Isolation of cDNA clones corresponding to genes differentially expressed in pericarp of mume (Prunus mume) in response to ripening, ethylene and wounding signals

Ripening of climacteric fruit is a complex developmental process that includes many changes in gene expression. Some ripening-regulated genes are responsive to ethylene and/or wounding signals. Wounding increased Pm-ACS1 expression in Prunus mume (Japanese apricot), but was negatively regulated by ethylene. However, exposure of freshly harvested mature green mume fruit to ethylene induced PmACS1 . Fifteen complementary DNA clones corresponding to messenger RNAs differentially expressed in the pericarp of P. mume fruit in response to ripening, ethylene and wounding signals were isolated by differential display. Quantitative real-time PCR analysis distinctly showed that these genes are differentially regulated. Genes that were upregulated during fruit ripening include Pm15 (cinnamyl-alcohol dehydrogenase), Pm21 (2-oxoacid-dependent dioxygenase), Pm22 (1-acyl- sn -glycerol-3-phosphate acyltransferase), Pm27 (unknown function), Pm38 (alcohol dehydrogenase), Pm41 (no homology), Pm52 (no homology), Pm65 (pectate lyase), Pm68 (expansin), Pm69 (serine carboxypeptidase) and Pm94 (alcohol acyltransferase). Expression of most of these genes was also inducible by ethylene and some of them were inducible by wounding. Pm3 (water channel protein, MIP) and Pm8 (unknown function) were downregulated during ripening. Expression of Pm71 (no homology) and Pm74 (NAC family protein) did not increase during ripening or in response to ethylene, but was upregulated in response to wounding. The possible physiological roles of these genes during ripening and in response to ethylene and wounding are discussed.     

Redefining disease? The nosologic implications of molecular genetic knowledge

How will developments in genetic knowledge affect the classification of disease? Leaders in genetics have suggested that knowledge of the role of genes in disease can determine nosology. Diseases might be defined by genotype, thus avoiding the limitations of more empirical approaches to categorization. Other commentators caution against disease definitions that are detached from the look and feel of disease, and argue for an interplay between genotypic and phenotypic information. Still others attribute nosologic change to social processes. We draw on an analysis of the scientific literature, our conversations with genetics clinicians, and reviews of patient organization Web sites to offer a revised interpretation of the nosologic implications of molecular genetic knowledge. We review the recent histories of three diseases--hemophilia, Rett syndrome, and cystic fibrosis--to argue that nosologic change cannot be explained by either biologic theories of disease etiology or sociologic theories of social tendencies. Although new genetic information challenges disease classifications and is highly influential in their redesign, genetic information can be used in diverse ways to reconstruct disease categories and is not the only influence in these revisions. Ironically, genetic information is likely to play a central role in producing a new, but still empirical, classification scheme.     

Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.     

Synthesis and antimycobacterial activity of calpinactam derivatives

Synthesis of calpinactam 1, a fungal antimycobacterial metabolite, utilizing solid-phase peptide synthesis is described. To explore the structure–activity relationships of 1, its derivatives with different amino acids were also synthesized on the basis of the same synthetic strategy. These derivatives were examined for antimycobacterial activity against Mycobacterium smegmatis. Among them, only peptide 6d having d-Ala in place of d-Glu showed moderate activity.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCl were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)–acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.     

Conformation of the HMG17-nucleosome complex

The nucleosome core binds more than two molecules of HMG17 at low ionic strength (8.9 mM Tris-HCl/8.9 mM boric acid/0.25 mM Na2EDTA, pH 8.3). Circular dichroism of the complexes showed only minor conformational changes of the nucleosome core DNA on binding of HMG17, with no detectable change in the histone secondary structure. The fluorescence of N-(3-pyrene) maleimide bound to -SH groups at Cys-110 of H3 histones in the core particle suggested that the structure of the histone octamer assembly changed little upon binding of HMG17 to the nucleosome. These observations support the idea that even a high level of HMG17 binding, e.g., four HMGs per nucleosome, alone, does not open up the core particle.