the IRE1α-EXOSC3 complex. The analysis indicates that IRE1α and EXOSC3 exhibit potential interaction with the involvement of ScanNet, predicting binding pockets between the two proteins. Further, the interaction was validated via co-immunoprecipitation and yeast two-hybrid assays, thus suggesting EXOSC3 as a component of the UPRosome complex. Hence, this functional crosstalk might influence the dynamic functional output of IRE1α.Communicated by Ramaswamy H. Sarma.

Organic-inorganic composite of polypropylene fumarate and nanohydroxyapatite as carrier of antibiotics for the treatment of bone infections.

Current treatment approaches in clinics to treat the infectious lesions have partial success thus demanding the need for development of advanced treatment modalities. In this study we fabricated an organic-inorganic composite of polypropylene fumarate (PPF) and nanohydroxyapatite (nHAP) by photo-crosslinking as a carrier of two clinically used antibiotics, ciprofloxacin (CIP) and rifampicin (RFP) for the treatment of bone infections. Carboxy terminal-PPF was first synthesized by cis-trans isomerization of maleic anhydride which was then photo-crosslinked using diethylfumarate (DEF) as crosslinker and bis-acylphosphine oxide (BAPO) as photo-initiator under UV lights (P). A composite of PPF and nHAP was fabricated by incorporating 40 % of nHAP in the polymeric matrix of PPF (PH) which was then characterized for different physicochemical parameters. CIP was added along with nHAP to fabricated CIPloaded composite scaffolds (PHC) which was then coated with RFP to synthesize RFP coated CIP-loaded scaffolds (PHCR). It was observed that there was a temporal separation in the in vitro release of two antibiotics after coating PHC with RFP with 80.48 ± 0.40 % release of CIP from PHC and 62.43 ± 0.21 % release of CIP from PHCR for a period of 60 days. Moreover, in vitro protein adsorption was also found to be maximum in PHCR (154.95 ± 0.07 µg/mL) as observed in PHC (75.42 ± 0.06 µg/mL), PH (24.47 ± 0.08 µg/mL) and P alone (4.47 ± 0.02 µg/mL). The scaffolds were also evaluated using in vivo infection model to assess their capacity in reducing the bacterial burden at the infection site. The outcome of this study suggests that RFP coated CIP-loaded PPF composite scaffolds could reduce bacterial burden and simultaneously augment bone healing during infection related fractures.

Quaternary structure analysis of IRE1.

IRE1 belongs to a type I transmembrane protein family harboring two functional domains, cytoplasmic domain with kinase and RNAse catalytic activity, and the luminal domain, which is involved in the sensing of unfolded proteins. IRE1 molecule undergoes dimerization in the lumenal domain, which functionally activates the catalytic C-terminal domain. IRE1 activation is directly related to transition between monomeric and dimeric forms. We have deduced two quaternary structures from the published crystal structure of IRE1. One structure with a large stable interface that requires large activation and deactivation energy to active IRE1. The other quaternary structure has low dissociation energy and is more suitable for IRE1 oligomeric transition.

The Bcl-2 family protein bid interacts with the ER stress sensor IRE1 to differentially modulate its RNase activity.

IRE1 is a transmembrane signalling protein that activates the unfolded protein response under endoplasmic reticulum stress. IRE1 is endowed with kinase and endoribonuclease activities. The ribonuclease activity of IRE1 can switch substrate specificities to carry out atypical splicing of Xbp1 mRNA or trigger the degradation of specific mRNAs. The mechanisms regulating the distinct ribonuclease activities of IRE1 have yet to be fully understood. Here, we report the Bcl-2 family protein Bid as a novel recruit of the IRE1 complex, which directly interacts with the cytoplasmic domain of IRE1. Bid binding to IRE1 leads to a decrease in IRE1 phosphorylation in a way that it can only perform Xbp1 splicing while mRNA degradation activity is repressed. The RNase outputs of IRE1 have been found to regulate the homeostatic-apoptotic switch. This study, thus, provides insight into IRE1-mediated cell survival.

Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11.

Mucor circinelloides serves as a model organism to investigate the lipid metabolism in oleaginous microorganisms. It is considered as an important producer of γ-linolenic acid (GLA) that has vital medicinal benefits. In this study, we used WJ11, a high lipid-producing strain of M. circinelloides (36% w/w lipid, cell dry weight, CDW), to examine the role in lipid accumulation of two mitochondrial malic enzyme (ME) genes malC and malD. The homologous overexpression of both malC and malD genes enhanced the total lipid content of WJ11 by 41.16 and 32.34%, respectively. In parallel, the total content of GLA was enhanced by 16.73 and 46.76% in malC and malD overexpressing strains, respectively, because of the elevation of total lipid content. The fact that GLA content was enhanced more in the strain with lower lipid content increase and vice versa, indicated that engineering of mitochondrial MEs altered the fatty acid profile. Our results reveal that mitochondrial ME plays an important role in lipid metabolism and suggest that future approaches may involve simultaneous overexpression of distinct ME genes to boost lipid accumulation even further.

Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes.

BACKGROUND: Organic cation transporter 1 primarily governs the action of metformin in the liver. There are considerable inter-individual variations in metformin response. In light of this, it is crucial to obtain a greater understanding of the influence of OCT1 expression or polymorphism in the context of variable responses elicited by metformin treatment. RESULTS: We observed that the variable response to metformin in the responders and non-responders is independent of isoform variation and mRNA expression of OCT-1. We also observed an insignificant difference in the serum metformin levels of the patient groups. Further, molecular docking provided us with an insight into the hotspot regions of OCT-1 for metformin binding. Genotyping of these regions revealed SNPs 156T>C and 1222A>G in both the groups, while as 181C>T and 1201G>A were found only in non-responders. The 181T>C and 1222A>G changes were further found to alter OCT-1 structure in silico and affect metformin transport in vitro which was illustrated by their effect on the activation of AMPK, the marker for metformin activity. CONCLUSION: Taken together, our results corroborate the role of OCT-1 in the transport of metformin and also point at OCT1 genetic variations possibly affecting the transport of metformin into the cells and hence its subsequent action in responders and non-responders.

Proteolysis dependent cell cycle regulation in Caulobacter crescentus.

Caulobacter crescentus, a Gram-negative alpha-proteobacterium, has surfaced as a powerful model system for unraveling molecular networks that control the bacterial cell cycle. A straightforward synchronization protocol and existence of many well-defined developmental markers has allowed the identification of various molecular circuits that control the underlying differentiation processes executed at the level of transcription, translation, protein localization and dynamic proteolysis. The oligomeric AAA+ protease ClpXP is a well-characterized example of an enzyme that exerts post-translational control over a number of pathways. Also, the proteolytic pathways of its candidate proteins are reported to play significant roles in regulating cell cycle and protein quality control. A detailed evaluation of the impact of its proteolysis on various regulatory networks of the cell has uncovered various significant cellular roles of this protease in C. crescentus. A deeper insight into the effects of regulatory proteolysis with emphasis on cell cycle progression could shed light on how cells respond to environmental cues and implement developmental switches. Perturbation of this network of molecular machines is also associated with diseases such as bacterial infections. Thus, research holds immense implications in clinical translation and health, representing a promising area for clinical advances in the diagnosis, therapeutics and prognosis.

Clinical Manifestations of Hyperandrogenism and Ovulatory Dysfunction Are Not Associated with His1058 C/T SNP (rs1799817) Polymorphism of Insulin Receptor Gene Tyrosine Kinase Domain in Kashmiri Women with PCOS.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrine metabolic disorder affecting premenopausal women. Besides primary features like anovulation, hyperandrogenism, and polycystic ovaries, women with PCOS present with multiple metabolic, cardiovascular, and psychological disorders. The etiology is multifactorial and the different genetic variants are suggested to play an important role in pathogenesis. Insulin resistance is a ubiquitous finding in PCOS and SNPs in genes involved in the insulin signaling pathway are possible candidates that can explain the development of clinical manifestations of PCOS. AIM: We aimed to investigate the association of INSR His1058 C/T (rs1799817) single nucleotide polymorphism with PCOS in Kashmiri women. The genotypic-phenotypic correlation of the tested SNP with hyperandrogenism, ovulatory dysfunction, and metabolic markers was evaluated. RESULTS: The allele frequency (OR = 1.00, 95% CI = 0.67-1.48, χ 2 = 0.01, P=0.99) and genotype distribution (χ 2 = 3.73, P=0.15) in INSR C/T polymorphism were comparable with controls. No significant association was found with PCOS in dominant (P=0.194), recessive (P=0.442), and homo vs. het. (P=0.5) genotype models. Genotype-phenotype correlation analysis revealed that variant TT genotype had significantly higher HOMA (P=0.029) and reduced insulin sensitivity QUICKI (P=0.037) values. There was no significant variation in the prevalence of hirsutism, acne, alopecia, menstrual disturbances, acanthosis nigricans, and obesity (all P > 0.05) in different INSR C/T genotypes. CONCLUSION: The INSR C/T SNP (rs1799817) does not increase the risk of PCOS in Kashmiri women. This SNP is unlikely to play a significant role in the development and manifestation of clinical symptoms of polycystic ovary syndrome.

The molecular mechanism and functional diversity of UPR signaling sensor IRE1.

The endoplasmic reticulum is primarily responsible for protein folding and maturation. However, the organelle is subject to varied stress conditions from time to time, which lead to the activation of a signaling program known as the Unfolded Protein Response (UPR) pathway. This pathway, upon sensing any disturbance in the protein-folding milieu sends signals to the nucleus and cytoplasm in order to restore homeostasis. One of the prime UPR signaling sensors is Inositol-requiring enzyme 1 (IRE1); an ER membrane embedded protein with dual enzyme activities, kinase and endoribonuclease. The ribonuclease activity of IRE1 results in Xbp1 splicing in mammals or Hac1 splicing in yeast. However, IRE1 can switch its substrate specificity to the mRNAs that are co-transnationally transported to the ER, a phenomenon known as Regulated IRE1 Dependent Decay (RIDD). IRE1 is also reported to act as a principal molecule that coordinates with other proteins and signaling pathways, which in turn might be responsible for its regulation. The current review highlights studies on IRE1 explaining the structural features and molecular mechanism behind its ribonuclease outputs. The emphasis is also laid on the molecular effectors, which directly or indirectly interact with IRE1 to either modulate its function or connect it to other pathways. This is important in understanding the functional pleiotropy of IRE1, by which it can switch its activity from pro-survival to pro-apoptotic, thus determining the fate of cells.

Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner.

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A.IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.

Aggregation of M3 (E376D) variant of alpha1- antitrypsin.

Alpha1-antitrypsin (α1AT) is an abundant serine-protease inhibitor in circulation. It has an important role in neutralizing the neutrophil elastase activity. Different pathogenic point mutations like Z(E342K)-α1AT have been implicated in the development of liver cirrhosis and Chronic Obstructive Pulmonary Disease (COPD), the latter being a cluster of progressive lung diseases including chronic bronchitis and emphysema. M3-α1AT (376Glu > Asp) is another variant of α1AT which so far is largely being considered as normal though increased frequency of the variant has been reported in many human diseases including COPD. We also observed increased frequency of M3-α1AT in COPD cases in Kashmiri population. The frequency of heterozygous (AC) genotype in cases and controls was 58.57% and 27.61% (odds-ratio 6.53 (2.27-15.21); p < 0.0001) respectively, while homozygous CC genotype was found to be 21.42% and 6.66% (odds-ratio 10.56 (3.63-18.64); p < 0.0001) respectively. Comparative in vitro investigations that include trypsin‒antitrypsin assay, Circular Dichroism spectroscopy and dynamic light scattering performed on wild-type (M-α1AT), M3-α1AT, and Z-α1AT proteins along with the molecular dynamics simulations revealed that M3-α1AT has properties similar to Z-α1AT capable of forming aggregates of varied size. Our maiden observations suggest that M3-α1AT may contribute to the pathogenesis of COPD and other disorders by mechanisms that warrant further investigations.

AKT Regulates Mitotic Progression of Mammalian Cells by Phosphorylating MASTL, Leading to Protein Phosphatase 2A Inactivation.

Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl) kinase, has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A (PP2A), it plays a crucial role in activating one of the most important mitotic kinases, known as cyclin-dependent kinase 1 (CDK1). MASTL has been seen to be upregulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop, but the complete mechanism of its activation is still unclear. Here, we report that AKT phosphorylates MASTL at residue T299, which plays a critical role in its activation. Our results suggest that AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which, in turn, promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation, since AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT plays an important role in the progression of mitosis in mammalian cells and that it does so through the phosphorylation and activation of MASTL.

Elevated fasting insulin is associated with cardiovascular and metabolic risk in women with polycystic ovary syndrome.

AIMS: PCOS is associated with various immediate and long term health complications. The aim of this study was to investigate the association of serum fasting insulin concentration with cardiovascular and metabolic risk factors in women with polycystic ovary syndrome. METHODS: A total of 349 women, 249 women with polycystic ovary syndrome and 100 age-matched healthy controls, were recruited in this case-control study. Fasting insulin and various other biochemical, hormonal and clinical parameters were measured in all participants. The correlation of insulin with cardiometabolic risk factors was evaluated in PCOS women with normal and high serum insulin concentration. RESULTS: Fasting Insulin, BMI, WHR, FAI, LH: FSH, HOMA, QUICKI were significantly higher in PCOS women compared with healthy controls (p < 0.01). Fasting insulin showed a positive correlation with more cardiovascular and metabolic risk factors in PCOS compared to controls. The BMI, BAI, LAP, HOMA IR, QUICKI and FAI were significantly higher (all p < 0.05) in PCOS patients with higher insulin levels than with PCOS women with normal levels. CONCLUSION: Fasting insulin is an important determinant in the pathogenesis of obesity and hyperandrogenism in PCOS. It is associated with an increased risk of cardiovascular and metabolic disorders in women with PCOS.

A network map of netrin receptor UNC5B-mediated signaling.

UNC-5 Homolog B (UNC5B) is a member of the dependence receptor family. This family of receptors can induce two opposite intracellular signaling cascades depending on the presence or absence of the ligand and is thus capable of driving two opposing processes. UNC5B signaling has been implicated in several cancers, where it induces cell death in the absence of its ligand Netrin-1 and promotes cell survival in its presence. In addition, inhibition of Netrin-1 ligand has been reported to decrease invasiveness and angiogenesis in tumors. UNC5B signaling pathway has also been reported to be involved in several processes such as neural development, developmental angiogenesis and inflammatory processes. However, literature pertaining to UNC5B signaling is scarce and scattered. Considering the importance of UNC5B signaling, we developed a resource of signaling events mediated by UNC5B. Using data mined from published literature, we compiled an integrated pathway map consisting of 88 UNC5B-mediated signaling events and 55 proteins. These signaling events include 27 protein-protein interaction events, 33 catalytic events involving various post-translational modifications, 9 events of UNC5B-mediated protein activation/inhibition, 27 gene regulation events and 2 events of translocation. This pathway resource has been made available to the research community through NetPath ( http://www.netpath.org /), a manually curated resource of signaling pathways (Database URL: http://www.netpath.org/pathways?path_id=NetPath_172 ). The current resource provides a foundation for the understanding of UNC5B-mediated cellular responses. The development of resource will serve researchers to explore the mechanisms of UNC-5B signaling in cancers.

SERPINA1 Hepatocyte-Specific Promoter Polymorphism Associate with Chronic Obstructive Pulmonary Disease in a Study of Kashmiri Ancestry Individuals.

PURPOSE: Different mutations in coding and non-coding sequences of the SERPINA1 gene have been implicated in the pathogenesis of COPD. However, - 10T/C mutation in the hepatocyte-directed promoter region has not been associated with COPD pathogenesis so far. Here, we report an increased frequency of - 10C genotype that is associated with decreased levels of serum alpha1-antitrypsin (α1AT) in COPD patients. METHODS: The quantification of serum α1AT was done by ELISA, the phenol-chloroform method was used for DNA extraction, PCR products were directly sequenced. The IBM SPSS Statistics v21 software was used for statistical analyses of the data. RESULTS: The mean serum α1AT level was found to be 1.203+0.239 and 3.162+0.160 g/L in COPD cases and in control, respectively. The - 10C allele is associated with an increased risk of COPD [OR, 3.50 (95%CI, 1.86-6.58); p < 0.001]. The combined variant genotype (TT+CC) was significantly found associated with an increased risk of COPD [OR, 3.20 (95% CI, 1.47-6.96); p = 0.003]. A significant association of the family history with COPD (overall p value= 0.0331) suggests that genetics may play an important role in the pathogenesis of COPD. CONCLUSION: The polymorphism associated with hepatocyte-specific promoter region (- 10T/C) is likely to be associated with the pathogenesis of COPD. It is quite possible that the change of the base in the hepatocyte-specific promoter of the SERPINA1 gene can modulate its strength, thereby driving the reduced expression of α1AT.

Bioefficacy potential of different genotypes of walnut Juglans regia L.

The purpose of investigation was to assess the phytochemical and nutraceutical of walnut in leaf extracts through diverse quantitative and qualitative phytochemical tests followed by array of assays. The screening of 50 elite walnut genotypes which exhibited wide range of discrepancy in terms of phytochemicals as well as their anti-oxidant potential was done. Walnut genotypes displayed maximum divergence in quercetin content (2.86-5.78 mg/100 g) as represented by cluster analysis. The phenolic rich genotypes exhibiting total phenols (37.61-46.47 mg/g GAE) having higher DPPH potential (IP of 32.82-73.50) where as genotypes that accumulate flavonoids/flavanols (5.52-28.48 mg/g QE and 4.11-21.76 mg/g QE showed immense FRAP activity (418.92-1067.94 µM Fe2+/g FW). There was positive correlation between the phenolics content and anti-oxidant potential. The results showed oil content of 50.1-85.08% and kernel percentage 25.21-81.92% of all walnut genotypes. To evaluate the anti-proliferative potential of walnut genotypes, Trypan blue exclusion test, MTT assay and Griess assay was used. Each assay was repeated with different positive controls against a panel of human cancer cell lines viz THP-1, U2OS, IMR-32 and HBL-100 and then compared with the walnut extracts for their efficiency in anti-proliferative activity. The SPS 1 walnut extract at concentration of 500 µg/ml exhibited 10% cell viability and with 1000 µg/ml walnut extract there was consequent decline towards (6.25%) viability. The results indicated that walnut leaf constitutes an excellent source of effective natural antioxidants and chemo-preventive agents that can act as anti cancer agents.

Polyoma small T upregulates the expression of cytoskeletal proteins in mammalian cells during mitosis.

Mammalian cells expressing murine polyoma small T antigen are known to undergo prolonged mitotic arrest followed by extensive cell death. However, the detailed mechanism of this process is not fully understood. While studying the mechanism related to small T induced mitotic arrest in mammalian cells, we observed that the expression of various cytoskeletal proteins was unusually altered in polyoma small T expressing cell line. Since most of the cytoskeletal proteins are reoriented during mitosis and are involved in spindle formation, so it was pertinent to investigate the expression of these genes in PyST expressing cell line. In this study, we evaluated the expression of tubulin, vinculin and actin. We report that polyoma small T antigen leads to upregulation of tubulin and vinculin in a time dependent manner with tubulin expression being most significantly affected. Intriguingly, we demonstrate that dividing cells normally change the expression of these proteins during mitotic progression. The alteration in cytoskeletal elements specifically occurs during mitosis as cells arrested in replicative phase did not show any change. Together these results reveal that the protein levels of tubulin and vinculin do not remain constant throughout cell cycle but change during mitosis and in polyoma small T expressing cells.

Alpha-1-antitrypsin deficiency: Genetic variations, clinical manifestations and therapeutic interventions.

Alpha-1-antitrypsin (AAT) is an acute phase secretory glycoprotein that inhibits neutrophil proteases like elastase and is considered as the archetype of a family of structurally related serine-protease inhibitors termed serpins. Serum AAT predominantly originates from liver and increases three to five fold during host response to tissue injury and inflammation. The AAT deficiency is unique among the protein-misfolding diseases in that it causes target organ injury by both loss-of-function and gain-of-toxic function mechanisms. Lack of its antiprotease activity is associated with premature development of pulmonary emphysema and loss-of-function due to accumulation of resultant aggregates in chronic obstructive pulmonary disease (COPD). This' in turn' markedly reduces the amount of AAT that is available to protect lungs against proteolytic attack by the enzyme neutrophil elastase. The coalescence of AAT deficiency, its reduced efficacy, and cigarette smoking or poor ventilation conditions have devastating effect on lung function. On the other hand, the accumulation of retained mutant proteins in the endoplasmic reticulum of hepatocytes in a polymerized form rather than secreted into the blood in its monomeric form is associated with chronic liver disease and predisposition to hepatocellular carcinoma (HCC) by gain- of- toxic function. Liver injury resulting from this gain-of-toxic function mechanism in which mutant AAT retained in the ER initiates a series of pathologic events, eventually culminating at liver cirrhosis and HCC. Here in this review, we underline the structural, genetic, polymorphic, biochemical and pathological advances made in the field of AAT deficiency and further comprehensively emphasize on the therapeutic interventions available for the patient.