Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.     

Role of Lectins in Plant-Microorganism Interactions: III. Influence of Rhizosphere/Rhizoplane Culture Conditions on the Soybean Lectin-binding Properties of Rhizobia

The influence of rhizosphere/rhizoplane culture conditions on the ability of various rhizobia to bind soybean seed lectin (SBL) was examined. Eleven strains of the soybean symbiont, Rhizobium japonicum, and six strains of various heterologous Rhizobium species were cultured in root exudate of soybean (Glycine max [L.] Merr.) and in association with roots of soybean seedlings which were growing either hydroponically or in montmorillonite clay soil amendment (Turface). All 11 of the R. japonicum strains developed biochemically specific receptors for the lectin when cultured under these conditions, whereas six of the 11 did not develop such receptors when cultured in synthetic salts medium. Two cowpea strains also developed receptors for SBL. The other four heterologous strains of rhizobia gave no evidence of biochemically specific SBL binding in either synthetic salts media or rhizosphere/rhizoplane cultures. These results demonstrate that the environment provided by plant roots is an important factor in the development of specific lectin receptors on the cell surface of R. japonicum.     

Unexpected divergence of enzyme function and sequence: "N-acylamino acid racemase" is o-succinylbenzoate synthase

A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp. is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1). Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome. Both proteins efficiently catalyze the o-succinylbenzoate synthase reaction in menaquinone biosynthesis (kcat/Km = 2.5 x 10(5) and 7.5 x 10(5) M-1 s-1, respectively), suggesting that this is their "correct" metabolic function. Their membership in the mechanistically diverse enolase superfamily provides an explanation for the catalytic promiscuity of the protein from Amycolaptosis. The adventitious promiscuity may provide an example of a protein poised for evolution of a new enzymatic function in the enolase superfamily. This study demonstrates that the correct assignment of function to new proteins in functional and structural genomics may require an understanding of the metabolism of the organism.     

Roles for riboflavin in the Sinorhizobium-alfalfa association

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.     

Pharmacophore and Virtual Screening of JAK3 inhibitors

Janus kinase 3 (JAK3) is a non-receptor tyrosine kinases family of protein which is comprised of JAK1, JAK2, JAK3 and TYK2. It plays an important role in immune function and lymphoid development and it only resides in the hematopoietic system. Therefore, selective targeting JAK3 is a rational approach in developing new therapeutic molecule. In this study, about 116 JAK3 inhibitors were collected from the literature and were used to build four-point pharmacophore model using Phase (Schrodinger module). The statistically significant pharmacophore hypothesis of AAHR.92 with r2 value of 0.942 was used as 3D query to search against 3D database namely Zincpharmer. A total of 2, 27,483 compounds obtained as hit were subjected to high throughput virtual screening (HTVS module of Schrodinger). Among the hits, ten compounds with good G-score ranging from -12.96 to -11.18 with good binding energy to JAK3 were identified.     

Induction of three-dimensional assembly of human liver cells by simulated microgravity

Summary The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.     

Anti-cancer potential of selenium-chitosan-polyethylene glycol-carvacrol nanocomposites in multiple myeloma U266 cells

Multiple myeloma (MM) is an incurable cancer that is characterized by malignant plasma cell proliferation. Approximately 10% of all blood cancers are MM, and there is no standard curative therapy. In this work, we intended to synthesize, characterize, and assess the anticancer effects of selenium/chitosan/polyethylene glycol–carvacrol nanocomposites (SCP-Car-NCs) on MM U266 cells in vitro. Various characterization techniques were used to characterize the synthesized SCP-Car-NCs. Several in vitro free radical scavenging experiments were conducted to test the ability of synthesized SCP-Car-NCs to scavenge the different free radicals. The cytotoxicity of SCP-Car-NCs was assessed on Vero and U266 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. By using various fluorescence staining techniques, the amount of reactive oxygen species (ROS) generation, MMP, and apoptosis were measured. Using commercial test kits, the levels of oxidative stress and apoptotic biomarkers in control and treated U266 cells were assessed. The highest peak in the UV spectral analysis was found to be at 271 nm, demonstrating the development of SCP-Car-NCs. Fourier transform infrared analysis showed that the synthesized SCP-Car-NCs contained a variety of stretching and bonding. The X-ray diffraction study confirmed the crystallinity of SCP-Car-NCs. The dynamic light scattering analysis showed that the SCP-Car-NCs had an average size of 171 nm. The different free radicals, such as the 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, and peroxyl radicals, were significantly scavenged by the SCP-Car-NCs. According to the MTT assay results, the SCP-Car-NCs decreased the viability of U266 cells while having no impact on the proliferation of Vero cells. The SCP-Car-NCs significantly boosted ROS production, decreased the MMP level, and promoted apoptosis, as evidenced by the fluorescence staining experiments. In U266 cells treated with SCP-Car-NCs, the level of thiobarbituric acid reactive substances increased while superoxide dismutases and glutathione levels were reduced. In the SCP-Car-NCs treated U266 cells, it was found that the Bax, caspase-3, and −9 activities had increased while the Bcl-2 level had decreased. In conclusion, our findings show that SCP-Car-NCs treatment reduced the viability and increased apoptosis in the U266 cells, providing a new insight on SCP-Car-NCs' potential for usage in the future to treat MM.