Anticancer and α-chymotrypsin inhibiting diterpenes and triterpenes from Salvia leriifolia

Salvialeriol (1), a new abietane-type diterpene, was isolated from Salvia leriifolia Benth. (Salvia leriaefolia), along with two known abietane-type diterpenoids, 6-hydroxysalvinolone (2) and deacetylnemorone (3), and two known triterpenes, 2-acetoxylupeol (4), and lupine-2,3-diol (5). Compounds 2–5 are reported here for the first time from this species. Compound 4 was previously reported as a synthetic derivative of 5 and this is the first report of its isolation from a natural source. Compounds 2, 3 and 5 exhibited a potent antiproliferative activity against the prostate cancer cell lines (PC3) with IC₅₀ of 3.9 ± 0.1, 6.2 ± 0.1 and 2.8 ± 0.1 μM, respectively, and cervical cancer cell lines (HeLa) with IC₅₀ of 8.0 ± 0.3, 2.6 ± 0.1 and 2.7 ± 0.1 μM, respectively. Whereas compounds 1 and 4 showed moderate antiproliferative activities against the cell lines. Compounds 1–5 were also evaluated for the inhibition of α-chymotrypsin, a protease enzyme, and 2 exhibited a competitive inhibition of the enzyme (IC₅₀ = 188.8 μM).     

AMP-activated protein kinase and metabolic regulation in cold-hardy insects

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypometabolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated protein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurosta solidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPK activity was almost 2-fold higher in winter larvae (February) compared with animals collected in September. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphorylation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema during midwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significant increase in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurosta and Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increased in the winter. Pyruvate dehydrogenase (PDH) E1α subunit site 1 phosphorylation was 2-fold higher in extracts of Eurosta larvae collected in February versus September while PDH activity decreased by about 50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glycogen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February compared with September and also in these animals, triglyceride lipase activity increased by 70% during winter. Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a more catabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.     

Genetic association of IDE,POU2F1, PON1, IL1α and IL1β with type 2 diabetes in Pakistani population

A number of genes are known to be involved in glucose homeostasis. Mutations and polymorphisms in candidate genes may effect insulin production, action or resistance. This study was designed to report the association of genetic polymorphism with the type 2 diabetes (T2D) in Pakistani population. A total of 458 subjects (case n = 288, control n = 170) participated in the study. Nine single nucleotide polymorphisms were investigated in genes IDE (rs6583813 C>T, rs7910977 C>T), POU2F1 (rs3767434 A>T, rs10918682 A>T, rs2146727 A>G), WFS1 (rs734312 A>G), PON1 (rs854560 T>A), IL1α (rs1800587 C>T) and IL1β (rs1143634 C>T). Genotyping was performed by DNA sequencing after nested polymerase chain reaction of targeted regions. Results indicated that rs7910977 in IDE showed significant association with the development of T2D [P = 0.012, OR 1.677 (95 % CI 1.112–2.438)]. The rs10918682 in POU2F1 was associated with T2D [P < 0.001, OR 3.606 (95 % CI 2.165–6.005)]. The rs854560 in PON1was associated with incidences of T2D and increased the risk of cardiovascular complications [P = 0.031, OR 0.663 (95 % CI 0.455–0.965)] in diabetics. The rs734312 from WFS1 gene was associated with diabetes at genotype level (P < 0.01). Haplotype analysis of rs1800587–rs1143634 depicted CC haplotype increased the susceptibility to diabetes (P < 0.05). Haplotype GAA from rs2146727–10918682–rs3767434 was protective against diabetes (P < 0.01) and GGA exhibited the association with T2D (P < 0.01). Haplotype CT from rs6583813–rs7910977 was protective against diabetes (P = 0.02). Our study provided evidence to IDE, PON1, WFS1, POU2F1, IL1α and IL1β associated with T2D in Pakistanis.     

Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.     

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

Background  

Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.     

Genetic association of lipid metabolism related SNPs with myocardial infarction in the Pakistani population

Myocardial infarction (MI) is the major cardiovascular disease. This can be caused by mutual interaction of environmental and genetic factors. The current study was designed to investigate the role of lipid metabolism related genetic polymorphisms with the onset of MI in Punjabi population of Pakistan. A total of 384 subjects was studied from April 2011 to July 2012. To determine the genetic associations with MI, the single nucleotide polymorphisms (SNPs) were genotyped by sequencing, as well as one label extension method. Out of eight SNPs in four candidate genes, seven genetic variants were significantly (P < 0.05) associated with elevated risk of MI. In current study two SNPs rs662799 risk allele G (P = 0.03) and rs3135506 risk allele C (P = 0.05) of APOA5 were found to be associated with significant higher risk of triglyceride levels, irrespective of age, sex, obesity, diabetes, hypertension and smoking. Gene variants (rs1558861, rs662799 and rs10750097) in APOA5 showed almost complete linkage disequilibrium and their minor allele frequencies (0.34, 0.28, and 0.41 respectively) were more prevalent (P < 0.05) in cases than controls. We further revealed risk haplotypes (C-T-G-A, G-C-A-G; P = 0.001) and protective haplotypes (G-T-A-G, C-C-G-A; P = 0.005) between these four SNPs for the progression of MI. Current study confirms the correlation between lipid metabolism related SNPs with MI and supports the role of APOA5 in raising plasma triglyceride levels in Pakistanis. However further studies are needed for delineating the role of these SNPs.