Isolation and partial characterization of ribonuclease inhibitor from goat liver

Ribonuclease inhibitor (RI), a 50 kDa protein, has been found both in mammalian and nonmammalian tissues. We have isolated RI from goat liver and partial characterization has been accomplished. For the isolation of RI, DEAE cellulose column chromatography followed by affinity chromatography using CNBr activated Sepharose 4B was performed. The inhibition of ribonucleolytic activity of Ribonuclease A has been checked by an agarose gel based assay. The antiangiogenic property of the protein was tested by the chorioallantoic membrane (CAM) assay. Results indicate inhibition of angiogenesis.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

A Probabilistic Model of RNA Conformational Space

The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling procedure. Both are only partly solved problems. Here, we focus on the problem of conformational sampling. The current state of the art solution is based on fragment assembly methods, which construct plausible conformations by stringing together short fragments obtained from experimental structures. However, the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows efficient sampling of RNA conformations in continuous space, and with associated probabilities. We show that the model captures several key features of RNA structure, such as its rotameric nature and the distribution of the helix lengths. Furthermore, the model readily generates native-like 3-D conformations for 9 out of 10 test structures, solely using coarse-grained base-pairing information. In conclusion, the method provides a theoretical and practical solution for a major bottleneck on the way to routine prediction and simulation of RNA structure and dynamics in atomic detail.     

Aflatoxin B1-induced toxicity in HepG2 cells inhibited by carotenoids: morphology, apoptosis and DNA damage

Aflatoxin B1 (AFB1) is a fungal toxin that has been associated with primary hepatocellular carcinoma (HCC) in humans. This study was undertaken to determine the cellular and molecular mechanisms by which the antioxidants beta-carotene and lycopene inhibit AFB1-induced toxic changes in human hepatocytes (HepG2 cells). An in vitro system was optimized to test the chemoprotective effects of lycopene and beta-carotene on HepG2 cells exposed to different concentrations of AFB1. Ultrastructurally, HepG2 cells cultured in the presence of AFB1 showed mitochondrial damage, nuclear condensation and a loss of cell-to-cell contact; the latter was reflected in the observation of dysfunctional gap junctions, resulting in a loss of cell-to-cell communication. At the genomic level, AFB1 formed AFB1-N7-guanine adducts, caused apoptotic cell death and suppressed p53 protein expression. In the presence of the carotenoids, survival of cells exposed to AFB1 was increased, and there was also a significant increase in cellular mitochondrial activity. Our results demonstrate that HepG2 cells pretreated with lycopene and beta-carotene are protected from the toxic effects of AFB1 at both the cellular and molecular levels.     

Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.     

Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase

Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease. In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells. The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells. ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media. In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines. Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner. The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity. These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells. The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.     

The role of the DRY motif of human MC4R for receptor activation

We constructed several mutant human MC4R cDNAs by site directed mutagenesis and expressed these receptors in COS-1 cells. The conserved DRY motif among GPCRs was mutated to generate eight mutants. While no MC4R ligand binding was detected in any of the mutants, one mutant, D146A, resulted in higher cAMP production in cells than the wild-type receptor without ligand stimulation.     

Kinin-B1 receptors in ischaemia-induced pancreatitis: functional importance and cellular localisation

In this study we compare the role of kinin-B1 and B2 receptors during ischaemia/reperfusion of rat pancreas. Our investigations were prompted by the observation that infusion of a kinin-B2 receptor antagonist produced significant improvement in acute experimental pancreatitis. In an acute model with two hours of ischaemia/two hours of reperfusion, application of the kinin-B1 receptor antagonist (CP-0298) alone, or in combination with kinin-B2 receptor antagonist (CP-0597), significantly reduced the number of adherent leukocytes in post-capillary venules. In a chronic model with five days of reperfusion, the continuous application of kinin-B1 receptor antagonist or a combination of kinin-B1 and B2 receptor antagonists markedly reduced the survival rate. In kinin-receptor binding studies kinin-B1 receptor showed a 22-fold increase in expression during the time of ischaemia/reperfusion. Carboxypeptidase M activity was up-regulated 10-fold following two hours of ischaemia and two hours of reperfusion, provided the appropriate specific ligand, des-Arg10-kallidin and/or des-Arg9-bradykinin, was used. The occurrence of kinin-B1 receptor binding sites on acinar cell membranes was demonstrated by micro-autoradiography. With a specific antibody, the localisation of kinin-B1 receptor protein was confirmed at the same sites. In conclusion, we have demonstrated the up-regulation of the pancreatic acinar cell kinin-B1 receptors during ischaemia/reperfusion. The novel functional finding was that antagonism of the kinin-B1 receptors decreased the survival rate in an experimental model of pancreatitis.     

Perception of and agricultural adjustment to floods in Jamuna floodplain,Bangladesh

Based on a sample survey collected from randomly selected farmers inhabiting the Jamuna floodplain of Bangladesh, this study deals with the perception of and agricultural adjustments to both normal and abnormal floods. The study found that the respondents' perception of flood is conditioned by the key role played by floods in their lives. The study further observed that the respondents successfully cope with the normal floods. In cases of abnormal floods, they practiced several adjustments to reduce damages.