On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual''s level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.¹ These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,² lipid-^{3, 4} and RAGE-induced⁵ inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.     

Control of cellulose hydrolysis by fungi

Summary The extent of filter paper degradation by extracellular preparations fromT. reesei and its mutants with a decreasing level of -glucosidase and an increasing level of endoglucanase has been determined. The ability to degrade cellulose is restricted by the level of endoglucanase and not by -glucosidase.     

A comparative study of the epidermis of the common carp and the three Indian major carp

A comparative study has been made of the mucogenic epidermis of the common carp, Cyprinus carpio var. communis, and the three Indian major carps, Catla catla, Labeo rohita and Cirrhina mrigala: on the basis of epidermis structural organization, these species are easily differentiated. The epithelial cells in the superficial layer, as in most fishes, show secretory activity, evidenced by positive histochemical reactions, which is high in C. carpio var. communis, moderate in C. catla and low in L. rohita and C. mrigala. The epithelial cells in the underlying two or three layers also give positive reactions, though their intensity is relatively weak. The mucous cells in C. carpio var. communis are distributed in large numbers arranged in several superimposed layers in the outer regions of the epidermis, whereas in C. catla they are fewer in number and are widely separated in the surface layers as well as in the deeper layers of the epidermis; in both species the mucous cells appear rounded, large, and open on the surface by wide pores. In contrast, in L. rohita and C. mrigala the mucous cells are smaller, restricted mainly to the superficial layer, close together in a single row, and open on the surface by narrow pores. The overall density of mucous cells in L. rohita and C. mrigala, as in C. catla, is much lower than in C. carpio var. communis. In the epidermis of C. carpio var. communis there are a large number of mucous cells, and the few club cells are restricted to the deeper layers. In contrast, in the epidermis of the three Indian major carp the overall density of the mucous cells is much lower and the club cells are very numerous. It is suggested that the high density of club cells compensates an overall low density of mucous cells as an adaptation for an effective defence mechanism. Increased mucus production in the epidermis of C. carpio var. communis, as evidenced by a large number of mucous cells in outer regions and high secretory activity of superficial layer epithelial cells, is associated with increased precipitation of mud held in suspension, needed as an adaptation to the species’peculiar bottom-scooping habits. The varied density of the taste buds in the epidermis of the four carp is associated with their feeding habits.     

A photomap of the salivary gland chromosomes of Anopheles stephensi liston (Culicidae: Diptera)

A photomap of the banding pattern of the salivary gland chromosomes of Anopheles stephensi Liston, which is first of its kind, has been prepared. The salivary chromosome complement consists of five arms, the shortest of which represents the telocentric X-chromosome, and the remaining four the autosomal arms. A comparison has been made of the banding pattern of this species with other species of the subgenus Cellia.     

Fatty acid activation of guanylate cyclase from fibroblasts and liver.

Sodium arachidonate and sodium oleate increased particulate guanylate cyclase activity from homogenates of Balb 3T3 cells or rat liver. The fatty acids were about equipotent and were maximally effective at about 100 μm concentrations. Higher concentrations were less effective or inhibitory. Activation was similar in an air or nitrogen atmosphere and was unaltered by KCN, aspirin, or indomethacin. The dose-response curve was shifted to the right when arachidonate was preincubated prior to its addition to guanylate cyclase assays. Agents that facilitate fatty acid oxidation and the formation of malonyldialdehyde during preincubation such as glutathione, hemoglobin, Mn²⁺, Fe³⁺, or lipoxygenase shifted the dose-response curve further to the right. In contrast, agents that decreased or prevented arachidonate oxidation and malonyldialdehyde formation during preincubation such as butylated hydroxyanisole, propyl gallate, hydroquinone, and diphenylfuran prevented the shift in the dose-response curve or in some instances shifted the dose-response curve to the left. Activation of guanylate cyclase by arachidonate was reversed by the addition of lipoxygenase to incubations. These studies indicate that unsaturated fatty acids and not their oxidation products activate particulate enzyme from Balb 3T3 cells. The mechanism of fatty acid activation appears to be different from activation by nitro compounds. Fatty acids but not nitro compounds activated fibroblast preparations, and the effect of fatty acids in contrast to the activation by nitroprusside in liver preparations was not prevented with Lubrol PX.     

Chemical equilibrium and E value of Zn (ZnE) in inceptisols and entisols of tropical india

Summary The chemical equilibrium and E value for Zn was calculated in 9 soils of Haryana (India) which represented mainly entisol and inceptisols. The rate of isotopic exchange between⁶⁵Zn and native soil Zn was quite rapid and radioactivity reduced to about 0.2 per cent of initial activity after 3 days but total Zn concentration in soil solution did not decrease. In 5 out of 9 soils equilibrium was established in 2 to 3 days. In typic ustochrepts having high clay content (3,4) equilibrium was attained in two days. In typic udipsamments (2), typic camborthids (8) and aquic vertic ustichrepts (9) it took 3 days to set equilibrium. Typic ustochrepts (5) took 5 days for equilibrium whereas typic ustipssaments (6) and typic camborthids of high O.C. did not attain equilibrium even in 7 days. This indicated that the chemistry and availability of Zn in soil would depend on soil types. When Zn_E was estimated by applying activity after equilibrium with carrier Zn and by applying activity with carrier Zn before equilibrium was set, there was no agreement in Zn_E in two methods. Increasing Zn_E with increasing Zn dose was observed by both methods only in alkaline typic ustochrept (4). In some soils higher E values than added amounts were observed, whereas, in soil 8 negative E values were obtained. The E values become erratic and are over estimated where complex reactions take place due to high pH, high O.C. and high complex forming elements.     

Mechanical Signaling on the Single Protein Level Studied Using Steered Molecular Dynamics

Efficient communication between the cell and its external environment is of the utmost importance to the function of multicellular organisms. While signaling events can be generally characterized as information exchange by means of controlled energy conversion, research efforts have hitherto mainly been concerned with mechanisms involving chemical and electrical energy transfer. Here, we review recent computational efforts addressing the function of mechanical force in signal transduction. Specifically, we focus on the role of steered molecular dynamics (SMD) simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain. We start by giving a brief introduction to the SMD technique and general properties of mechanically stable protein folds, followed by specific examples illustrating three general regimes of signal transfer utilizing mechanical energy: purely mechanical, mechanical to chemical, and chemical to mechanical. Whenever possible the physiological importance of the example at hand is stressed to highlight the diversity of the processes in which mechanical signaling plays a key role. We also provide an overview of future challenges and perspectives for this rapidly developing field.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.