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.     

Endogenous opiates: 1986

This is the ninth installment of our annual review of research involving the endogenous opiate peptides. It is restricted to the non-analgesic and behavioral studies of the opiate peptides published in 1986. The specific topics this year include stress; tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic processes; mental illness; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; activity; sex, pregnancy, and development; and some other behaviors.     

The chlorophyll a/b-binding protein inserts into the thylakoids independent of its cognate transit peptide

In order to determine if the cognate transit peptide of the light-harvesting chlorophyll a/b-binding protein (LHCP) is essential for LHCP import into the chloroplast and proper localization to the thylakoids, it was replaced with the transit peptide of the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase, a stromal protein. Wheat LHCP and S genes were fused to make a chimeric gene coding for the hybrid precursor, which was synthesized in vitro and incubated with purified pea chloroplasts. My results show that LHCP is translocated into chloroplasts by the S transit peptide. The hybrid precursor was processed; and most importantly, mature LHCP did not remain in the stroma, but was inserted into thylakoid membranes, where it normally functions. Density gradient centrifugation showed no LHCP in the envelope fraction. Hence, the transit peptide of LHCP is not required for intraorganellar routing, and LHCP itself contains an internal signal for localization to the correct membrane compartment.     

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet.     

Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain

We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N-terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N-terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction.     

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.

---

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.

---

---

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.     

Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.     

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