T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Growth of Odontesthes bonariiensis (Atherinidae) larvae feeding on suboptimal zooplankton densities

Synopsis In this study we investigate the effect of food availability (zooplankton biomass) on the growth of Odontesthes bonariensis (Atherinidae) larvae. The larvae were stocked in four 45 m² outdoor tanks at relatively high densities (100 and 200 larvae m^–2). Because of the high stocking densities, the zooplankton biomass was depleted in all tanks. However, the patterns of food limitation, and particularly periods of severe food shortage, differed in tanks stocked at different densities. We could therefore, observe the effect of food limitation in larvae that differed in weight and age. The effects of variables suspected to influence O. bonariensis growth rates (age and weight of larvae, available zooplankton biomass, mean individual weight of available preys, total ingested prey weight, and mean weight of ingested preys) were investigated using standard multiple regression methods, and a model assuming: (1) an allometric relationship between maximum growth rates and weight of larvae, and (2) an inverse relationship between growth depression and the available zooplankton biomass. Both methods were consistent in showing that only the weight of larvae, and the availability of zooplankton prey had significant effects on the growth of O. bonariensis. The model's results additionally suggest that, if the observed growth rates are scaled by the maximum growth rate corresponding to the larva weight, the effect of zooplankton biomass is largely independent of age and weight of larvae.     

A study of 28 Elymus species using repetitive DNA sequences.

Four repetitive DNA sequences cloned from the barley (Hordeum vulgare) genome and common for different Triticeae species were used for a molecular study of phylogenetic relationships among 28 Elymus species. Two wild Hordeum species (H genome), two Pseudoroegneria species (S genome), Agropyron cristatum (P genome), and Australopyrum velutinum (W genome) were included as genomic representatives for the genomes that supposedly were involved in the evolution of the genus Elymus. Our results are essentially congruent with the genomic classification system. This study demonstrates that Elymus is not a monophyletic genus. Based on an analysis of Southern blot hybridization we could discriminate between SY and SH species owing to the strong specific hybridization pattern of the H genome. Hexaploid SYH species gave a hybridization pattern similar to SH species for the same reason. The results support the genomic composition of Elymus batalinii as SYP and also indicated the presence of at least one H genome in Elymus enysii with a hitherto unknown genomic constitution. Elymus erianthus had a hybridization pattern distinctly different from all other species in the investigation. Key words : Elymus, RFLP, phylogeny, repetitive DNA.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Probability of mast cells to mediate anaphylaxis in skeletal muscle

Are there enough mast cells in denervated skeletal muscle to account for autopharmacological mediation of the antigen potentials (APs) elicited by microtaps? Through rough qualitative estimations, some authors have suggested a positive answer to this question. However, in view of measurements performed in this investigation of both the density of mast cells and the diffusion coefficient of antigens, the probability of such mediated effects was found to be relatively low:P=0.016 for egg albumin andP=0.004 for ferritin. Therefore, most APs induced by microtaps should be attributed to the direct effect of antigen over the sensitized muscle fibers. Yet, both the density of mast cells found in this work and the known amount of histamine they are capable of releasing when challenged with antigen, support the hypothesis regarding the involvement of these cells when antigen is massively superfused so as to induce Schultz-Dale reactions in muscle strips. Under this circumstance, the direct and mediated mechanisms may coexist.     

Root/Shoot Allocation and Root Architecture in Seedlings: Variation among Forest Sites, Microhabitats, and Ecological Groups1

I analyzed patterns of variation in root mass allocation and root morphology among seedlings of woody species in relation to environmental factors in four Neotropical forests. Among forests, I explored the response of root traits to sites varying in water or nutrient availability. Within each forest, I explored the plastic response of species to different microhabitats: gaps and understory. Additionally, I explored evidence for life history correlation of root and shoot traits by comparing species differing in their successional group (light‐demanding [22 spp.] or shade tolerant [27 spp.]) and germination type (species with photosynthetic cotyledons or species with reserve cotyledons). At each forest site, young seedlings from 10 to 20 species were excavated. A total of 55 species was collected in understory conditions and 31 of them were also collected in gaps. From each seedling, six morphological ratios were determined. Allocation to roots was higher in forest sites with the lowest soil resources. Roots were finer and longer in the most infertile site, while roots were deeper in the site with the longest dry season. Seedling traits did not differ between germination types. Shade tolerant species allocated more to roots and developed thicker roots than light‐demanding species. Light‐demanding species showed stronger plastic responses to habitat than shade tolerant species, and species with photo‐synthetic cotyledons showed lower plasticity than species with reserve cotyledons. Overall, these results suggest that among Neotropical species, root allocation and root morphology of seedlings reflect plant adjustments to water or nutrient availability at geographic and microhabitat scales. In addition, life history specialization to light environments is suggested by differences among groups of species in their allocation to roots and in their root morphology.     

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt^c). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt^c activity toward LexA have little or no effect on the coprt^c activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.