Skeletal and muscular pelvic morphology of hillstream loaches (Cypriniformes: Balitoridae)

The rheophilic hillstream loaches (Balitoridae) of South and Southeast Asia possess a range of pelvic girdle morphologies, which may be attributed to adaptations for locomotion against rapidly flowing water. Specifically, the connectivity of the pelvic plate (basipterygium) to the vertebral column via a sacral rib, and the relative size and shape of the sacral rib, fall within a spectrum of three discrete morphotypes: long, narrow rib that meets the basipterygium; thicker, slightly curved rib meeting the basipterygium; and robust crested rib interlocking with the basipterygium. Species in this third category with more robust sacral rib connections between the basipterygium and vertebral column are capable of walking out of water with a tetrapod-like lateral-sequence, diagonal-couplet gait. This behavior has not been observed in species lacking direct skeletal connection between the vertebrae and the pelvis. The phylogenetic positions of the morphotypes were visualized by matching the morphological features onto a novel hypothesis of relationships for the family Balitoridae. The morphotypes determined through skeletal morphology were correlated with patterns observed in the pelvic muscle morphology of these fishes. Transitions towards increasingly robust pelvic girdle attachment were coincident with a more anterior origin on the basipterygium and more lateral insertion of the muscles on the fin rays, along with a reduction of the superficial abductors and adductors with more posterior insertions. These modifications are expected to provide a mechanical advantage for generating force against the ground. Inclusion of the enigmatic cave-adapted balitorid Cryptotora thamicola into the most data-rich balitorid phylogeny reveals its closest relatives, providing insight into the origin of the skeletal connection between the axial skeleton and basipterygium.     

Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR. It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer. We have called this the repression binding site. In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold. Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay. The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2. DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate. We have named this second binding site the activation binding site. Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate. Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site. However, binding of CatR to the activation binding site required an intact repression binding site.     

Synthesis and QSAR studies on hypotensive 1-[3-(4-substituted phenylthio) propyl]-4-(substituted phenyl) piperazines

A series of 1-[3-(4-substituted phenylthio) propyl]-4-(substituted phenyl) piperazines has been synthesized and evaluated for hypotensive activity. The QSAR studies indicate that resonance and hydrophobic parameters of the aryl substituents are important for hypotensive activity. The similar role of resonance parameter in describing the variance of 5-HT(2A) receptor binding affinities of these compounds suggests a possible role of 5-HT(2A) receptors in mediating the hypotensive action of title compounds.     

Disruption of transforming growth factor beta-regulated laminin receptor function by expression of antisense laminin, a chain RNA in human colon cancer cells

Transforming growth factor beta1 (TGFbeta) simultaneously induces the expression of fibronectin, fibronectin receptor, laminin, and laminin receptor (alpha6beta1 integrin) in the human colon cancer cell line Moser (Int J Cancer, 57:742, 1994). Induction of fibronectin and induction of fibronectin receptor by TGFB are tightly coupled, and disrupting fibronectin induction disrupts the induction of fibronectin receptor and cellular adhesion to fibronectin (J Cellular Physiol, 170:138, 1997). We recently demonstrated the efficacy of using antisense chain-specific laminin RNA expression vectors to disrupt the induction by TGFP of the multichain laminin molecule (J Cellular Physiol, 178:296, 1999). We now show in this report that Moser cells used alpha6 and beta1 integrins to adhere to laminin, and, as is the fibronectin and fibronectin receptor system, disrupting the induction by TGFbeta of the ligand laminin by the expression of antisense laminin A chain RNA disrupted the induction of 125I-laminin binding and cellular adhesion to laminin. Disrupting laminin induction also blocked the induction of alpha6 and beta1 integrin laminin receptor by TGFbeta. We conclude that disrupting the induction of the ligand laminin by TGFbeta disrupts TGFbeta-regulated laminin receptor function by suppressing the induction of alpha6 and beta1 integrins. Therefore, targeted disruption of the ligand laminin may be an effective means in disrupting the function of both the ligand and its receptor in cells that utilize the laminin and laminin receptor system in malignant cell behavior.     

The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis

Ubiquitin-mediated protein modification plays a key role in many cellular signal transduction pathways. The Arabidopsis gene XBAT32 encodes a protein containing an ankyrin repeat domain at the N-terminal half and a RING finger motif. The XBAT32 protein is capable of ubiquitinating itself. Mutation in XBAT32 causes a number of phenotypes including severe defects in lateral root production and in the expression of the cell division marker CYCB1;1::GUS . The XBAT32 gene is expressed abundantly in the vascular system of the primary root, but not in newly formed lateral root primordia. Treatment with auxin increases the expression of XBAT32 in the primary root and partially rescues the lateral root defect in xbat32 - 1 mutant plants. Thus, XBAT32 is a novel ubiquitin ligase required for lateral root initiation.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.     

Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes

Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be significantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows--cellular yield per mouse, their protein content, lysosomal acid hydrolase levels and capability to phagocyte, all were significantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD levels but no change in total intracellular ascorbic acid levels. Further, though ascorbic acid interaction enhanced the phagocytic capability of MPM as compared to resident cells, no significant boosting of phagocytic process could be observed when each of three stimulators coupled with ascorbic acid was used for macrophage elicitation.     

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.