Physiologic expression of two superantigens in the BDF1 mouse.

The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits.

Although Freund's adjuvant has been used for decades as an immune enhancer in rabbits, adverse physiologic side effects have prompted the search for more suitable alternatives. We used osteocalcin, a bovine bone protein (M.W. 5,800), as the test antigen to evaluate four adjuvant regimens: a) primary inoculation with complete Freund's adjuvant (CFA) followed by three boosts with incomplete Freund's adjuvant (IFA), b) four serial inoculations with RIBI MPL+TDM+CWS adjuvant, c) four serial inoculations with TiterMax #R-1, and d) primary inoculation (only) with TiterMax #R-1. The antibody yield associated with the CFA/IFA regimen (mean OD = 2.152) was at least sixfold that of either TiterMax (mean OD = 0.358) or RIBI (mean OD = 0.239) multiple injection regimens. No antibody response was observed after the single injection of TiterMax antigen emulsion. Maximal antibody production occurred rapidly in response to Freund's adjuvant (day 31) as compared with TiterMax (day 74) and RIBI (day 66).     

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.     

Predicting the distribution of plant communities using annual rainfall and elevation: an example from southern Africa

Abstract. Using the results of a total floristic survey of two veld types (Arid Sweet Bushveld and Mixed Bushveld) in the northeastern Transvaal, South Africa, we linked median annual rainfall from a surface response model to each of 139 samples. The samples had been classified floristically into 15 plant communities. These communities represent two broad divisions, corresponding with the concepts embodied in the two veld types. Using contingency tables, we defined the conditions of median annual rainfall and elevation for each of the veld types. Using a geographic analysis system we predicted the distribution of the veld types in an area of 120 000 km² outside the study area. The predicted distribution was validated by comparison with a digitized version of the Acocks map. We conclude that the defined conditions of median annual rainfall and elevation provide confident criteria for the definition of these veld types.     

Effect of crab effluent and scent of damaged conspecifics on feeding,growth, and shell morphology of the Atlantic dogwhelk Nucella lapillus (L.)

Juvenile Nucella lapillus of two different shell phenotypes, exposed shore and protected shore, were maintained in running seawater under each of three experimental conditions for 94 d: a) laboratory control, b) exposed to the effluent of crabs (Cancer pagurus) fed frozen fish (fish-crab), and c) exposed to the effluent of crabs fed live conspecific snails (snail-crab). Rates of barnacle consumption and rates of body weight change varied significantly between phenotypes and among experimental conditions. Individuals from the protected-shore consumed consistently fewer barnacles and grew consistently less than those from the exposed shore. Body weight increases in the fish-crab treatments were from 25 to 50% less than those in the controls and body weights in the snail-crab treatment either did not change or actually decreased. The perceived risk of predation thus appears to have a dramatic effect on the rates of feeding and growth of N. lapillus.At the end of the experiment, size-adjusted final shell weights for both phenotypes were consistently higher than controls (no crab) in both the fish-crab and snail-crab treatments. In addition, apertural tooth height, thickness of the lip, and retractability (i.e. the extent to which a snail could withdraw into its shell), with few exceptions all varied in an adaptive manner in response to the various risk treatments. Similar changes in the shell form of starved snails exposed to the same stimuli suggest very strongly that the morphological responses of both phenotypes were not just due to differences in rates of growth. These differences, at least in part, represented a direct cueing of the shell form of Nucella lapillus to differences in the perceived risk of predation. Somewhat surprisingly, the extent of phenotypic plasticity appeared to differ between the populations examined. Both field and laboratory evidence suggest that the exposed-shore population was much more labile morphologically than the protected-shore population.In many instances, particularly among starved snails, the development of antipredatory shell traits was greater in the fish-crab treatment than in the snail-crab treatment. Because the scent of crabs was present in both treatments, these results suggest a) that, at the frequency/concentration used in the experiments, the scent of damaged conspecifics may have been a supernormal stimulus and b) that the morphological response in these treatments might have been greater if the stimulus had been provided at a lower level.     

Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock.

The relationship between vascular tone and the induction by endotoxin of a nitric oxide (NO) synthase was studied in vitro in rings of rat thoracic aorta. In rings with and without endothelium there was a time-dependent induction of NO synthase accompanied by both spontaneous and L-arginine-induced relaxation and by reduced contractility to phenylephrine. These effects, which were attributable to the presence of endotoxin in the Krebs' buffer, were attenuated by cycloheximide, polymyxin B and inhibitors of NO synthase. Furthermore, dexamethasone inhibited the induction of NO synthase and the consequent effects on vascular tone. These findings indicate that prevention of the induction of NO synthase by glucocorticoids may be an important component of their therapeutic action.