An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Closure of a genetic linkage map of human chromosome 7q with centromere and telomere polymorphisms.

We have constructed a 2.4-cM resolution genetic linkage map for chromosome 7q that is bounded by centromere and telomere polymorphisms and contains 66 loci (88 polymorphic systems), 38 of which are uniquely placed with odds for order of at least 1000:1. Ten genes are included in the map and 11 markers have heterozygosities of at least 70%. This map is the first to incorporate several highly informative markers derived from a telomere YAC clone HTY146 (locus D7S427), including HTY146c3 (HET 92%). The telomere locus markers span at least 200 kb of the 7q terminus and no crossovers within the physical confines of the locus were observed in approximately 240 jointly informative meioses. The sex-equal map length is 158 cM and the largest genetic interval between uniquely localized markers in this map is 11 cM. The female and male map lengths are 181 and 133 cM, respectively. The map is based on the CEPH reference pedigrees and includes over 4000 new genotypes, our previously reported data plus 29 allele systems from the published CEPH version 5 database, and was constructed using the program package CRI-MAP. This genetic linkage map can be considered a baseline map for 7q, and will be useful for defining the extent of chromosome deletions previously reported for breast and prostate cancers, for developing additional genetic maps such as index marker and 1-cM maps, and ultimately for developing a fully integrated genetic and physical map for this chromosome.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

Differential Expression of Two Soybean (Glycine max L.) Proline-Rich Protein Genes after Wounding

We have investigated the wound-induced expression of two members of the soybean (Glycine max L.) proline-rich cell wall protein gene family and show that SbPRP1 and SbPRP2 exhibit unique patterns of expression after physical damage. SbPRP1 mRNA can be detected in the hook of soybean seedlings within 2 h after wounding and is present at high levels in the hook and elongating hypocotyl 20 h after wounding. In contrast, SbPRP2 mRNA increases transiently and rapidly throughout the soybean seedling after wounding. SbPRP2 is also induced by wounding in soybean leaves, but the pattern of mRNA accumulation in leaves is distinct from that seen in seedlings and reaches high levels of expression 20 h after physical damage. SbPRP2 mRNA levels were also found to increase in the mature hypocotyl and roots of seedlings in response to treatment with 10 [mu]M indoleacetic acid and naphthalene-1-acetic acid. These data indicate that the wound-induced expression of PRPs in soybean is tissue specific and that the regulation of these genes after physical damage may operate through different signal transduction pathways.     

Persistence of poliovirus 1 in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent.

Land disposal of sewage sludge and effluent is becoming a common practice in the United States. The fertilizer content and humus value of such wastes are useful for agricultural purposes, and the recycling of sewage onto the land eliminates many of our stream pollution problems. The potential exists for crops grown in such irrigated soil to be contaminated by viruses that may be present in the sewage. Studies were initiated to determine viral persistence in soil and on crops grown under natural conditions in field plots that had been flooded to a depth of 1 inch (2.54 cm) with poliovirus 1-inoculated sewage wastes. Lettuce and radishes were planted in sludge- or effluent-flooded soil. In one study, the vegetables were planted 1 day before flooding, and in another they were planted 3 days after the plots were flooded. Survival of poliovirus 1 in soil irrigated with inoculated sewage sludge and effluent was determined during two summer growing seasons and one winter period. The longest period of survival was during the winter, when virus was detected after 96 days. During the summer, the longest survival period was 11 days. Poliovirus 1 was recovered from the mature vegetables 23 days after flooding of the plots had ceased. Lettuce and radishes are usually harvested 3 to 4 weeks after planting.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.