Cytokines modulate endothelial cell intracellular signal transduction required for VCAM-1-dependent lymphocyte transendothelial migration

Vascular cell adhesion molecule-1 (VCAM-1) activates endothelial cell NADPH oxidase which catalyzes production of reactive oxygen species (ROS). This activity is required for VCAM-1-dependent lymphocyte migration. The focus of our study was to determine whether these VCAM-1-dependent functions are modulated by cytokines. TGF-beta1 or IFN-gamma pretreatment of mouse endothelial cell lines inhibited VCAM-1-dependent B and T cell transendothelial migration without affecting initial lymphocyte adhesion. Neutralizing anti-TGF-beta1 blocked the effects of TGF-beta1 pretreatment of endothelial cells, whereas addition of anti-TGF-beta1 after TGF-beta1 pretreatment of the endothelial cells did not block TGF-beta1-mediated inhibition. Neutralizing anti-IFN-gamma also blocked the inhibitory effects of IFN-gamma. TGF-beta1 and IFN-gamma blocked migration by inhibiting the VCAM-1-stimulated production of low levels of ROS (0.1-0.9 microM H2O2). These results demonstrate that both TGF-beta1 and IFN-gamma directly affect the endothelial cells' ability to promote lymphocyte migration. IL-4 had differing effects on T and B cells during transmigration. IL-4 augmented T cell migration across the endothelial cell lines but did not affect T cell adhesion. Conversely, IL-4 increased B cell adhesion to the endothelial cell lines without affecting migration. In summary, cytokines can directly modulate microvascular endothelial cell intracellular signaling, demonstrating a new level of cytokine regulation of lymphocyte diapedesis.     

Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation

We have compared the mechanisms that contribute to endothelium-derived hyperpolarizing factor (EDHF)-type responses induced by ACh and the Ca(2+) ionophore A-23187 in the rabbit iliac artery. Relaxations to both agents were associated with ~1.5-fold elevations in smooth muscle cAMP levels and were attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA) and potentiated by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Mechanical responses were inhibited by coadministration of the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin, both in the absence and presence of IBMX, but were unaffected by blockade of ATP-sensitive K(+) channels with the sulphonylurea glibenclamide. Relaxations and elevations in cAMP evoked by ACh were abolished by 18alpha-glycyrrhetinic acid, which disrupts gap junction plaques, whereas the corresponding responses to A-23187 were unaffected by this agent. Consistently, in "sandwich" bioassay experiments, A-23187, but not ACh, elicited extracellular release of a factor that evoked relaxations that were inhibited by DDA and potentiated by IBMX. These findings provide evidence that EDHF-type relaxations of rabbit iliac arteries evoked by ACh and A-23187 depend on cAMP accumulation in smooth muscle, but involve signaling via myoendothelial gap junctions and the extracellular space, respectively.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

A 5.9-kb tandem repeat at the euchromatin-heterochromatin boundary of the X chromosome of Drosophila melanogaster

We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.     

Responses of dormant heather (Calluna vulgaris) seeds to light, temperature, chemical and advancement treatments

Two seed lots of Calluna vulgaris were obtainedfrom English Nature (seed of Cornish provenance) (EN) and John ChambersWildflower Seeds (JCWS). In laboratory tests, under continuous light untreatedseeds of both seed lots were partially dormant at temperatures between14–35 °C, but JCWS seeds were more deeply dormant thanENseeds. The optimum temperature for germination for both lots was ca 18°C. Germination of EN seeds was much lower in the dark than inthe light at all temperatures; JCWS seeds did not germinate in the dark. In thelight at 22 °C, dormancy of both seed lots was broken whenseeds were incubated in GA_4/7 solution(2×10^–4 M). Dormancy ofJCWSseeds at 22 °C in the light was broken when seeds wereincubated in four different smoke solutions but more so when used incombinationwith GA_4/7. Soaking seeds for 4h insmoke/GA_4/7solutions before sowing improved both the speed andpercentage germination in pot experiments on a mist bench in the glasshouse byat least 10-fold. Soaking with GA_4/7 alone produced a 5-fold increasein germination but seedlings were more etiolated than with thesmoke/GA_4/7 mixtures. A seed advancement treatment modified from thatused commercially on sugar beet seeds also promoted germination in bothlaboratory and glasshouse tests. This entailed soaking seeds in 0.2% thiramsuspension for 4h followed by incubation in excess solution at 22°C for 4 days. This treatment was not as effective as thesmoke/GA_4/7 seed soaks.     

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.     

Effect of IGF-I therapy on VLDL apolipoprotein B100 metabolism in type 1 diabetes mellitus

Abnormal lipid metabolism may be related to the increased cardiovascular risk in type 1 diabetes. Secretion and clearance rates of very low density lipoprotein (VLDL) apolipoprotein B100 (apoB) determine plasma lipid concentrations. Type 1 diabetes is characterized by increased growth hormone (GH) secretion and decreased insulin-like growth factor (IGF) I concentrations. High-dose IGF-I therapy improves the lipid profile in type 1 diabetes. This study examined the effect of low-dose (40 microg.kg(-1).day(-1)) IGF-I therapy on VLDL apoB metabolism, VLDL composition, and the GH-IGF-I axis during euglycemia in type 1 diabetes. Using a stable isotope technique, VLDL apoB kinetics were estimated before and after 1 wk of IGF-I therapy in 12 patients with type 1 diabetes in a double-blind, placebo-controlled trial. Fasting plasma triglyceride (P < 0.03), VLDL-triglyceride concentrations (P < 0.05), and the VLDL-triglyceride-to-VLDL apoB ratio (P < 0.002) significantly decreased after IGF-I therapy, whereas VLDL apoB kinetics were not significantly affected by IGF-I therapy. IGF-I therapy resulted in a significant increase in IGF-I and a significant reduction in GH concentrations. The mean overnight insulin concentrations during euglycemia decreased by 25% after IGF-I therapy. These results indicate that low-dose IGF-I therapy restores the GH-IGF-I axis in type 1 diabetes. IGF-I therapy changes fasting triglyceride concentrations and VLDL composition probably because of an increase in insulin sensitivity.     

Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT

We have separated the effect of insulin on glucose distribution/transport, glucose disposal, and endogenous production (EGP) during an intravenous glucose tolerance test (IVGTT) by use of a dual-tracer dilution methodology. Six healthy lean male subjects (age 33 +/- 3 yr, body mass index 22.7 +/- 0.6 kg/m(2)) underwent a 4-h IVGTT (0.3 g/kg glucose enriched with 3-6% D-[U-(13)C]glucose and 5-10% 3-O-methyl-D-glucose) preceded by a 2-h investigation under basal conditions (5 mg/kg of D-[U-(13)C]glucose and 8 mg/kg of 3-O-methyl-D-glucose). A new model described the kinetics of the two glucose tracers and native glucose with the use of a two-compartment structure for glucose and a one-compartment structure for insulin effects. Insulin sensitivities of distribution/transport, disposal, and EGP were similar (11.5 +/- 3.8 vs. 10.4 +/- 3.9 vs. 11.1 +/- 2.7 x 10(-2) ml small middle dot kg(-1) small middle dot min(-1) per mU/l; P = nonsignificant, ANOVA). When expressed in terms of ability to lower glucose concentration, stimulation of disposal and stimulation of distribution/transport accounted each independently for 25 and 30%, respectively, of the overall effect. Suppression of EGP was more effective (P < 0.01, ANOVA) and accounted for 50% of the overall effect. EGP was suppressed by 70% (52-82%) (95% confidence interval relative to basal) within 60 min of the IVGTT; glucose distribution/transport was least responsive to insulin and was maximally activated by 62% (34-96%) above basal at 80 min compared with maximum 279% (116-565%) activation of glucose disposal at 20 min. The deactivation of glucose distribution/transport was slower than that of glucose disposal and EGP (P < 0.02) with half-times of 207 (84-510), 12 (7-22), and 29 (16-54) min, respectively. The minimal-model insulin sensitivity was tightly correlated with and linearly related to sensitivity of EGP (r = 0.96, P < 0.005) and correlated positively but nonsignificantly with distribution/transport sensitivity (r = 0.73, P = 0.10) and disposal sensitivity (r = 0.55, P = 0.26). We conclude that, in healthy subjects during an IVGTT, the two peripheral insulin effects account jointly for approximately one-half of the overall insulin-stimulated glucose lowering, each effect contributing equally. Suppression of EGP matches the effect in the periphery.