Genomic organization of the rat aromatic L-amino acid decarboxylase (AADC) locus: Partial analysis reveals divergence from the Drosophila dopa decarboxylase (DDC) gene structure

Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution.     

Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase

A cDNA clone, named ppmMDH-1 and covering a part of the porcine mitochondrial malate dehydrogenase (mMDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37) mRNA, was isolated from a porcine liver cDNA library with a mixture of 24 oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known sequence of porcine mMDH amino acid residues 288-293. ppmMDH-1 covered the coding region for porcine mMDH amino acid residues 17-314 and the 3' untranslated region. Subsequently, mouse mMDH cDNA clones were isolated from a mouse liver cDNA library with the ppmMDH-1 cDNA as a probe. One of the clones, named pmmMDH-1 and containing a cDNA insert of about 1350 base pairs, was selected for sequence analysis, and the primary structure of the mouse precursor form of mMDH (pre-mMDH) was deduced from its cDNA sequence. The sequenced coding regions for the porcine and mouse mMDH mRNAs showed about 85% homology. When the deduced amino acid sequence of the mouse pre-mMDH was compared with that of the porcine mMDH, they shared a 95% homology, and the mouse pre-mMDH yielded a leader sequence consisting of 24 amino acid residues and a mature mMDH, consisting of 314 amino acid residues. The leader sequence contained three basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The mouse mMDH leader sequence was compared with those of three other rodent mitochondrial matrix proteins.     

Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: Studies in vivo and in vitro

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.     

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Rhodium-catalyzed synthesis of 2(5H)-furanones from terminal alkynes and non-substituted alkynes under water-gas shift reaction conditions

Rhodium-catalyzed synthesis of 2(5H)-furanones from alkynes under water-gas shift reaction conditions was studied. By improving the reaction conditions for internal alkynes reported previously, the reaction could be extended to terminal alkynes. Terminal alkynes are selectively converted into 3- and 4-substituted 2(5H)-furanones (2 and 3). When acetylene itself is used, 2(5H)-furanone (2n) is obtained in a good yield. Examination of reaction solutions by IR spectroscopy and some other experimental findings suggest that the active species would be an alkyne-coordinated monomeric rhodium anion. A new reaction path is proposed.     

Pyridoxal kinase immunoreactivity in rabbit brain

Murine polyclonal antibody against purified bovine brain pyridoxal kinase (EC 2.7.1.35) was generated and showed cross-reactivity with rabbit brain pyridoxal kinase. This antibody was used to immunohistochemically examine the distribution of pyridoxal kinase in the rabbit brain. The cytoplasm of neuronal cells and neuroglial cells in the cerebral cortex, hippocampal region, brain nuclei and cerebellar cortex showed positive staining with various degrees of intensity. The neuronal cells and surrounding fibers in some brain nuclei, such as the area tegmentalis ventralis or the substantia nigra, showed intense staining. The neuronal cells of the hippocampal region showed somewhat weak reactivity, but some with intense reactivity were found sparsely distributed and positive staining fiber networks of a very low density were also observed.     

Progressive search in tandem mass spectrometry

A cell exhibits a variety of responses to internal and external cues. These responses are possible, in part, due to the presence of an elaborate gene regulatory network (GRN) in every single cell. In the past 20 years, many groups worked on reconstructing the topological structure of GRNs from large-scale gene expression data using a variety of inference algorithms. Insights gained about participating players in GRNs may ultimately lead to therapeutic benefits. Mutual information (MI) is a widely used metric within this inference/reconstruction pipeline as it can detect any correlation (linear and non-linear) between any number of variables (n-dimensions). However, the use of MI with continuous data (for example, normalized fluorescence intensity measurement of gene expression levels) is sensitive to data size, correlation strength and underlying distributions, and often requires laborious and, at times, ad hoc optimization. In this work, we first show that estimating MI of a bi- and tri-variate Gaussian distribution using k-nearest neighbor (kNN) MI estimation results in significant error reduction as compared to commonly used methods based on fixed binning. Second, we demonstrate that implementing the MI-based kNN Kraskov–Stoögbauer–Grassberger (KSG) algorithm leads to a significant improvement in GRN reconstruction for popular inference algorithms, such as Context Likelihood of Relatedness (CLR). Finally, through extensive in-silico benchmarking we show that a new inference algorithm CMIA (Conditional Mutual Information Augmentation), inspired by CLR, in combination with the KSG-MI estimator, outperforms commonly used methods. Using three canonical datasets containing 15 synthetic networks, the newly developed method for GRN reconstruction—which combines CMIA, and the KSG-MI estimator—achieves an improvement of 20–35% in precision-recall measures over the current gold standard in the field. This new method will enable researchers to discover new gene interactions or better choose gene candidates for experimental validations.