Human helper T-cell clones that recognize different influenza hemagglutinin determinants are restricted by different HLA-D region epitopes

Human T-lymphocyte clones (TLCs) were generated against the hemagglutinin (HA) of A/Texas/1/77 influenza virus by limiting dilution. TLCs were then screened for antigen specificity on chemically synthesized peptides representing the HA1 molecule. It has been hypothesized that different T cells that recognize the identical antigenic determinant are controlled by (restricted by) the same class II epitope. Two TLCs, HA1.4 and HA1.7, both recognized the same HA peptide and in proliferation studies exhibited identical restriction patterns. Two other clones, HA 1.9 and HA 2.43, recognized different HA determinants and also had distinct restriction patterns. Proliferation inhibition studies with monoclonal antibodies against human class II molecules demonstrated three unique patterns of blocking with the clones, suggesting that clones may be restricted to a unique class II epitope depending on the HA determinant recognized. These data can be interpreted as supporting the argument that human immune responses to influenza hemagglutinin are under Ir gene control exerted at the level of the viral antigenic determinant recognized in association with particular D-region restricting elements. The determinant selection and clonal deletion theories are compared for their capacity to best explain these findings.Abbreviations used in this paper ³HTdR tritiated methyl thymidine - MHC major histocompatibility complex - HLA human MHC - PBLs peripheral blood lymphocytes - APCs antigen-presenting cells - TLCs T-lymphocyte clones - TCGF T-cell growth factor - MoAbs monoclonal antibodies     

Distribution of Daily Breathing Rates For Use in California's Air Toxics Hot Spots Program Risk Assessments

Data were combined from a study measuring breathing rates at various activities and two activity pattern studies to generate breathing rate distributions for children and adults. The children and adult breathing rate distributions were combined using a Monte Carlo technique to generate a breathing rate distribution for a lifetime spanning ages 0 to 70. The children's breathing rate distribution has a mean, standard deviation, median and 95^th percentile of 452, 67.7, 441, and 581 L/kg-day, respectively. The adult breathing rate distribution has a mean, standard deviation, median and 95^th percentile of 232, 64.6, 209, and 381 L/kg-day, respectively. The simulated 70-year distribution has a mean, standard deviation, median and 95^th percentile of 271, 57.9, 253, and 393 L/kg-day, respectively. The adult breathing rate distribution is based on 24-hour recall activity data that would not necessarily capture average activity patterns and therefore breathing rates. We utilized the human energy expenditure literature to validate the breathing rate distribution. We conclude that the breathing rate distribution is reasonable for chronic long-term risk assessment in California's Air Toxics Hot Spots program.     

Tautomeric forms study of 1H-(2′-pyridyl)-3-methyl-5-hydroxypyrazole and 1H-(2′-pyridyl)-3-phenyl-5-hydroxypyrazole. Synthesis,structure, and cytotoxic activity of their complexes with palladium(II) ions

In this article the synthesis of new 1H-(2′-pyridyl)-3-methyl-5-hydroxypyrazole and 1H-(2′-pyridyl)-3-phenyl-5-hydroxypyrazole complexes with palladium(II) ions is reported. The structures of obtained compounds have been characterized by X-ray crystallography and DFT (density functional theory) calculations. The cytotoxicity of complexes and ligands has been examined for two human leukemia cell lines (HL-60 and NALM-6) and one human melanoma cell line (WM-115). The palladium(II) complex with 1H-(2′-pyridyl)-3-phenyl-5-hydroxypyrazole has been shown to possess greater activity than carboplatin against the WM-115 melanoma cell line. Additionally, the ligands’ tautomeric forms existence in different solvents (chloroform, methanol, DMSO) has been characterized by ¹H nuclear magnetic resonance (NMR) analysis and DFT calculations. The obtained results have been compared with those from other studies of similar compounds.     

Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation

The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.     

The patterns and distribution of euchrysine-binding grains in nonstimulated human peripheral T and non-T lymphocytes.

The patterns of supravital staining with euchrysine, a fluorescent stain thought to bind selectively to lysosomal membranes, were evaluated in resting human lymphocytes separated on the basis of their ability to form spontaneous rosettes with sheep red blood cells in thymus-dependent (T) and thymus-independent (non-T) subpopulations. Two basic staining patterns were found in unseparated lymphocyte populations: type I, small fluorescent granules in a conglomerate form, usually located in a single spot close to the cell membrane; type II, discrete fluorescent granules dispersed over the entire cytoplasm. The overwhelming majority of non-T lymphocytes displayed the type II pattern. Within the T-cell subpopulations both type I and type II patterns were found in proportions which were subject to donor-to-donor variability.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Uveal melanoma cell-based vaccines express MHC II molecules that traffic via the endocytic and secretory pathways and activate CD8+ cytotoxic, tumor-specific T cells

We are exploring cell-based vaccines as a treatment for the 50% of patients with large primary uveal melanomas who develop lethal metastatic disease. MHC II uveal melanoma vaccines are MHC class I⁺ uveal melanoma cells transduced with CD80 genes and MHC II genes syngeneic to the recipient. Previous studies demonstrated that the vaccines activate tumor-specific CD4⁺ T cells from patients with metastatic uveal melanoma. We have hypothesized that vaccine potency is due to the absence of the MHC II-associated invariant chain (Ii). In the absence of Ii, newly synthesized MHC II molecules traffic intracellularly via a non-traditional pathway where they encounter and bind novel tumor peptides. Using confocal microscopy, we now confirm this hypothesis and demonstrate that MHC II molecules are present in both the endosomal and secretory pathways in vaccine cells. We also demonstrate that uveal melanoma MHC II vaccines activate uveal melanoma-specific, cytolytic CD8⁺ T cells that do not lyse normal fibroblasts or other tumor cells. Surprisingly, the CD8⁺ T cells are cytolytic for HLA-A syngeneic and MHC I-mismatched uveal melanomas. Collectively, these studies demonstrate that MHC II uveal melanoma vaccines are potent activators of tumor-specific CD4⁺ and CD8⁺ T cells and suggest that the non-conventional intracellular trafficking pattern of MHC II may contribute to their enhanced immunogenicity. Since MHC I compatibility is unnecessary for the activation of cytolytic CD8⁺ T cells, the vaccines could be used in uveal melanoma patients without regard to MHC I genotype.     

The relation between cell division rate and 3 HTdR incorporation in organ culture

Rates of incorporation of labelled thymidine (RIT), radioautographic labelling index for DNA synthesis (LI) and mitotic incidence following colcemid metaphase arrest (MI) were measured in organ cultures of newborn and adult rat lung. In adult cultures these three parameters correlated well, being low after explantation and reaching a maximum after two to three days. In newborn cultures RIT fell several fold over the first 24 hours after explantation and, in this respect, did not correlate with LI and MI. The changes in RIT over the first 24 hours appear to be due to changes in the degree of competition between endogenous TdR and exogenous labelled TdR, probably caused by leakage of the intracellular thymidine pool following explantation. The report emphasizes the need to check RIT data against radioautographic evidence before accepting it as an index of DNA synthesis.     

Mycotoxin induction of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures

Thegenotoxic activity of four mycotoxins has been studied. A high level of somatic mutagenesis in imaginal disks of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures was induced only by the strong carcinogen aflatoxin B₁. Patulin somewhat elevated the level of somatic mutations in D. melanogaster, but did not elicit DNA repair synthesis. Citrinin and stachybotryotoxin were inactive in both systems.Abbreviations AFB₁ aflatoxin B₁ - DMSO dimethylsulfoxide - ³HTdR tritiated thymidine - SCE sisterchromatid exchange - UDS unscheduled DNA synthesis     

Cation transport in different volume populations of genetically low K+ lamb red cells

During the first three months after birth lambs produce sequentially three erthryocyte populations of different mean volume as demonstrated by electric sizing methods (Valet, Franz, and Lauf, J. Cell. Physiol. 94 (1978) 215). We separated by centrifugal elutriation the small volume population (type II) red cells of a genotypically low K⁺ (LK) lamb from the population containing the larger volume type I and III cells, an admixture of fetal (I) and adult (III) erythrocytes. The cells were separated at various time intervals after birth and analyzed with respect to their volumes, cation contents, and cation flux properties by means of ⁸⁶Rb uptake. The effect of anti-L on K⁺ pump and leak fluxes was ascertained in unseparated and separated red cells. It was found that the small red cells of population II, transiently present for several weeks, were fully developed LK cells with K⁺ pumps responding characteristically to the stimulatory action of anti-L. In constrast, the larger cells of population I and III were of high K⁺ (HK) nature at early time points, the K⁺ pump activities approximately ten times higher than adult LK cells. These cells constitute an admixture of type I fetal HK cells, and type III reticulocytes which are precursors for the final type III adult LK cells, since anti-L had a small stimulatory effect. At later times, however, only adult type III LK cells predominated. The data directly support our earlier finding that the HK-LK transition in genotypically LK lambs is primarily governed by cellular replacement.     

A single structural type in the regular surface layer of Aeromonas salmonicida

The surface protein array of Aeromonas salmonicida (or A-layer) appears, in negatively stained preparations, as two distinct patterns, type I and type II. Type I patterns were restricted to, and predominated in, darkly stained areas, whereas lighter staining regions exclusively displayed type II patterns. The type I morphology was faithfully reproduced in computer-simulated superimpositions of type II patterns, as was the intermediate transition zone frequently seen between the two patterns. Variations in the lattice constant of both patterns, presumably due to artifactual flattening, demonstrated that these patterns could not be distinguished on this basis. The conceptual model presented points to the type II pattern as the only single A-layer structural type. We propose the use of the terms type 1/type II to exclusively describe the morphological patterns that appear upon negative staining and the open/closed nomenclature to describe the conformations that a single structural type can adopt.     

X-ray Sensitivity of HeLa S3 Cells in the G2 Phase: Comparison of Two Methods of Synchronization

The sensitivity of HeLa S3 cells to 220 kv X-rays was measured in terms of cell survival (colony development) during the G2 phase of the cell generation cycle, employing two procedures designed to free G2 cultures from contaminating cells from other phases of the cycle. Treatment of synchronous cultures (obtained initially by mitotic selection) with high specific activity tritiated thymidine (HSA-³HTdR) selectively eliminated S phase cells, while addition of vinblastine permitted removal of cells as they entered mitosis. It was found that HeLa S3 cells become increasingly sensitive as they progress through G2. The pattern of sensitivity fluctuations observed in synchronous HeLa S3 populations selected by the foregoing method was compared with that found in synchronous cultures prepared by the HSA-³HTdR method of Whitmore. The latter method had been used previously with mouse L cells, which were found to undergo a different pattern of sensitivity fluctuations. The two methods yield similar results for HeLa cells in the S and G2 phases of the cycle. It may be concluded, therefore, that the discrepancies between HeLa and mouse L cells do not arise from methodological factors, but represent fundamental differences between the cell types.     

13C-NMR study of CO2-fixation during the heterotrophic growth in Chlorobium thiosulfatophilum

The functioning of the biosynthetic pathways of the amino acids alanine, glycine, aspartic acid, glutamic acid and tyrosine, and of nucleosides in the photosynthetic bacterium Chlorobium thiosulfatophilum during heterotrophic growth on ¹³CO₂ and unlabelled acetate was investigated using ¹³C-NMR as the method for determination of the labelling patterns of the separated substances. On the basis of the analysis of the multiplet structure of the spectra of the tightly-coupled systems, the conclusion was drawn that the Calvin cycle does not function in the experimental conditions used. The labelling pattern of the glutamic acid indicated that about 30% of the amino acid molecules were synthesized through the reactions of the reductive carboxylic acid cycle, the remaining 70% being derived from oxaloacetate and exogenous acetate through the reactions of the Krebs cycle. Labelling patterns of the nucleosides were in agreement with their known biosynthetic pathways.     

Chromosomal localizations by in situ hybridization of the repetitious human DNA families and evidence of their satellite DNA equivalents

Four of the five major repetitious human DNA families, have been mapped by the in situ hybridization technique at their T_OPT values. Two of the lighter density DNA families have autoradiographic grain patterns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density DNA families have autoradiographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic T_OPT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag⁺-Cs₂SO₄ gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.     

Spatial and temporal organization of actin during hydration,activation, and germination of pollen inPyrus communis L.: a population study

Summary Dynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.     

Y chromosome replication and chromosome arrangement in germ line cells and sperm of the rat

The chronology of Y chromosome replication in meiosis of male adult rats was investigated. ³HTdR was injected into the testes and animals were sacrificed at 2-hour intervals from 2 to 24 hour after the injection; and at 2-day intervals from 2 to 64 days after the injection. Autoradiograms from germ line cell spreads were prepared. The study of spermatogonial metaphases showed that the Y chromosome is the last to begin and end DNA synthesis. Consequently, by detecting such a pattern of replication it was possible to trace the asynchronous Y from spermatogonia to sperm. Assuming that Y chromosomes are early replicating in preimplantation embryos of mammals it is proposed that Y chromosome of rats shift from late to early replicating in the first divisions of the fertilized egg. Moreover, the analysis of the patterns of sperm labeling allow one to infer that chromosomes are end-to-end associated in sperm nuclei, and that the Y chromosome and perhaps autosomes as well occupy a constant position in sperm of rats.     

Determining natal dispersal patterns in a population of North American pikas (Ochotona princeps) using direct mark-resight and indirect genetic methods

A combination of direct (mark-resight) and indirect geneticmethods were used to investigate natal dispersal patterns andgenetic population structure in a population of North Americanpikas, Ochotona princeps. Pikas are small lagomorphs found intalus habitat of alpine areas throughout western North America.Adult pikas are individually territorial and rarely disperse.I used multilocus DNA fingerprinting to identify the parentsof juvenile animals. The settlement pattern of marked juvenilesand the pattern of relatedness of pikas across the study sitewas then examined within the study area. Although juvenilesborn at the study site exhibited a philopatric settlement pattern,an isolation-by-distance analysis did not reveal clusters ofhighly related individuals within the population. The F_ST estimatesuggests little genetic differentiation between populations2 km apart, and average DNA fingerprinting band-sharing amongadults was similar to values reported for outbred species. Anaverage of 34% of the adult population was replaced each winterby immigrants. DNA fingerprinting band-sharing analysis suggeststhat these immigrants had dispersed short, intermediate, andlong distances. These findings differ from earlier studies whichused observations of marked animals only to characterize dispersalpatterns. Direct observations of marked juveniles had documenteda philopatric settlement pattern, little or no dispersal outof natal populations, and no direct evidence of long distancemovement. Of the three major hypotheses proposed to explainthe evolution of dispersal in birds and mammals, competitionfor resources, competition for mates, and inbreeding avoidance,the results of this study support a competition for resourceshypothesis, where the key resource is territory     

Method For Binary Classification and Risk Assessment of Individuals With Familial Polyposis Based On [3H]Tdr Labelling of Epithelial Cells In Colonic Crypts

Abstract An analysis has been developed to improve the quantitation of abnormal patterns of tritiated thymidine ([³H]TdR) labelling of colonic epithelial cells, in biopsy specimens removed from human subjects at varying degrees of risk for colon cancer. After pulse incubation of specimens of colonic mucosa with [³H]TdR, each subject's microautoradiographic epithelial cell labelling distribution was segregated into eleven compartments over entire colonic crypts. the findings of each subject were then analysed to determine their relative degree of similarity to the findings for two reference populations of interest, i.e. a high-risk and a low-risk population; the individual was then classified as being closer to one or the other of the reference populations. the analysis developed is based upon a comparison of multinomial probabilities for the distributions of the labelled cells within the crypts, and permits the routine categorization of uneven distributions of labelled cells. For each subject, certain linear scores, a prognostic index based on them, and a related presumptive risk, were calculated. the sensitivity with which individuals known to be symptomatic for polyposis, and the specificity with which individuals known to be at lower risk were determined, were 73 and 93% respectively. the results suggest that this method of distinguishing among integer distributions of [³H]TdR- labelled cells in biopsies of colonic mucosa, may provide a useful basis for identifying individuals with familial polyposis, by separating their labelling patterns from those of low-risk subjects.     

The effect of x-rays on labelling pattern of M 1 and M 2 chromosomes in Chinese hamster cells

Chinese hamster cells (line CHEF-125) were cultured for 1 or 4 h in the presence of tritiated thymidine (³HTdR). Immediately after the end of the treatment with ³HTdR or 4 h afterwards, some cultures were irradiated with X-rays, while others served as controls.Analysis of colchicine-C anaphase of M₁ and M₂ cells showed that: (a) in the M₁ chromosomes the X-rays produced a significant departure from a 1:1 ratio of the number of silver grains counted on the sister chromatids; and (b) in the M₂ chromosomes the X-rays increased significantly the frequency of sister-chromatid exchanges and of isolabelling regions.Subchromatid exchanges involving a single polynucleotide strand may be induced by X-rays. These exchanges would cause inequality of labelling over M₁ sister chromatids and isolabelling in the M₂ chromosomes.