Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998     

Comprehensive molecular phylogeny of the sub-family Dipterocarpoideae (Dipterocarpaceae) based on chloroplast DNA sequences

Dipterocarpoideae, the largest sub-family of well-known plant family Dipterocarpaceae, dominates in South Asian rain forests. Although several previous studies addressed the phylogeny of the Dipterocarpaceae family, relationships among many of its genera from the Dipterocarpoideae sub-family are still not well understood. In particular, little is known about the relationships of the genera Vateriopsis, Stemonoporus, Vateria and inconsistence remains between phylogenetic results and taxonomic classifications of Shorea and Hopea species. We studied molecular phylogeny of the sub-family Dipterocarpoideae using the trnL-trnF spacer, trnL intron and the matK gene sequences of chloroplast DNA (cpDNA). This study is the first comprehensive phylogeny reconstruction for the sub-family Dipterocarpoideae based on cpDNA, as it includes most genera (14) and a large number of species (79) with most species endemic to Sri Lanka, as well as one species from Seychelles and one species from the genus Monotes from Madagascar. Phylogenetic trees were constructed using the Neighbor Joining (NJ) and Maximum Likelihood (ML) methods using combined set of sequences including all three cpDNA regions. The topologies of the NJ and ML trees were to a certain extent, consistent with the current taxonomy of Dipterocarpoideae based on morphology and with previous molecular phylogenies based on cpDNA. Furthermore, our results provided new evidence regarding the relationships of the following genera: Vateriopsis and Stemonoporus and about the validity of the previous morphology based classifications of Shorea species. In addition, the topology of our trees was consistent with the classification of Shorea species proposed by Maury (1978), Maury-Lechon (1979) and Symington (1943). Finally, our results provided evidence for the affinity of the genus Monotes to Asian Dipterocarpoideae rather than to Tiliaceae and indicated that it is a good candidate for outgroup species for future studies of the former sub-family.     

The diversity of mycorrhizal fungi in Japanese Cephalanthera species

The diversity of mycorrhizal fungi among four Japanese Cephalanthera (Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three Cephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese Cephalanthera species for a specific fungal group of Thelephoraceae.     

Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Induction of endogenous uncoupling protein 3 suppresses mitochondrial oxidant emission during fatty acid-supported respiration

Uncoupling protein 3 (UCP3) expression increases dramatically in skeletal muscle under metabolic states associated with elevated lipid metabolism, yet the function of UCP3 in a physiological context remains controversial. Here, in situ mitochondrial H(2)O(2) emission and respiration were measured in permeabilized fiber bundles prepared from both rat and mouse (wild-type) gastrocnemius muscle after a single bout of exercise plus 18 h of recovery (Ex/R) that induced a approximately 2-4-fold increase in UCP3 protein. Elevated uncoupling activity (i.e. GDP inhibitable) was evident in Ex/R fibers only upon the addition of palmitate (known activator of UCP3) or under substrate conditions eliciting substantial rates of H(2)O(2) production (i.e. respiration supported by succinate or palmitoyl-L-carnitine/malate but not pyruvate/malate), indicative of UCP3 activation by endogenous reactive oxygen species. In mice completely lacking UCP3 (ucp3(-/-)), Ex/R failed to induce uncoupling activity. Surprisingly, when UCP3 activity was inhibited by GDP (rats) or in the absence of UCP3 (ucp3(-/-)), H(2)O(2) emission was significantly (p < 0.05) higher in Ex/R versus non-exercised control fibers. Collectively, these findings demonstrate that the oxidant emitting potential of mitochondria is increased in skeletal muscle during recovery from exercise, possibly as a consequence of prolonged reliance on lipid metabolism and/or altered mitochondrial biochemistry/morphology and that induction of UCP3 in vivo mediates an increase in uncoupling activity that restores mitochondrial H(2)O(2) emission to non-exercised, control levels.     

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998