Synthesis and cytotoxic activity of 3-O-acyl/3-hydrazine /2-bromo/20,29-dibromo betulinic acid derivatives

A series of 3-O-acyl, 3-hydrazine, 2-bromo, and 20,29-dibromo betulinic acid derivatives (1-27) have been synthesized and screened for in vitro cytotoxic activity on human cancer cell lines MOLT-4, JurkatE6.1, CEM.CM3, BRISTOL8, U937, DU145, PA-1, A549, and L132. A number of compounds have shown ED(50)<1 microg/mL against the cancer cell lines tested and have shown better cytotoxicity than betulinic acid. Compounds 13, 19, 20, 23, and 27 were the best derivatives and were selected as lead molecules for further development. The structure-activity relationship has been described.     

In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

Synthesis and in vitro pharmacological studies of new C(2) modified salvinorin A analogues

Salvinorin A is the most potent naturally occurring opioid agonist yet discovered with high selectivity and affinity for kappa-opioid receptor. To explore its structure and activity relationships, a series of salvinorin A derivatives modified at the C2 position were prepared and studied. These salvinorin A derivatives were screened for binding and functional activities at the human kappa-opioid receptor. Compound 4, containing a methoxymethyl group at the 2-position, was a full kappa-agonist with an EC50 value at 0.6 nM, which is about 7 times more potent than salvinorin A.     

Influence of antioxidant (L- ascorbic acid) on tolbutamide induced hypoglycaemia/antihyperglycaemia in normal and diabetic rats

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats. METHODS: L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. RESULTS: L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control. CONCLUSION: Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats.     

High-quality RNA from cells isolated by laser capture microdissection

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.     

Fluvastatin Mediated Breast Cancer Cell Death: A Proteomic Approach to Identify Differentially Regulated Proteins in MDA-MB-231 Cells

Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.     

Light and scanning electron microscopic studies of flower development inLindenbergia macrostachya Benth. (Scrophulariaceae)

All the floral primordia are homologous to leaves in their development inLindenbergia macrostachya. The sepals follow an anterior to posterior sequence of initiation. The petals and stamens are initiated almost simultaneously but sequentially in order of petals followed by stamens. There is no sign of development of fifth posterior stamen. p ]The calyx tube is formed by interprimordial growth followed by zonal growth. The combined interprimordial growth between the petal primordia and growth on the abaxial side of stamen primordia results in the formation of upper corolla tube whereas lower corolla tube is formed only by zonal growth. The zonal growth extends below the bases of stamen primordia also due to which they become epipetalous. The placentae arise from the carpellary margins, move inwards and get fused in the lower half and remain free in the upper part of the ovary. Thus the ovary appears biloeular with axile plaeentation in the lower haler and unilocular with parietal placentation in the upper half.     

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host–bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.