Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Phenology of plants in relation to ambient environment in a subalpine forest of Uttarakhand,western Himalaya

Observations on phenology of some representative trees, shrubs, under-shrubs and herbs in a subalpine forest of Uttarakhand, western Himalaya were recorded. With the commencement of favorable growth season in April, occurrence of leaf fall was indicatory growth phenomenon in Quercus semecarpifolia, Q. floribunda and Abies spectabilis. However, active vegetative growth in herbaceous species starts onward April and fruit maturation and seed dehiscence are completed from mid of September to October. In general, vegetative growth and reproductive stages in majority of the studied species seems to be dependent on adequate moisture content and also flowering and fruiting in subalpine plants correlate ambient temperature.     

The role of mass spectrometry in the characterization of biologic protein products

Introduction: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products.

Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented.

Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.     

Antihyperglycaemic activity of aqueous extract of Embelia ribes Burm in streptozotocin-induced diabetic rats

Forty days of orally feeding the aqueous E. ribes extract (100 and 200 mg/kg) to streptozotocin (40 mg/kg, iv, single dose) induced diabetic rats produced significant decrease in heart rate, systolic blood pressure, blood glucose, blood glycosylated hemoglobin, serum lactate dehydrogenase, creatine kinase and increase in blood glutathione levels as compared to pathogenic diabetic rats. Further, the extract significantly decreased the levels of pancreatic lipid peroxides and increased the levels of pancreatic superoxide dismutase, catalase and glutathione. The results suggest that aqueous E. ribes extract exhibits a significant blood glucose and blood pressure lowering potential. Further, it enhances endogenous antioxidant defense against free radicals produced under hyperglycaemic conditions, thereby, seemingly protects the pancreatic beta-cells against loss in streptozotocin induced diabetic rats.     

Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis

Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.Colorectal carcinoma is one of the most common forms of cancer seen in the developed world (10). The incidence in developing countries appears to be somewhat lower, but with high rates of mortality similar to those seen in developed countries (43). Treatment usually involves surgery or palliative chemotherapy, and recent research focuses on investigating molecular signatures associated with colorectal tumorigenesis as well as means of early detection in order to avoid high and rapid fatalities (61). Increased activity and expression of the tyrosine kinase c-src is frequently seen in colorectal cancer (5, 12), and activation of c-src in early stages of neoplastic transformation (32) is an indicator of a poor clinical prognosis (26). In general, c-src activity is as much as 16-fold higher in cancerous cells than in the adjacent colonic mucosa (12). Information on potential molecular mechanisms for the role of c-src in colonic tumorigenesis includes evidence that c-src activity increases during mitosis of human colon carcinoma cells (39), and increased c-src activity promotes cell motility and invasion during intestinal cell migration (34).A large number of substrates for c-src have been identified in different cell types, and a consensus site for tyrosine phosphorylation by c-src has been defined (50). Prediction of these sites in proteins present in colorectal carcinoma cells may assist in identifying novel targets for c-src action. The modular structure of c-src and other members of the src family kinases (SFKs), consisting of the SH4, unique, SH3, SH2, and kinase domains, allows these kinases to interact with a diverse group of proteins, creating a highly complex signal transduction network (48, 52). Consequently, SFKs have been demonstrated to be key downstream elements in signaling pathways emerging from cell surface receptors (52).Guanylyl cyclase C (GC-C) plays an important role in maintaining fluid ion homeostasis (54) and genomic integrity in intestinal cells (29) and serves as the receptor for the diarrheagenic heat-stable enterotoxin (ST) as well as endogenous ligands guanylin and uroguanylin (57). Binding of ST to the receptor leads to increased intracellular cyclic GMP (cGMP) accumulation and activation of the cystic fibrosis transmembrane conductance regulator, resulting in fluid and ion efflux that manifests as traveler''s diarrhea (45, 54). Recently it has been noted that regions of the world with the highest incidence of enterotoxigenic Escherichia coli-associated diarrhea exhibit the lowest incidence of colon cancer (43). In this context, studies have shown that activation of GC-C by its ligands inhibits human colonic cell proliferation and adenoma formation in mice, demonstrating a critical role for GC-C in inhibiting colon cancer initiation and progression (29, 33). For example, uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon carcinoma cells via cGMP produced by GC-C (46). Interestingly, expression of the endogenous paracrine hormones for GC-C, guanylin and uroguanylin, is lost at the initiation of transformation, and therefore this reduced signaling via GC-C may allow cells to proliferate more rapidly, thereby leading to carcinogenesis (14).Expression of GC-C is largely restricted to the luminal side of intestinal cells in humans, and consequently, monitoring GC-C expression in circulating tumor cells in the peripheral blood of patients has been suggested to be a good marker for early detection of the primary tumor and/or metastasis of colon cancer (11, 28, 36). The molecular mechanisms by which GC-C activity is regulated during the initiation and progression of carcinogenesis remain undefined, and studies to investigate whether signaling events prevalent in colorectal cancer can modulate GC-C function have not been performed. Human colorectal carcinoma cell lines express GC-C and can therefore be used to study these molecular aspects of GC-C regulation and downstream signaling events (3, 4, 22).Phosphorylation is a rapid and reversible form of covalent modification frequently found in signaling systems. In our earlier studies, we have coexpressed domains of GC-C with the tyrosine kinase EphB1/Elk and observed tyrosine phosphorylation of GC-C (7). We report here that GC-C is a substrate for c-src tyrosine kinase and identify Tyr820 as the site for phosphorylation in GC-C. Following phosphorylation, pTyr820 serves as a site for interaction with the SH2 domain of c-src, resulting in further activation of c-src. Most importantly, tyrosine phosphorylation of GC-C inhibits cGMP production and prevents the cytostatic effects of GC-C induced by ligand interaction. Our studies therefore show the existence of a new signal transduction cross talk between c-src and GC-C in colonic cells, resulting in a feed-forward mechanism to further activate c-src, with important implications in cancer cell proliferation and disease progression.     

Aryloxy cyclohexyl imidazoles: A novel class of antileishmanial agents

Thirteen novel aryloxy cyclohexane-based mono and bis imidazoles were synthesized and evaluated in vitro as antileishmanials against Leishmania donovani and cytotoxicity assessed. These compounds were better than the existing drugs, sodium stibogluconate and pentamidine in respect to IC₅₀ and SI values. Promising compounds were tested further in vivo. Among all, the bis methylimidazole with 2-fluoro, 4-nitro aryloxy group (9) exhibited significant in vivo inhibition of 77.9%, thus providing new structural lead for antileishmanials.