The antigen-binding T cell factor PCl-F sensitizes mast cells for in vitro release of serotonin. Comparison with monoclonal IgE antibody

Picryl chloride factor (PC1-F) is an antigen (TNP hapten)-binding T cell factor that initiates PC1 contact sensitivity (CS). PC1-F initiates PC1 CS by mediating an early 2-h skin swelling reaction that is due to local release of the vasoactive amine serotonin (5-HT) by mast cells, and perhaps other 5-HT-containing cells. Experiments were conducted to determine if PC1-F could sensitize normal mast cells in vitro for subsequent release of 3H-5-HT that had been taken up previously. It was found that PC1-F could sensitize mast cells, inasmuch as incubation with PC1-F, followed by washing, resulted in the ability to release 5-HT by challenge with Ag (TNP-bovine serum albumin), or by an anti-factor mAb called 14-30. As with release induced by anti-TNP IgE mAb PC1-F-induced release required phosphatidyl serine. Mast cell sensitization and activation for 5-HT release by PC1-F was not due to contamination of PC1-F with IgE antibody, because IgE (and not PC1-F) was sensitive to reduction and alkylation. Also, affinity columns linked with 14-30 or anti-IgE showed that the mast cell sensitizing and activating property of PC1-F was clearly separate from that of IgE. PC1-F-induced release was not IgE dependent, because mast cells that were acid-stripped and largely depleted of surface IgE, could then be sensitized by PC1-F. In vivo experiments demonstrated that local challenge with 14-30 antibody induced a 2-h ear swelling reaction in actively contact sensitized mice, or adoptive recipients of sensitized cells, and in normal mice that received PC1-F i.v. These findings suggest that in vitro sensitization of mast cells with PC1-F, and subsequent in vitro release of 5-HT induced by challenge with 14-30 antibodies, correlates with the initiation of PC1 CS in vivo. Therefore, in the initiation of CS by PC1-F, mast cells can be one source of 5-HT, to cause the early, vasoactive phase of CS.     

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.     

Haemostatic variables associated with diabetes and its complications.

To study the possible role of an "increased thrombotic tendency" in the vascular complications of diabetes several tests of haemostatic function were carried out on 91 men and 63 women with diabetes aged 35-54 years and the results compared with findings in 686 men and 393 women of the same age in the Northwick Park Heart Study. Mean values for factors VII and X, fibrinogen, and platelet adhesiveness were higher in the diabetics, but mean fibrinolytic activity and whole blood platelet counts were lower. Antithrombin III values were also higher in the diabetics, which may have constituted a protective response to other changes favouring the onset of vascular disease. Diabetics with retinopathy had higher factor VII and antithrombin III values, and those with proteinuria had higher values for factor VII, fibrinogen, and platelet adhesiveness than those without these complications. These findings suggest a potentially important association between a thrombogenic tendency and vascular disease in diabetes. Nevertheless, prospective data are needed to clarify whether the haemostatic abnormalities precede the onset of clinically manifest vascular complications or are a consequence of them.     

Use ofH-2 mutations to quantitate alloreactivity: Alloreactivity is strongest against H-2 antigens which are closest to self

Lymph-node cells fromH-2 allogeneic, intra-H-2 recombinant andH-2 mutant congenic strains were sensitized in limiting dilution cultures to quantitate the cytotoxic T-lymphocyte precursor frequencies (CTL.Pf) against antigens encoded by different regions of theH-2 complex. When fourH-2K ^b mutants of C57BL/6 (B6) were tested, we observed anti-B6 CTL.Pf that were as high or higher than those of recombinant strains which differ from B6 at theK end of theH-2 complex. Relative to strains completelyH–2 allogeneic to B6, the CTL.Pf inH-2 ^bm1,H-2 ^bm3 andH-2 ^bm5 averaged 40–50 percent, andH-2 ^bm8 averaged 140 percent. Recombinant strains B10.A (4R) and B10.D2 (R103), which differ from B6 at theK end of theH-2 complex, averaged 60 percent of the completelyH-2 allogeneic value. Since the mutant and wild-type gene products have no serological and minimal structural differences relative to other alleles atH-2K, these results indicate that the CTL.Pf does not increase with increasing H-2 antigenic disparity between any two strains. Rather, the data suggests that the T-cell receptor repertoire recognizes those H-2 molecules or determinants closest to self.     

Lectin-like activity from Persea americana

An extract from the seeds of Persea americana possessed an erythro-agglutinating activity. The agglutinin was devoid of specificity for carbohydrates, but interacted readily with basic proteins or basic polyamino acids. The interaction between the agglutinin and egg-white lysozyme was not inhibited by chaotropic salts, but was sensitive to relatively low concentrations of urea. An affinity chromatographic procedure was developed in an effort to purify the agglutinin. Products from the chromatographic procedure were found not to contain higher specific agglutinating activities than the crude extract. Amino acid acid analyses of the extract showed the presence of relatively high proportions of glutamic and aspartic acids. In addition, the extract contained phosphorus and a visible chromophore. The agglutinin was resistant to detergents and denaturants, and proteases, nucleases, and other enzymes. The results suggest that, as opposed to other plant agglutinins, the active component from Persea is not a protein. Similarly, in contrast to many lectins, the agglutinin from Persea was not mitogenic for mouse lymphocytes. The agglutinin partially inhibited the mitogenesis of lymphocytes when the cells were treated with concanavalin A, or with bacterial lipopolysaccharide.     

Preparation and Standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

The Center for Biologics Evaluation and Research within the U.S. Food and Drug Administration has prepared a new U.S. Standard Pertussis Vaccine. Whole cell pertussis vaccine concentrate was diluted in 5% (w/v) lactose and lyophilized. The preparation was tested for toxicity, sterility heterogeneity and residual moisture. Based on data from an international sollaborative study involving 11 laboratories, the potency was estimated in relation to the U.S. Master Standard Pertussis Vaccine, Lot 4 and the International Standard for Pertussis Vaccine, Lot 2. The potency of the preparation was defined to be 90 units per ampoule. When reconstituted and stored according to instructions, no significant change in potency was observed in the 14 days following reconstitution. This material was shown to be suitable for a pertussis vaccine standard and accordingly it was designated as U.S. Standard Pertussis Vaccine, Lot 11 on March 22, 1994.     

INTRASPECIFIC PHYLOGEOGRAPHY OF NORTH AMERICAN HIGHLAND FISHES: A TEST OF THE PLEISTOCENE VICARIANCE HYPOTHESIS

The highland fish fauna of eastern North America consists of Appalachian and Ozark centers of endemism separated by the intervening Glacial Till Plains. Clades within these areas are more closely related phylogenetically to each other than to clades occurring in the intervening formerly glaciated region, suggesting that the Pleistocene glaciations fragmented a widespread highland region and its associated fauna. Alternatively, it is possible that these faunal assemblages predate the glaciations or that recent dispersals may have been more important than vicariance in determining faunal compositions. We examined the relationships among mitochondrial DNA (mtDNA) haplotypes within five clades of highland fishes, each with a distribution suggestive of a Pleistocene vicariance event. Darters of the subgenera Litocara and Odontopholis have distributions and mtDNA relationships that are consistent with the Pleistocene integration and burial of the Teays-Mahomet valley, a major drainage of the early Pleistocene. The distribution and mtDNA relationships among subspecies of Erimystax dissimilis are not consistent with Pleistocene vicariance, but relationships among Appalachian haplotypes are consistent with the late Pleistocene integration of the modern Ohio River system. Both Cottus carolinae and the Fundulus catenatus species group have representatives in the Mobile basin consistent with pre-Pleistocene divergences. Three haplotype clusters were found in C. carolinae, corresponding to the Appalachian, Ozark, and upper Kanawha River populations. However, Appalachian and Ozark F. catenatus populations are paraphyletic with respect to each other. This, coupled with a relatively low degree of sequence divergence, suggests that no long-term barriers to gene flow exist for C. carolinae and F. catenatus. These three distinct phylogeographic patterns indicate that Pleistocene vicariance is not the only explanation for the Appalachian-Ozark distribution of highland fish communities.     

Expression of cryptopleurine resistance in Saccharomyces cerevisiae.

An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.