TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.     

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.     

RFLP maps of potato and their alignment with the homoeologous tomato genome

Summary An RFLP linkage map of the potato is presented which comprises 304 loci derived from 230 DNA probes and one morphological marker (tuber skin color). The self-incompatibility locus of potato was mapped to chromosome I, which is homoeologous to tomato chromosome I. By mapping chromosome-specific tomato RFLP markers in potato and, vice versa, potato markers in tomato, the different potato and tomato RFLP maps were aligned to each other and the similarity of the potato and tomato genome was confirmed. The numbers given to the 12 potato chromosomes are now in accordance with the established tomato nomenclature. Comparisons between potato RFLP maps derived from different genetic backgrounds revealed conservation of marker order but differences in chromosome and total map length. In particular, significant reduction of map length was observed in interspecific compared to intraspecific crosses. The distribution of regions with distorted segregation ratios in the genome was analyzed for four potato parents. The most prominent distortion of recombination was found to be caused by the self-incompatibility locus.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids

The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively. C. paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R. glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced. The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments. A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.     

Localization of substance P-like immunoreactive fibers in the thoracic spinal cord of guinea pig

Summary A dorsal-horn fiber system is revealed in the thoracic spinal cord of guinea pig by means of substance P immunocytochemistry. This system has repeated craniocaudal and/or caudo-cranial extensions and possesses five main components: (1) a superficial network, situated beneath the dorsolateral surface of the spinal cord. This network is connected with the dorsal root fibers and the accumulations of substance P-like immunoreactive (SP-LI) fibers in the Lissauer's tract; (2) an accumulation of SP-LI fibers in the Lissauer's tract at the border of the dorsal horn; (3) two collateral SP-LI fascicles (one lateral and one medial) emerging from the SP-LI fiber accumulation in the Lissauer's tract; (4) a transversal fascicle running through laminae III–V, and (5) an SP-LI network in the region of the lateral spinal cord nucleus. These components of the dorsal-horn fiber system show widespread connections with ipsi-and contralateral spinal cord areas, connecting them in cranio-caudal and/or caudo-cranial directions. The SP-LI dorsal-horn system has close relationship with groups of preganglionic sympathetic cells in the intermediate zone of the spinal cord, respective with the vegetative network of this zone. It is suggested that some fibers of the dorsal-horn system that originate from dorsal-root ganglia may represent primary sensory or visceral afferents. It is likely that the dorsal-horn fiber system and the vegetative network of the thoracic spinal cord may represent the morphological basis for the integration of (1) the central and peripheral vegetative nervous systems, and (2) the somatic and vegetative nervous system.     

Distribution of intrapleural and intravenous Corynebacterium parvum in humans; 99mTc−, and 131I-labeled bacteria

Summary The distribution of Corynebacterium parvum labeled with ¹³¹iodine or ^99mtechnetium was studied in 17 patients with bronchogenic carcinoma. The labeled bacteria were given intravenously or intrapleurally and monitored by whole-body gamma tracking and samples of blood and urine. Even though the rate of physical decay is quite different for ¹³¹iodine and ^99mtechnetium, the tracking time of labeled bacteria was limited to 24 h after injection for both radioactive isotopes. Technetium labeling was preferred because of greater imaging resolution and less radiation dose to the patient. Following intravenous administration, labeled C. parvum was found predominantly in the liver and spleen, and in a lesser amount in the lung. Radioactivity was confined to the pleural cavity after intrapleural injection. These results suggest the combined intravenous and intrapleural route of adjuvant immunosupportive agents such as C. parvum for operable lung cancer patients.     

Influence of a 9-double bond on stereospecific microbial 4,5-reductions

By stereospecific microbial reduction with Rhodosporidium rubrum or Rhodotorula glutinis, 17 alpha-cyano-methyl-4-estren-17 beta-ol-3-one was metabolized to 17 alpha-cyanomethyl-5 alpha-estrane-3 beta,17 beta-diol (50%) and 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha,17 beta-diol (30%). By Clostridium paraputrificum the same substrate was reduced stereospecifically to 17 alpha-cyanomethyl-5 beta-estrane-3 alpha, 17 beta-diol (70%). When the corresponding 9-dehydrogenated compound 17 alpha-cyanomethyl-4,9-estradien-17 beta-ol-3-one (STS 557, a new progestagen) was fermented, yeasts failed in 5 alpha-reducing the 4-double bond. Still Clostridium paraputrificum formed the expected 5 beta-reduced metabolite 17 alpha-cyanomethyl-5 beta-estr-9-ene-3 alpha,17 beta-diol (60%). Structures were elucidated by n.m.r. and mass spectra and partly by circular dichroism. By oxidation of the metabolites, the corresponding 3-oxo compounds 17 alpha-cyanomethyl-5 alpha-estran-17 beta-ol-3-one, 17 alpha-cyanomethyl-5 beta-estran-17 beta-ol-3-one and 17 alpha-cyanomethyl-5 beta-estr-9-en-17 beta-ol-3-one were prepared. The evident influence of the 9-double bond on reduction of the 4-en-3-oxo compound STS 557 preventing 5 alpha-reduction but permitting 5 beta-reduction is discussed in view of the distinctly diminished metabolism of this progestagen in mammals.     

Monoclonal cytotoxic T lymphocyte hybridomas capable of specific killing activity, antigenic responsiveness, and inducible interleukin secretion

We have recently described the production of cytotoxic T lymphocyte (CTL) hybridomas that grow continuously in culture, exhibiting constitutive, allospecific (anti-H-2b) killing activity. We now report on the response of these monoclonal CTL hybridomas to specific antigen (H-2Db) and to mitogenic lectins. Both specific antigen and T cell mitogens enhance hybridoma-mediated specific target cell killing. In addition, stimulated, but not unstimulated hybridoma cells secrete considerable amounts of IL 2 into the culture medium. Repeated cloning of the hybridomas provides strong evidence that both killing activity and IL 2 secretion can be attributed to one cell. Unfractionated Con A supernatants, containing IL 2 and other factors known to influence T cell responsiveness, or IL 2-containing media of stimulated hybridomas affect neither the growth nor the lytic activity of the hybridomas. Anti-LFA-1 monoclonal antibody, a potent inhibitor of CTL and CTL hybridoma-mediated target cell lysis, abolishes antigen- or mitogen-induced IL 2 secretion by the CTL hybridomas. Involvement of a single hybridoma receptor in antigen recognition (afferent and efferent) and in initiating IL 2 secretion is proposed. The CTL hybridomas displaying retarded killing activity before the antigenic or mitogenic stimulation appear to represent an intermediate stage in CTL differentiation, reminiscent of "memory" CTL.