Genetic variation of a fimbrial protein from Porphyromonas gingivalis and its distribution in patients with periodontal diseases

Pg-II fim from various strains of Porphyromonas gingivalis was classified on the basis of each nucleotide sequence, while the distribution of Pg-II fim types in 141 subgingival plaque samples was analyzed using PCR assays. Pg-II fim was divided into two types as follows: strains OMZ409, HG405, 381, ATCC 33277 and BH18/10 (type 1) and strains OMZ314 and HW24D-1 (type 2). The presence of P. gingivalis was demonstrated in 2.8% of healthy subjects and 56.1% of patients with periodontal diseases, and Pg-II fim was detected in 91.8% of the P. gingivalis-positive subjects. We also analyzed the distribution of the Pg-II fim types among Pg-II fim-positive patients, with the following results: type 1 (38.2%), type 2 (56.4%) and types 1 and 2 (5.4%). These findings strongly suggest that P. gingivalis organisms possessing Pg-II fim type 2 was principally detected in patients with periodontal diseases.     

Transportation mechanism for vanillin uptake through fungal plasma membrane

Protoplasts of the basidiomycete, Fomitopsis palustris (formerly Tyromyces palustris), were utilized to study a function of the fungal plasma membrane. Fungal protoplasts exhibited metabolic activities as seen with intact mycelial cells. Furthermore, the uptake of certain compounds into the protoplast cells was quantitatively observed by using non-radioactive compounds. Vanillin was converted to vanillyl alcohol and vanillic acid as major products and to protocatechuic acid and 1,2,4-trihydroxybenzene as trace products by protoplasts prepared from F. palustris. Extracellular culture medium showed no activity responsible for the redox reactions of vanillin. Only vanillic acid was detected in the intracellular fraction of protoplasts. However, the addition of disulfiram, an aldehyde dehydrogenase inhibitor, caused an intracellular accumulation of vanillin, strongly suggesting that vanillin is taken up by the cell, followed by oxidation to vanillic acid. The addition of carbonylcyanide m-chlorophenylhydrazone, which dissipates the pH gradient across the plasma membrane, inhibited the uptake of either vanillin or vanillic acid into the cell. Thus, the fungus seems to possess transporter devices for both vanillin and vanillic acid for their uptake. Since vanillyl alcohol was only observed extracellularly, the reduction of vanillin was thought to be catalyzed by a membrane system.     

Dimeric transthyretin variant assembles into spherical neurotoxins

Familial amyloidotic polyneuropathy is a hereditary autosomal-dominant disease in which the deposited transthyretin fibrils are derived from amyloidogenic mutation. We investigated structure and stability of a human Ser112Ile transthyretin variant and showed that the Ser112Ile variant exists as a dimer having nonnative tertiary structure at physiological pH. In addition, the dimeric Ser112Ile assembles into a spherical aggregate and exerts cytotoxicity in a human neuroblastoma cell line. Our results suggest the importance of an unstable dimeric structure in forming spherical aggregates that will induce cell death.     

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.     

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.     

Expression of zebrafish glutamate receptor delta2 in neurons with cerebellum-like wiring

Mammalian glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays key roles in cerebellar plasticity, motor learning, and neural wiring. Here, we isolated cDNA encoding the zebrafish ortholog of mammalian GluRdelta2. We found that in adult zebrafish brain, glurdelta2 mRNA was expressed not only in cerebellar Purkinje cells, but also in the crest cells of the medial octavolateral nucleus (MON) and the type I neurons of the optic tectum. Immunohistochemical analysis revealed that zebrafish GluRdelta2 proteins were selectively localized in the apical dendrites of these neurons. Interestingly, the crest cells of the MON and the type I neurons of the optic tectum receive large numbers of parallel fiber inputs at the apical dendrites and sensory inputs at the proximal or basal dendrites. These results suggest that the expression of zebrafish GluRdelta2 is selective for cerebellum-like neural wiring with large numbers of parallel fiber inputs.     

Nonylphenol induces the death of neural stem cells due to activation of the caspase cascade and regulation of the cell cycle

Endocrine disruptors (EDs) are a great concern throughout the world, because they have adverse effects on human health and wildlife. In the present study, we investigated the effects of EDs on the proliferation and survival of murine neural stem cells (NSCs). In contrast to bisphenol A, phthalic acid benzyl n-butyl ester, phthalic acid di-n-butyl ester and phthalic acid di(2-ethylhexyl) ester, the treatment of NSCs with 4-nonylphenol for 24 h inhibited cell growth in a concentration-dependent manner. In addition, treatment with 4-nonylphenol resulted in nuclear condensation and DNA fragmentation (morphological changes due to apoptosis) in NSCs after 12 h of exposure, and activated caspase-3 after 6 h and 9 h of exposure. Furthermore, an exposure to 4-nonylphenol led to the accumulation of cells at the G2/M phase interface and down-regulated the protein levels of cyclin A and B1, which are the major regulatory proteins at the G2 to M transition of the cell cycle. Together, these results indicate that, in contrast to other EDs, 4-nonylphenol may exhibit a potent cytotoxicity through apoptosis via the caspase cascade and cell cycle arrest at the G2/M phase, and suggest that 4-nonylphenol may affect neurogenesis in the CNS.     

Motor activity and gene expression in rats with neonatal 6-hydroxydopamine lesions

A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders.