Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg²⁺ to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca²⁺ to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca²⁺ before exerting its antisenescence action.     

Surface antigenic determinants of mammalian "hepadnaviruses" defined by group- and class-specific monoclonal antibodies

The hepatitis B-like viruses (human hepatitis B virus, woodchuck hepatitis virus, ground squirrel hepatitis virus, and duck hepatitis B virus) are hepatotropic DNA viruses which have been referred to collectively as "hepadnaviruses." Using a murine monoclonal antibody (101-2) to the surface antigen of woodchuck hepatitis virus, we have shown that the surface antigens of mammalian hepadnaviruses (HBsAg, WHsAg, and GSHsAg) are antigenically related via a common determinant (HV/101). Furthermore, analysis with other monoclonal antibodies to WHsAg revealed that WHsAg and GHsAg are antigenically distinct, although the antigens had more determinants in common with each other than with HBsAg. The hepadnavirus group-specific antibody (101-2) reacted with HBsAg subtypic variants in a group-specific rather than subtype-specific manner. In conjunction with observations with an HBsAg-specific, group-reactive monoclonal antibody (BX259), the present data suggest that there are at least two group-reactive epitopes of HBsAg: one which is virus specific (HBV/259) and one which is common to two other mammalian hepadnaviruses (HV/101).     

Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration associated with hyperornithinemia with an autosomal recessive mode of inheritance. Cultured skin fibroblasts from five affected patients showed a virtual absence of ornithine ketoacid transaminase (OKT) (L-ornithine:2-oxoacid aminotransferase E.C.2.6.1.13) activity. Fibroblasts from four carrier parents showed a 42%-65% reduction in OKT activity. Increasing the concentration of pyridoxal phosphate (vitamin B6 in the assay media resulted in partial restoration of OKT activity in fibroblasts from one out of five patients studied. We conclude that OKT deficiency is closely associated with the genetic defect in gyrate atrophy of the choroid and retina and that genetic heterogeneity exists in this disease.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.     

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.     

Detection of the intracellular ras p21 oncogene product by flow cytometry

Rapid identification of the expression of oncogene products in specific cell types could potentially be useful in the diagnosis and treatment of human malignancy. We have now observed that through the use of lysolecithin permeabilization and fluorescence-activated flow cytometry, cells expressing high levels of the v-Ha-ras oncogene product, p21, can readily be distinguished from the nontransformed parent cells in a rapid and quantitative manner.     

Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.     

Synthesis of substituted 4(Z)-(methoxyimino)pentyl-1-piperidines as dual NK1/NK2 inhibitors

The NK1 and NK2 receptor activity of a series of 5-[(3,5-bis(trifluoromethyl)phenyl)methoxy]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl-1-piperidines was evaluated. Compounds 11d, 11e, 11f, 12a, and 12k were found to be our most potent inhibitors.     

Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus.

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.