Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

Papovaviral sialoadenitis in athymic nude rats

A wasting disease was found in 32 athymic nude rats. The rats had parotid sialoadenitis with intranuclear inclusion bodies in ductal and acinar epithelial cells. Other common lesions included bronchitis, bronchiolitis and secondary bacterial pneumonia. Less commonly, rhinitis and Harderian adenitis were seen. Intranuclear inclusions were also seen in bronchial epithelium of 1 rat, Harderian gland acini of 1 rat and laryngeal glands of 2 rats. Viral particles, averaging 45 nm in diameter, sometimes in crystalline arrays, were found in the nucleus of parotid epithelial cells. By the use of the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique, antibodies to disrupted SV40 virus (the group specific antigen of the polyomavirus (miopapovavirus) genus of the papovavirus family) reacted with intranuclear inclusions and cytoplasm of parotid epithelium and inclusions in lung and Harderian gland. The viral antigen did not cross react with antibodies to mouse polyoma, mouse K or disrupted bovine papilloma viruses.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.     

Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat

Six transplantable large granular lymphocyte (LGL) tumor lines in F344 rats were examined for natural killer (NK) and antibody-dependent cell-mediated cytotoxicity. Tumor cells from all six lines were highly cytotoxic, even at low effector to target ratios, when tested against NK-susceptible targets, but were unreactive against an NK-resistant target (C58NT)D) and a macrophage-susceptible target (P815). Three lines showed significant levels of lysis against antibody-coated tumor cells. After in vivo transplantation, the levels of cytotoxicity steadily increased in three lines and decreased in one. The cytotoxic activity of one line (RNK-16) remained high through 12 transplant generations. Tumor cells injected i.p. spread via the lymphatics to regional lymph nodes, mediastinal nodes, blood, and eventually the bone marrow. Leukemia occurred concurrently with organ enlargement and increased levels of NK. Studies in (F344 X W/Fu)F1 rats clearly demonstrated that the cytotoxic cells from leukemic animals were the transplanted tumor cells themselves and not merely the activation of normal host LGL. These results demonstrate that naturally occurring, transplantable LGL leukemias are an easily obtainable and excellent source of materials for those studies requiring a large number of functionally active LGL.     

Monocyte suppression of antigen-specific lymphocyte responses in diffuse cutaneous leishmaniasis patients from the Dominican Republic

Patients from the Dominican Republic with diffuse cutaneous leishmaniasis showed in vivo and in vitro anergy to leishmanial antigen. Relatives of these DCL patients living in the same endemic area frequently showed skin test and lymphocyte reactivity to leishmanial antigens. This further supports the concept of specific anergy in patients with diffuse cutaneous leishmaniasis. Adherent suppressor cells modulate the antigen-specific lymphocyte proliferative response. Suppressor cells could also be isolated by Percoll gradient centrifugation. Co-culturing of lymphocytes and monocytes from HLA-identical leishmanin responders and nonresponders also identified the suppressor cell as a monocyte. In one patient, this suppression disappeared when clinical cure had been accomplished.     

Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera.

Recombinant plasmids containing the genomes of both bovine papillomavirus type I and minute virus of mice (MVM) were constructed and used to transform mouse C127 cells. Transformed lines that express MVM gene products with high efficiency were isolated and characterized. These transformants synthesize large amounts of MVM structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. These lines were, however, genetically unstable; they slowly generated subpopulations that failed to express MVM-specific proteins, and they possessed episomal DNA in which both MVM and bovine papillomavirus sequences were deleted or rearranged, or both. Clonal isolates of these transformants were also superinfectible by infectious MVM virus. Therefore, in spite of their instability, they should be useful host cell lines for transcomplementing mutations introduced into the MVM genome and for growing defective viruses as virions.     

Brain temperature measurements in rats: a comparison of microwave and ambient temperature exposures

In an effort to understand microwave heating better, regional brain and core temperatures of rats exposed to microwave radiation (2450 MHz) or elevated air temperatures were measured in two studies. In general, we have found no substantial evidence for temperature differentials, or "hot spots," in the brain of these animals. In the first study, after a 30-min exposure, no temperature differences between brain regions either after microwave or ambient air exposure were found. However, a highly significant correlation between brain and core temperatures was found and this correlation was the same for both microwave and ambient air heating. In the second study, time-temperature profiles were measured in rats exposed to either 30 mW/cm2 or 36.2 degrees C. In this study, the 30-min exposure period was divided into seven intervals and the change in temperature during each period was analyzed. Only the cortex showed significantly different heating rates between the air heating and microwave heating; however, this difference disappeared after the initial 5 min of exposure.     

Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli

Summary The effect of dissolved carbon dioxide concentration in the anaerobic growth of Escherichia coli was investigated. E. coli was grown anaerobically with the dissolved CO₂ concentration controlled over the range from 8x10^-6 M to 3.7x10^-2 M in the liquid phase. The maximum specific growth rate was 0.75h^-1 at 1.3x10^-3 M CO₂ and the maximum yield of cells on glucose was 0.32 at 1.75x10^-4 M CO₂. The maximum specific growth rate occurs close to the concentration of CO₂ prevalent in the mammalian gut where E. coli naturally resides.Alberta Research Council contribution, paper 1364     

Characterisation of an mRNA encoding a human ribosomal protein homologous to the yeast L44 ribosomal protein

We describe the isolation and characterisation of a full-length cDNA sequence (pZH-21) of a human ribosomal protein (rp) mRNA isolated from a cDNA library constructed from the human ZR-75-1 mammary tumour cell-line. The predicted protein is highly basic and shows 72% homology at the amino acid (aa) level with yeast rp L44. Comparative RNA blotting of ZR-75-1 poly(A)+ RNA isolated from cells cultured in the presence of the anti-oestrogen tamoxifen demonstrates the presence of a number of mRNA species whose concentration is elevated co-ordinately 5-6-fold in the presence of 17beta-oestradiol. Insulin in the presence of tamoxifen, also enhanced rp mRNA levels suggesting increased levels are a reflection of cell proliferation as opposed to specific hormonal regulation. Genomic analysis demonstrates the presence of a family of related human sequences, and homology with rat and guinea pig rp genes, but not yeast DNA. The conservation of rp aa sequence, in the absence of detectable homology at the nucleotide (nt) level, points to an important common functional role of the L44 protein in ribosome structure and function in man and yeast.