Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Construction of interleukin-1 alpha mutants using unequal contamination of synthetic oligonucleotides.

Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants.     

Acute lethality after x-irradiation of Tribolium confusum adults

Recently emerged adults of Tribolium confusum, maintained at 30° in flour-yeast medium, were exposed to X-ray doses between 8 and 75 kR at dose rates of 225 or 1000 R/min. In all cases, there was no significant mortality for about 10 days, and all deaths attributable to the irradiation occurred during the next 10 to 15 days; after the lower doses, many insects survived this critical period and then lived on for many months. The relative dose-independence of the survival-time of decedents suggested that there is a specific mode of death, i.e., an as-yet-unrecognized acute lethal syndrome. Irradiated beetles incubated at 22° exhibited about twice as great a delay in the onset of mortality, and a mortality period with about twice the duration, as those at 30°, suggesting the necessity of a sequence of metabolic events in the development of lethality. Beetles on a cornstarch diet were at least as radioresistant as beetles on flour yeast; beetles at 22° were distinctly more sensitive than those kept at 30°. Older beetles, exposed 4 or 10 months after emergence, showed progressive increases in acute radiosensitivity. Implications of these findings for radiobiological investigations on adult insects are discussed.

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Lethalite aigüe apres irradiation aux rayons X des adultes de Tribolium Confusum

Résumé La plupart des insectes utilisés dans les expériences de radiobiologie ont une longévité relativement brève à l'état adulte. De ce fait, il est difficile ou impossible de distinguer la mortalité aigüe liée à des mécanismes spécifiques d'une diminution nonspécifique de la résistance à un stress.La longévité des adultes de Tribolium rend possible de séparer ces deux catégories de réponses, elle peut aussi permettre la mise en évidence des différents modes de léthalité aigüe.Des adultes récemment formés de Tribolium confusum, maintenus à 30° dans le milieu farine-levure, ont été exposés à des doses de rayons X de 8 à 75 kR aux taux de 225 ou 1000 R/min. Dans tous les cas, il n'y a pas eu de mortalité significative pendant 10 jours, et toutes les morts attribuables à l'irradiation ont eu lieu au cours des 10 à 15 jours suivants; après les doses les plus faibles, beaucoup d'insectes ont survécu à cette période critique et ont alors vécu de nombreux mois. L'indépendance relative par rapport à la dose de la durée de la survie des descendants suggère l'existence d'un mode spécifique de mortalité, ou d'un syndrome léthal aigu.Les insectes irradiés en élevage à 22° ont présenté un délai de début de mortalité environ le double et une durée de la période de mortalité environ double de ceux enregistrés à 30°. Ceci représente un Q₁₀ d'environ 2,3, qui suggère la nécessité d'une succession de phénomènes métaboliques dans le développement de la léthalité.Des insectes sur un régime à base d'amidon de maïs étaient au moins aussi radiorésistants que ceux sur farine et levure; les insectes à 22° furent sans conteste plus sensibles que ceux élevés à 30°. Des coléoptères plus âgés, exposés 4 ou 10 mois après leur formation, ont montré des augmentations progressives dans la radiosensibilité aigüe.Ces résultats ont deux conséquences importantes pour d'autres recherches radiobiologiques sur les insectes adultes. La mise en évidence de l'existence d'un syndrome léthal spécifique et de ses délais de manifestation devrait accélérer l'analyse des facteurs intervenant dans la mort des insectes irradiés; d'autre part dans toute expérimentation radiobiologique sur des insectes adultes, les imagos doivent être d'un âge uniforme et connu.

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This work was supported by U. S. Public Health Service, NIH Grant GM-10208. During 1964–1965, the senior author held a Special Fellowship (1-F3-GM23423), U. S. Public Health Service, at the Department of Zoology, University of Cambridge; he is indebted to Professor C. F. A. Pantin, F. R. S., and other members of the Department, and especially to Dr. George Salt, F. R. S., both for extending use of their facilities and for many illuminating discussions.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene -glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/ meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.