In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity. In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.

Sialylated glycoconjugates play important roles in various biological functions. The structures are also observed in brains and it has been proposed that sialylation may affect neural plasticity. To clarify the effects of sialylation in the brain, particular neurons that exhibit sialylation should first be determined. Using in situ hybridization, we performed systematic surveys of the localization of mRNAs encoding the six a2,3-sialyltransferases (ST3Gal I-VI) in the adult mouse brain with or without physiological stimulation. First, striking regionspecific patterns of expression were observed: While ST3Gal II, III, and V mRNAs were in neuronal cells throughout the brain, ST3Gal I, IV, and VI mRNAs were in restricted brain regions. Next, to assess whether the expression of the six mRNAs can be regulated, we examined the effect of kindling epileptogenesis on the six mRNA levels. Of the six subtypes, upregulation in the ST3Gal IV level in the thalamus was most pronounced; the number of ST3Gal IV-expressing neurons in the anterior thalamic nuclei increased from 2% to 21% in a time-dependent manner during epileptogenesis. Western blot analysis evaluated the increase of the end-products in the thalamus. These findings provide a molecular basis to clarify when and where sialylated glycoconjugates function accompanied by neural plasticity.

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Previous studies have indicated that the geniohyoid (GH) muscle is innervated by efferent axons from both the hypoglossal nerve (CN XII) and ansa cervicalis. To clarify the physiological significance of this dual innervation of the GH muscle, we examined properties of efferent innervations in rat GH muscle using electrophysiological, horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Recordings from the branch of the XII nerve that innervates the GH (GH.Br) revealed that bursts of impulses during fictitious swallowing were conducted via the XII nerve trunk, in which neuronal cell bodies were labeled in the ventrolateral subnucleus of the XII nucleus by HRP tracing. In contrast, in vivo experiments demonstrated that tonic discharges in GH.Br were conducted via the ansa cervicalis. However, HRP-labeled efferent neurons were observed in neither brainstem nor upper spinal cord, but sensory neurons were labeled in the most rostral cervical spinal ganglia via the ansa cervicalis. Tonic activity was abolished in vitro by mecamylamine, an antagonist of nicotinic acetylcholine receptors (nAChR), and by pirenzepine, an antagonist of muscarinic M1 receptors. Incubation of isolated XII nerve segments with antisera to vasoactive intestinal peptide, nAChR, and muscarinic M1 receptor yielded small numbers of labeled neurons with each antiserum. All labeled neurons displayed similar diameters and were located approximately 1.5 mm proximal to the bifurcation of the XII nerve into medial and lateral branches. Our findings indicate that GH muscle in the rat is innervated by both somatic and parasympathetic nervous systems.