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.     

Removal of Enteroviruses from Sewage by Bench-Scale Rotary-Tube Trickling Filters

The efficacy of a rotary-tube type of trickling filter for removing coxsackievirus A9, poliovirus 1, and echovirus 12 suspended in raw settled sewage was investigated. At filtration rates equivalent to about 10 MGD (million gallons per day)/acre (ca. 3,785 m3/day per acre), the filters removed 95% of the poliovirus, 83% of echovirus 12, and 94% of coxsackievirus A9. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals were remarkably similar, averaging 94, 92, 93, and 95%, respectively. At filtration rates equivalent to about 23 MGD/acre, 59% of the poliovirus, 63% of the echovirus 23, and 81% of the coxsackievirus A9 were removed. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals at this filtration rate were 68, 75, 72, and 56%, respectively. Viruses were assumed to be adsorbed to the biological slime growing in the filters, but attempts to disassociate the viruses from the slime were unsuccessful, indicating that the slime-virus complex is very stable or that the viruses were somehow inactivated. The data indicate that coliform and fecal streptococci reductions in this type sewage treatment process can be used as an index of virus reduction. Disinfection, however, must be used to ensure a virus-free final effluent.     

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.     

In situ, in-liquid, all-electrical detection of Salmonella typhimurium using lead titanate zirconate/gold-coated glass cantilevers at any dipping depth

Most biosensing techniques are indirect, slow, and require labeling. Even though silicon-based microcantilever sensors are sensitive and label-free, they are not suitable for in-liquid detection. More recently lead zirconate titanate (PZT) thin-film-based microcantilevers are shown to be sensitive and in situ. However, they require microfabrication and must be electrically insulated. In this study, we show that highly sensitive, in situ, Salmonella typhimurium detection can be achieved at 90% relative humidity using a lead zirconate titanate (PZT)/gold-coated glass cantilever 0.7 mm long with a non-piezoelectric 2.7 mm long gold-coated glass tip by partially dipping the gold-coated glass tip in the suspension at any depth without electrically insulating the PZT. In particular, we showed that at 90% relative humidity and with a dipping depth larger than 0.8 mm the PZT/gold-coated glass cantilever showed virtually no background resonance frequency up-shift due to water evaporation and exhibited a mass detection sensitivity of Δm/Δf = −5 × 10⁻¹¹ g/Hz. The concentration sensitivities of this PZT/gold-coated glass cantilever were 1 × 10³ and 500 cells/ml in 2 ml of liquid with a 1 and 1.5 mm dipping depth, respectively, both more than two orders of magnitude lower than the infectious dose and more than one order of magnitude lower than the detection limit of a commercial Raptor sensor.     

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.     

Relation of polyamine synthesis and titer to aging and senescence in oat leaves

Polyamine biosynthesis in senescing leaves of Avena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during light exposure of excised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the light, irrespective of senescence. S-Adenosyl-l-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and decline with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of light exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spermidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development.     

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.     

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.