Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene

The purpose of this study was to determine whether retrovirus-mediated transfer of the murine macrophage inducible nitric oxide synthase (iNOS) gene can inhibit tumorigenicity and metastasis of human renal cancer cells. Retroviral vectors encoding murine macrophage iNOS were constructed in the pLXSN retroviral vector with the iNOS gene under the control of a long terminal repeat promoter and a neomycin resistance gene under the control of an internal simian virus 40 promoter. Highly metastatic human renal carcinoma SN12PM6 cells were infected with control or iNOS retrovirus. Expression of iNOS was confirmed by Northern and Western blot analyses, and expression of the functional iNOS protein, i.e., production of nitric oxide (NO), was determined by measuring nitrite accumulation in culture supernatants. Noninfected or control cells produced large orthotopic tumors in the kidney of nude mice and a larger number of experimental lung metastases, whereas iNOS-infected cells produced small tumors in the kidneys and few to no lung metastases. The data indicate that the infection of human renal cancer cells by retroviruses harboring the murine iNOS gene can induce the production of high levels of NO, which is associated with autocytotoxicity, suppression of tumorigenicity, and abrogation of metastasis.     

The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella

The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.     

Acquired tracheomalacia--a case report

Divalproex sodium is an effective drug for the treatment of migraine. Most adverse drug events are transient and not of great clinical concern. Although rare, well-documented examples of liver toxicity have been reported in children under 2 years of age on polypharmacy. Additional cases occur in children under 10 who are receiving polypharmacy, particularly those who have intractable seizures and degenerative central nervous system disease. Clinicians who treat migraineurs with divalproex sodium do not need to be overly preoccupied with monitoring of drug levels and liver function tests. The most valuable test is clinical observation of the patient.     

Identity elements and aminoacylation of plant tRNATrp

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.     

The effect of surface species on the photoluminescence of porous silicon

The photoluminescence of porous Si immersed in alcohol exhibits a blue shift and a marked decrease in intensity relatively to dry, as-anodized wafers. However, when the immersed samples are treated with ultraviolet (UV) light for a few minutes, the photoluminescence (PL) peak shifts to longer wavelength. Fourier transform infrared spectroscopy reveals that alkoxy surface species and silicon hydride species backbonded to oxygen atoms appear on the UV-treated samples. Furthermore, the PL characteristics and surface species of the UV-treated samples can be recovered to those of as-anodized wafers by dipping in hydrofluoric acid. These results point out the importance of surface chemistry in the luminescence process of porous Si.     

Variation in human thymidylate synthase is associated with resistance to 5-fluoro-2'-deoxyuridine

Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is less sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lines. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different procedures. Relative to the enzyme from C cells, the enzyme from RCA cells demonstrated higher Km values for the substrates deoxyuridylate and 5,10-methylene-tetrahydrofolate, a lower rate of association of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissociation, and lower enhancement of covalent FdUMP binding by folate derivatives. The activities of the enzymes in situ and the catalytic efficiencies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS with reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, relative to the enzyme expressed in a FdUrd-sensitive cell line.     

Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines

Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF-beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF-alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.     

Analysis of suckling-induced changes in adenohypophyseal prolactin concentration in the lactating rat by three assay methods

Suckling-induced changes in adenohypophyseal prolactin (PRL) concentrations in lactating rats were measured in two experiments by three assays: a disc electrophoretic assay (DEA), a bioassay (BA) and a radioimmunoassay (RIA). Substantial discrepancies among the three assays were found in the absolute amounts of pituitary PRL measured and in the changes in adenohypophyseal PRL concentration that were recorded between suckling intervals. The results indicate that in dynamic states of secretory activity the correspondence among BA, RIA and DEA estimates of pituitary PRL concentration is poor.     

Erythrocyte spectrin peak II phosphorylation in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a rapidly progressive crippling disease of young boys that is inherited as an X-linked recessive trait. Previous studies have demonstrated the usefulness of erythrocyte studies in exploring membrane abnormalities in inheritied muscular dystrophy. Erythrocyte spectrin peak II protein (m.w. equivalent to 220,000) was more highly phosphorylated under initial rate conditions in DMD than in controls. The extent of peak II phosphorylation was greater in DMD erythrocytes and a Na+ stimulated peak II phosphorylation effect (Avruch and Fairbanks 1974) was not found to account for the differences between DMD and controls. The phosphorylated state of spectrin proteins in the membrane was evaluated and no differences in DMD could be measured. The maximal transfer of phosphate from differences in DMD could be measured. The maximal transfer of phosphate from [gamma-32P]ATP to spectrin peak II accounts for approximately 5-10% of the total phosphate content of spectrin.