Galectin-1-Induced Autophagy Facilitates Cisplatin Resistance of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in Taiwan. Although chemotherapy is the primary treatment for HCC patients, drug resistance often leads to clinical failure. Galectin-1 is a beta-galactoside binding lectin which is up-regulated in HCC patients and promotes tumor growth by mediating cancer cell adhesion, migration and proliferation, but its role in chemoresistance of HCC is poorly understood. In this study we found that galectin-1 is able to lead to chemoresistance against cisplatin treatment, and subsequent inhibition has reversed the effect of cell death in HCC cells. Moreover, galectin-1 was found to induce autophagic flux in HCC cells. Inhibition of autophagy by inhibitors or knockdown of Atg5 cancels galectin-1-induced cisplatin resistance in HCC cells. Increase of mitophagy triggered by galectin-1 was found to reduce the mitochondrial potential loss and apoptosis induced by cisplatin treatment. Finally, using an in situ hepatoma mouse model, we clearly demonstrated that inhibition of galectin-1 by thiodigalactoside could significantly augment the anti-HCC effect of cisplatin. Taken together, our findings offer a new insight into the chemoresistance galectin-1 causes against cisplatin treatment, and points to a potential approach to improve the efficacy of cisplatin in the treatment of HCC patients.     

Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Anopheles culicifacies complex and control of malaria

Of the 50 or so species and varieties of anopheline mosquito in India, about 20 are implicated as vectors of human malaria. Of these, perhaps the most important and widespread is Anopheles culicifacies s.l. For the first 10 years of widespread DDT spraying, An. culicifacies remained susceptible to this insecticide - indeed, some thought it lacked resistance genes altogether. By 1960 however, resistance to DDT was apparent. DDT is a cheap insecticide, and its use is still favoured for mosquito control wherever it remains effective. But by the end of the late 1970s it appeared that DDT resistance in An. culicifacies (and other species) was a major barrier to effective vector control in several areas - particularly in parts of the northwestern states. Yet in other areas DDT still seemed to be effective. There was also increasing evidence from other studies suggesting differences in An. culicifacies found in different areas - particularly differences in seasonal prevalence and man-biting activity. We now know - as Sarala Subbarao discusses here - that An. culicifacies s.l. represents a complex of at least four sibling species. But in this case, one of the most important findings is that DDT resistance is mainly associated with species B which proves to be a very poor vector of malaria. Such findings, made possible by careful cytogenetic studies, have very important consequences for malaria epidemiology and control policies.     

X-linkage of malic enzyme in Anopheles culicifacies species B

A survey for malic enzyme (Me) in laboratory strains of species A and species B of Anopheles culicifacies had uncovered two electrophoretic variants, slow and fast, in two strains of species B. Genetic analysis revealed the two variants to be codominant alleles segregating at a locus, Me, which is sex linked. Because of the XX-XY sex determining mechanism, in F1 females, two electromorphs, viz., slow and fast, were observed, whereas in males only one electromorph of maternal origin was seen. Linkage experiments with another X-linked mutant, white eye (w), indicated the map distance between the two loci to be 9.52 +/- 0.86.     

Prediction and comparison of the secondary structure of legume lectins

Secondary structure prediction for the 4 legume lectins: Concanavalin A, soybean agglutinin, favabean lectin and lentil lectin, was done by the method of Chou and Fasman. This prediction shows that these four lectins fall into a structurally distinct class of proteins, containing high amounts of β-sheet and β-turns. There is a notable similarity in the gross structure of these proteins; all four of them contain about 40–50% of β-sheet, 35–45 % β-turn and 0–10% of α-helix. When the secondary structure of corresponding residues in each pair of these lectins was compared, there was a striking similarity in the Concanavalin A-soybean agglutinin and favabean lectin-lentil lectin pairs, and considerably less similarity in the other pairs, suggesting that these legume lectins have probably evolved in a divergent manner from a common ancestor. A comparison of the predicted potential β-turn sites also supports the hypothesis of divergent evolution in this class of lectins.     

A site in a tRNA precursor that can be processed by the whole RNase P enzyme but not by the RNA alone

A precursor molecule for 10 Sb RNA, the RNA moiety of the RNA processing enzyme RNase P, was purified, characterized for enzymatic activity, and compared to 10 Sb RNA and to RNase P. In these studies the K RNA, a dimeric precursor of tRNAGln-tRNALeu, coded by bacteriophage T4, was used as a substrate. This precursor contains two RNase P cleavage sites, one at each 5' end of the two tRNAs. The precursor 10 Sb and 10 Sb RNAs have the capacity to cleave the precursor tRNA molecule but only at the 5' end of tRNALeu, not at the 5' end of tRNAGln. Even when a substrate was prepared that contained only one site for RNase P (the one next to tRNAGln), this substrate was not cleaved by the RNA alone while the whole enzyme was effective in processing this substrate. The possible function of the protein of RNase P in the enzymatic reaction is discussed.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Incorporating an Asymptotic Parameter into the Weibull Model to Describe Plant Disease Progress

The Weibull model is a flexible growth model that describes both general population growth and plant disease progress. However, lack of an asymptotic parameter has limited its wider application. In the present study, an asymptotic parameter K was introduced into the original Weibull model, written as; y = K {1 − exp [− ( t − a ) ^c ]}, in which a , b , c and K are location, scale, shape, and asymptotic parameters, respectively, y is the proportion of disease and t is time. A wide range of simulated disease progress data sets were generated using logistic, Gompertz and monomolecular models by specifying different parameter values, and fitted to both original and modified Weibull models. The modified model provided statistically better fits for all data than the original model. The modified model can thus improve the curve-fitting ability of the original model which often failed to converge, especially when the asymptote is less than 1.0. Actual disease progress data on wheat leaf rust and tomato root rot with different asymptotic values were also used to compare the original and modified Weibull models. The modified model provided a statistically better fit than the original model, and model estimates of asymptotic parameter K were nearly identical to the actual disease maxima reflecting the characteristics of the host-pathosystem. Comparison of logistic, Gompertz, and Weibull models including parameter K by fitting to the observed data on wheat leaf rust and tomato root rot revealed the applicability of the modified Weibull model, which in a majority of cases provided a statistically superior fit.     

Segregation of Cytoplasmic Incompatibility Properties in CULEX PIPIENS FATIGANS

Maternally inherited variants, which arose within a laboratory colony of Culex pipiens fatigans, have been studied by rearing cultures from single egg rafts. Segregation, i.e, variation of cytoplasmic incompatibility properties between the male progeny of individual females, was demonstrated. Also, from the daughters of individual females, sub-lines were derived within which all the males showed the same incompatibility or compatibility properties. Among the descendants of tetracycline-treated individuals were lines which superficially simulated these phenomena, but theses lines ultimately reverted to the cytoplasmic compatibility type of the strain which was submitted to the treatment. The types of variation s in cytoplasmic incompatibility properties that have been studied are discussed.     

Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.