The NADPH-dependent electron flow in chloroplasts of the higher plants

The NADPH-dependent reduction of some photosynthetic electron carriers in the dark, and the reduction of NADP+ associated with the glycolytic sequence and the oxidative pentose phosphate pathway in chloroplasts are reviewed. The postulated pathways of electron transports sensitive and insensitive to antimycin A are also evaluated. It is proposed that the electron flow, predominantly through cytochrome bf complex, may be also involved in the pathway of NADPH-dependent and antimycin A-insensitive back electron transport. An information on the chlororespiration in higher plants is also included. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mutation of serine residue 318 in the class C β-lactamase of Enterobacter cloacae 908R

The involvement of the serine residue 318 in the specificity of a class C beta-lactamase was investigated. Multiple site-directed mutants at this position were generated using a polymerase chain reaction technique. These mutants were then probed for their activity towards various beta-lactam compounds. One mutant, S318G was further purified and its physico-chemical and catalytic properties determined. It was shown that the observed minimal inhibitory concentration values of this mutant could be correlated to its kinetic properties using a 'diffusion-hydrolysis' model. However, the data showed that residue 318 has little influence on the specificity of class C beta-lactamases.     

Glycosylation of Aromatic Amines I: Characterization of Reaction Products and Kinetic Scheme

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D ¹H–¹H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of α and β glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1–6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.     

Detection by multiplex polymerase chain reaction and typing of Chlamydia trachomatis isolates

Abstract The multiplex polymerase chain reaction (PCR) was applied for the detection of the Chlamydia trachomatis chromosome and plasmid. The multiplex PCR demonstrated a sensitivity of 0.8 fg of chlamydial DNA, corresponding to the detection of about 5 copies of the plasmid. Analysis of 195 genital specimens collected randomly from a female population, showed that the multiplex PCR is more sensitive and rapid than culturing for detecting Chlamydia trachomatis . Moreover, sequencing of the II variable domain of the ompl gene, directly from DNA of the clinical specimens, appears to be a simple and rapid method for determining serovar isolates.     

Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum

Abstract A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum , from bacteria phylogenetically close to mollicutes ( Clostridium innocuum, C. ramosum and Bacillus subtilis ), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum .     

Individual and simultaneous treatment with antipsychotic aripiprazole and antidepressant trazodone inhibit sterol biosynthesis in the adult brain

Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners. Furthermore, when ARI and TRZ were combined, exposure resulted in an additive 7-dehydrocholesterol (7-DHC) increase, as well as desmosterol (DES) and cholesterol decreases in both cell types. In adult mice, at baseline, we found that the three investigated sterols showed significant differences in distribution across the eight assessed brain regions. Furthermore, experimental mice treated with ARI or TRZ, or a combination of both medications for 8 days, showed strong sterol disruption across all brain regions. We show ARI or TRZ alone elevated 7-DHC and decreased DES levels in all brain regions, but with regional differences. However, the combined utilization of these two medications for 8 days did not lead to additive changes in sterol disturbances. Based on the complex roles of 7-DHC derived oxysterols, we conclude that individual and potentially simultaneous use of medications with sterol biosynthesis-inhibiting properties might have undesired side effects on the adult brain, with as yet unknown long-term consequences on mental or physical health.     

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.     

The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.