Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD,the Cα‐dehydrogenase from Sphingobium sp. strain SYK‐6

Bacteria‐derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram‐negative bacterium Sphingobium sp. SYK‐6 possesses a Cα‐dehydrogenase (LigD) enzyme that has been shown to oxidize the α‐hydroxy functionalities in β–O–4‐linked dimers into α‐keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β–O–4‐linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol‐β‐coniferyl alcohol ether and syringylglycerol‐β‐sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon‐optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC‐MS/MS‐based metabolite profiling indicated that levels of oxidized guaiacyl (G) β–O–4‐coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1‐ to 2.8‐fold increased level of G‐type α‐keto‐β–O–4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.     

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.     

Experimental fascioliasis in the rat-like hamster,Tscherskia triton,and other rodent hosts

Experimental infection with Fasciola hepatica and parthenogenetic Fasciola sp. in laboratory animals have been conducted in rats and rabbits. Inoculation of less than 5 metacercariae into rat-like hamsters, Tscherskia triton, is sufficient to establish Fasciola infections. The prepatent period of F. hepatica and the parthenogenetic Fasciola sp. in T. triton was shorter than that in rats and rabbits, suggesting that T. triton is a suitable experimental model for these flukes. In contrast, F. gigantica infection in T. triton did not yield adult flukes; T. triton, is therefore, considered to be an unsuitable host for F. gigantica. The cotton rat, Sigmodon hispidus, was an unsuitable host for the parthenogenetic Fasciola sp.     

Leukotriene B4 receptor 1 expression on dendritic cells is required for the development of Th2 responses and allergen-induced airway hyperresponsiveness

Dendritic cells (DC) are important APCs that control allergen-induced airway responses by interacting directly with T cells. Leukotriene B(4) (LTB(4)), interacting with its high-affinity receptor, LTB(4) receptor 1 (BLT1), is known to attract and activate leukocytes during inflammation. We have previously shown that BLT1 expression on Ag-primed T cells is required for the development of airway hyperresponsiveness (AHR; Miyahara et al. 2005. Am. J. Respir. Crit. Care Med. 172: 161-167). However, the role for the LTB(4)-BLT1 pathway in DC function in allergen-induced airway responses has not been defined. Bone marrow-derived DCs (BMDC) were generated. Naive BALB/c mice received OVA-pulsed BLT1-deficient (BLT1(-/-)) BMDCs or wild-type BMDCs intratracheally and were then challenged with OVA for 3 days. Airway responses were monitored 48 h after the last allergen challenge. BLT1(-/-) BMDCs showed normal maturation judged from surface expression of CD markers. Compared with recipients of wild-type BMDCs, mice that received BLT1(-/-) BMDCs developed significantly lower AHR to inhaled methacholine, lower goblet cell metaplasia, and eosinophilic infiltration in the airways and decreased levels of Th2 type cytokines in the bronchoalveolar lavage fluid. Migration of BLT1(-/-) BMDCs into peribronchial lymph nodes was significantly impaired compared with BLT1(+/+) BMDCs after intratracheal instillation. These data suggest that BLT1 expression on DCs is required for migration of DCs to regional lymph nodes as well as in the development of AHR and airway inflammation.     

A Method for the Rapid Growth in Culture of Gametophytes of Equisetum arvense with Antheridia

Rapid growth in culture of Equisetum arvense gametophytes wasobtained using Murashige-Skoog's medium plus 3% (w/v) sucroseand continuous illumination. In darkness, growth was reducedand chlorophyll synthesis markedly inhibited. Antheridia formedon the top margin of gametophytes in light and darkness butarchegonia were not observed in either case. ¹Present address: Interdisciplinary Research Institute of EnvironmentalSciences, Higashi-yanagi-cho, Nishi-iru, Shichihon-matsu, Itsutsuji-dori,Kamigyo-ku, Kyoto, 602 Japan. (Received June 5, 1989; Accepted September 12, 1989)     

Three-dimensional solution structure of the sodium channel agonist/antagonist delta-conotoxin TxVIA

The three-dimensional solution structure of delta-conotoxin TxVIA, a 27-mer peptide agonist/antagonist of sodium channels, was determined by two-dimensional (1)H NMR spectroscopy with simulated annealing calculations. A total of 20 converged structures of delta-conotoxin TxVIA were obtained on the basis of 360 distance constraints obtained from nuclear Overhauser effect connectivities, 28 torsion angle constraints, and 27 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.35 +/- 0.07 A for the backbone atoms (N, C(alpha), C) and 0.98 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of delta-conotoxin TxVIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as those for other conotoxins. However, the three-dimensional structure of delta-conotoxin TxVIA has an unusual hydrophobic patch on one side of the molecule, which may play an important role in the sodium channel binding. These results provide a molecular basis for understanding the mechanism of sodium channel modulation through the toxin-channel interaction and insight into the discrimination of different ion channels.     

Identification of Vanabin-interacting protein 1 (VIP1) from blood cells of the vanadium-rich ascidian Ascidia sydneiensis samea

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Arginine specific endopeptidases modify the aggregation properties of a synthetic peptide derived from Alzheimer β/A4 amyloid

A synthetic peptide corresponding to the first 28 amino acids of the Alzheimer disease amyloid /A4 peptide (3.2 kDa) aggregated to a high molecular weight (15 kDa) on SDS/urea polyacrylamide gels. Proteinase K, V8 protease, trypsin, and endopeptidase Lys-C readily degraded the aggregate. By contrast, when digested by endopeptidase Arg-C, a new polypeptide aggregate of higher molecular weight (16 kDa) was observed on denaturing gels without degraded smaller products. The new aggregate was comprised of three peptides: an intact /A4(1–28) and partially degraded peptides /A4(1–5) plus /A4(6–28). The results were confirmed by treatment of /A4 with other arginine-specific proteases: the gamma subunit of nerve growth factor and clostripain. The results indicate that arginine-specific proteases, including a growth factor processing enzyme, can nick aggregated /A4(1–28) amyloid and alter the configuration to produce a complcomple aggregated form. If similar highly specific proteolytic mechanisms occur in the Alzheimer disease brain, the processing may promote the formation of high molecular weight aggregates that contribute to the development of relatively insoluble senile plaque core protein.     

Ndrg1-deficient mice exhibit a progressive demyelinating disorder of peripheral nerves

NDRG1 is an intracellular protein that is induced under a number of stress and pathological conditions, and it is thought to be associated with cell growth and differentiation. Recently, human NDRG1 was identified as a gene responsible for hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth disease type 4D), which is characterized by early-onset peripheral neuropathy, leading to severe disability in adulthood. In this study, we generated mice lacking Ndrg1 to analyze its function and elucidate the pathogenesis of Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5 weeks of age. However, myelination of Schwann cells in the sciatic nerve was normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness, especially in the hind limbs, but complicated motor skills were retained. In wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells rather than the myelin sheath. These results indicate that NDRG1 deficiency leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for maintenance of the myelin sheaths in peripheral nerves. These mice will be used for future analyses of the mechanisms of myelin maintenance.