The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

MSl3, a multicopy suppressor of mutants hyperactivated in the RAS-CAMP pathway, encodes a novel HSP70 protein of Saccharomyces cerevisiae

The MSI3 gene was isolated as a multicopy suppressor of the heat shock-sensitive phenotype of the iral mutation, which causes hyperactivation of the RAS-cAMP pathway. Overexpression of MSI3 also suppresses the heat shock-sensitive phenotype of the bcyl mutant. Determination of the DNA sequence of MSI3 revealed that MSI3 can encode a 77.4 kDa protein related to the HSP70 family. The amino acid sequence of Msi3p is about 30% identical to that of the Ssalp of Saccharomyces cerevisiae. This contrasts with the finding that members of the HSP70 family generally show at least 50% amino acid identity. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of MSI3. Moreover, the steady-state levels of the MSI3 mRNA and protein were increased upon heat shock. These results indicate that the MSI3 gene encodes a novel HSP70-like heat shock protein. Disruption of the MSI3 gene was associated with a temperature sensitive growth phenotype but unexpectedly, thermotolerance was enhanced in the disruptant.     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994     

Localization of DNA polymerase α on the nuclear membrane in sea urchin embryos

Subnuclear localization of DNA polymerase α was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase α. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm³) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase α associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase α was able to associate with nuclear DNA polymerase α from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase α associates in unfertilized eggs.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Asymmetric perception of twilight affecting diapause induction by the fall webworm Hyphantria cunea

In order to assess the natural photophase effective for controlling the pupal diapause of Hyphantria cunea, larvae were exposed in the long-day season to natural conditions of light (through a window) for a period of 14 hr, 50 min. This photophase included different portions of either the dawn or dusk twilight period. Since the critical photophase was found to be 14 hr 35 min under natural daylight as well as under conditions of artificial light, 50% diapause was expected when the twilight intensity reached the threshold level 15 min after the onset (dawn) or before the end (dusk) of the exposure. The threshold intensities of twilight thus determined showed a significant asymmetry, being about 1 and 10 lux at dawn and dusk, respectively. From this, it was inferred that the photophase under natural conditions would begin about 40 min before sunrise and end about 20 min after sunset. This asymmetry in sensitivity seems to be caused by the conditions (light or dark) to which the larval photo-receptive system has been exposed. The larvae that had been kept under artificial light of 180–200 lux for 14 hr were sensitive to a subsequent 1 hr exposure to 0.5 lux or greater and averted diapause, whereas those held under 9,000 lux failed to avert diapause even when the photophase was supplemented by light of 7.5 lux for 1 hr

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Résumé De facon à évaluer la photophase naturelle efficace pour maîtriser la diapause nymphale de Hyphantria cunea, les chenilles ont été exposées pendant la saison aux jours longs aux conditions naturelles d'éclairement (à travers une fenêtre) pendant une période de 14 h 50 min. Puisqu'il a été établi que la photophase critique est de 14 h 35 min sous éclairement naturel aussi bien qu'artificiel, 50% de diapause était prévu quand l'intensité crépusculaire atteignait le seuil 15 min après le début (aube) ou avant la fin (crépuscule) de l'exposition. Les intensités-seuil crépusculaires ainsi déterminées ont présenté une asymétrie significative, étant respectivement de 1 à 10 lux à l'aube et au crépuscule. Il a été déduit de ces observations que la photophase en conditions naturelles commencerait environ 40 min avant le lever du soleil et se terminerait environ 20 min après son coucher. L'asymétrie de cette sensibilité semble être due aux conditions (lumière ou obscurité) auxquelles le système photo-récepteur des chenilles a été exposé. Les chenilles qui ont été maintenues pendant 14 h sous éclairage artificiel de 180–200 lux sont sensibles à une exposition ultérieure de 0,5 lux ou plus et évitent la diapause, tandis que celles maintenues à 9000 lux ou plus n'évitent pas la diapause quand la photophase est prolongée par un éclairement de 7,5 lux pendant une heure.