Evidence for two newVibrio anguillarum K antigens

Surface polysaccharides from five strains ofVibrio anguillarum were studied by means of immunoelectrophoretic procedures. The study suggested existence of two new K antigens, displaying cross-reactivity, in strains derived from diseased feral fish. The importance of a detailed serologic characterization of isolates for ecologic and epidemiologic studies ofV. anguillarum is considered.     

Further antigenic analyses of the fish pathogenic bacteriumVibrio anguillarum

TenVibrio anguillarum strains were selected for an immunoelectrophoretic study. Evidence was provided for existence of two new K antigens which displayed cross-reactivity. The importance of an exact characterization of surface antigens inV. anguillarum is considered.     

Structure and expression of elongation factor 1 alpha in tomato.

A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues.     

Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcus flavefaciens

Highly methylated, long-chain celluloses strongly inhibited cellulose degradation by several species of cellulolytic bacteria of ruminal origin. Specifically, the inhibitory effects of methylcellulose on the growth of Ruminococcus flavefaciens FD1 were concentration dependent, with complete inhibition at 0.1% (wt/vol). However, methylcellulose did not inhibit growth on cellobiose or cellulooligosaccharides. Mixtures of methylated cellulooligosaccharides having an average degree of polymerization of 6.7 to 9.5 inhibited cellulose degradation, but those with an average degree of polymerization of 1.0 to 4.5 did not. Similar inhibitory effects by methylcellulose and, to a lesser extent, by methyl cellulooligosaccharides were observed on cellulase activity, as measured by hydrolysis of p-nitrophenyl-beta-d-cellobioside. R. flavefaciens cultures hydrolyzed cellulooligosaccharides to cellobiose and cellotriose as final end products. Cellopentaose and cellohexaose were cleaved to these end products, but cellotetraose was also formed from cellohexaose. Methylcellulose did not inhibit hydrolysis of cellulooligosaccharides. These data are consistent with the presence of separate cellulase (beta-1,4-glucanase) and cellulodextrinase activities in R. flavefaciens.     

Attempt to detect recombination between B-F and B-L genes within the chicken B complex by serological typing,in vitro MLR,and RFLP analyses

In search for recombinants within the chicken major histocompatibility B complex, 1155 animals from crosses between the congenic lines CB (B¹²) and CC (B⁴) were tested with alloantibodies and monoclonal antibodies for the B-F (class I), B-L (class II), and B-G (class IV) antigens and by mixed lymphocyte reaction. The absence of detectable recombination was confirmed by restriction fragment length polymorphism analysis with B-L and B-F probes. Together with previous reports, this indicates that the distance between the B-F and B-L loci is below 0.01 centimorgan.     

Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus

Several disulfide-linked glycoprotein complexes were identified in the envelope of human cytomegalovirus (HCMV). These glycoprotein complexes were fractionated by rate-zonal centrifugation in sucrose density gradients in the presence of detergents. Fractionated glycoproteins and complexes were immunoprecipitated with three different monoclonal antibodies specific for HCMV glycoproteins and a rabbit polyclonal antiserum prepared against detergent-extracted virion and dense-body envelope glycoproteins. Three distinct families of disulfide-linked glycoprotein complexes were observed and designated glycoprotein complex gcI, gcII, and gcIII. The gcI family, recognized by monoclonal antibody 41C2 under nonreducing conditions, consisted of three complexes with approximate molecular masses of 250 to 300, 190, and 160 kilodaltons (kDa). These complexes consistently sediment more rapidly than other HCMV glycoproteins or complexes in sucrose density gradients. Upon reduction of the gcI family, two size classes of glycoproteins with average molecular masses of 93 to 130 and 55 kDa were observed. The gcII family was recognized by monoclonal antibody 9E10. Under nonreducing conditions, as many as six electrophoretic forms were observed for gcII. When reduced, the major component of the gcII family was a heterogeneous glycoprotein designated gp47-52. The gcIII family was recognized by monoclonal antibody 1G6. It consisted of a complex of approximately 240 kDa without reduction of disulfide bonds. When reduced, two glycoprotein size classes with average molecular masses of 145 and 86 kDa were observed. Polyclonal antiserum R-7 reacted strongly with the gcI and gcIII families, but weakly with the gcII family.     

Sib pair linkage analysis of renin gene haplotypes in human essential hypertension

Summary Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1±10.9 years) from 57 families were selected for sustained hypertension (160.7 ± 22.9/99.5 ± 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 ± 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, Hinfi, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 ± 0.6 vs 1.36 ± 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Systemic Induction of Salicylic Acid Accumulation in Cucumber after Inoculation with Pseudomonas syringae pv syringae

Inoculation of one true leaf of cucumber (Cucumis sativus L.) plants with Pseudomonas syringae pathovar syringae results in the systemic appearance of salicylic acid in the phloem exudates from petioles above, below, and at the site of inoculation. Analysis of phloem exudates from the petioles of leaves 1 and 2 demonstrated that the earliest increases in salicylic acid occurred 8 hours after inoculation of leaf 1 in leaf 1 and 12 hours after inoculation of leaf 1 in leaf 2. Detaching leaf 1 at intervals after inoculation demonstrated that leaf 1 must remain attached for only 4 hours after inoculation to result in the systemic accumulation of salicylic acid. Because the levels of salicylic acid in phloem exudates from leaf 1 did not increase to detectable levels until at least 8 hours after inoculation with P. s. pathovar syringae, the induction of increased levels of salicylic acid throughout the plant are presumably the result of another chemical signal generated from leaf 1 within 4 hours after inoculation. Injection of salicylic acid into tissues at concentrations found in the exudates induced resistance to disease and increased peroxidase activity. Our results support a role for salicylic acid as an endogenous inducer of resistance, but our data also suggest that salicylic acid is not the primary systemic signal of induced resistance in cucumber.