Signal peptide of <Emphasis Type="Italic">Aureobasidium</Emphasis><Emphasis Type="Italic">pullulans</Emphasis> xylanase: use for extracellular production of a fungal xylanase by <Emphasis Type="Italic">Escherichia coli</Emphasis>

An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262–270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-d-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.     

Pineal-dependent locomotor activity of lamprey,Lampetra japonica,measured in relation to LD cycle and circadian rhythmicity

Summary Locomotor activity of the river lamprey, Lampetra japonica, was investigated under a light-dark (LD 1212) cycle and under continuous dark conditions. Intact lampreys were entrained to the light:dark cycle. They were active mainly in the early half of the dark period and inactive in light period. The light:dark entrainment continued in 72.7% of lampreys after the removal of bilateral eyes, but additional pinealectomy made the entrainment disappear in all lampreys. When lampreys were pinealectomized with their eyes intact, light: dark entrainment was abolished in most cases. The results indicate that the pineal organ of the lamprey is a photoreceptive organ responsible for synchronizing locomotor activity to LD cycle. Under continuous dark conditions, the locomotor activity began to free-run with a period of 21.3 ± 0.9 h (mean ± SD, n = 53). This circadian rhythmicity was not affected by the removal of lateral eyes but was abolished by pinealectomy. The pineal organ appears to function as an oscillator, or as one of the oscillators, for the circadian locomotor rhythm of lampreys.Abbreviations DD continuous dark - LD light:dark     

Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

Isolation and expression patterns of genes for three angiopoietin-like proteins, Angptl1, 2 and 6 in zebrafish

Angiopoietin-like proteins (Angptls) are known to possess biological activities not only in the vascular system, but in the other mammalian tissues; however, their expression patterns and function in embryogenesis have not been extensively characterized. Here, we identify three zebrafish genes (Zangptl1, Zangptl2 and Zangptl6) highly homologous to mammalian Angptl1/ARP1, Angptl2/ARP2 and Angptl6/AGF, and describe their adult and embryonic temporal and spatial expression patterns. Zangptl1 is expressed faintly in the somites, while Zangptl2 is first detected in the yolk sac extension, spinal cord and branchial arches and is later expressed in the liver primordium and pectoral fin buds. Zangptl6 is expressed in the notochord. In addition to its embryonic expression, Zangptl2 is induced in adult fish during fin regeneration.     

Effects of acute exercise on insulin binding to erythrocytes in normal men

Ten normal men were subjected to acute mild and moderate exercise tests, and the insulin binding to erythrocytes was determined before and immediately after exercise and also 10, 30 and 60 minutes after exercise in each test. The insulin binding significantly increased immediately after acute moderate exercise and did not increase immediately after acute mild exercise. In contrast, it decreased below the basal level at 30 and 60 minutes in each test. The changes in insulin receptor binding were due mainly to an alteration in insulin receptor affinity rather than a change in receptor number. These results suggest that isolated erythrocytes may be of some value for study of the effect of physical exercise on the insulin receptor.     

Origin of annual laminations in tufa deposits, southwest Japan

Laminated tufas and a tufa-depositing stream in SW Japan (Shirokawa, Ehime Prefecture) were studied monthly over a 3-yr period. A series of samples from the tufa clearly reveals the pattern of annual laminations. The annual layering pattern was primarily controlled by changes in the rate of calcite precipitation, as calculated from water chemistry. The concentration of dissolved CaCO₃, which correlates with the precipitation rate, was high in summer-autumn and low in winter-spring, owing to changes in the partial pressure of CO₂ in underground air. Regular seasonal changes in underground PCO₂ probably resulted from two temperature-dependent processes, the diffusion of soil CO₂ and the ventilation of underground air. These changes, in addition to water temperature changes, altered the precipitation rate, which has a clear seasonal pattern, especially in the lower stream. The seasonal precipitation rate was high in summer-autumn and low in winter-spring, which is consistent with the seasonal lamination pattern seen in the tufas. The textures of collected samples show that the laminations consist of densely calcified summer-autumn (June-October) laminae and lightly calcified winter-spring (November-May) laminae. We infer that the increased precipitation rate stimulated thick calcite encrustation on cyanobacterial filaments to produce the dense textures. This interpretation is supported by the lowered organic/inorganic carbon-production ratio in summer-autumn. Seasonal variations in cyanobacterial assemblages are present, but do not reflect the seasonal lamination pattern. Because the relevant processes are temperature dependent, the seasonal lamination pattern at Shirokawa is thought to generally apply to other laminated tufas deposited in temperate climates. However, a reversed pattern can result from local and climatic circumstances. Dense laminae were deposited in winter near the source of the spring at Shirokawa, because calcite precipitation was high owing to low underground PCO₂ in winter. Reversed patterns reported from northwestern Europe were probably influenced by seasonal rainfall, which is reflected in hydrological conditions.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry,Prunus cerasus and P. avium

This study describes a novel F-box protein gene in the S-locus of sour cherry (Prunus cerasus) and sweet cherry (P. avium). The gene showed an S-haplotype-specific sequence polymorphism and the expression was specific to pollen. Genomic DNA blot analysis of eight sweet cherry cultivars with the probe for the F-box protein gene under low stringency conditions yielded RFLP bands specific to the S-haplotypes of each cultivar. We discuss the possibility of the gene for the F-box protein being a candidate for the male determinant of gametophytic self-incompatibility in PRUNUS:     

Effects of OP-1206 alpha-CD on walking dysfunction in the rat neuropathic intermittent claudication model: comparison with nifedipine, ticlopidine and cilostazol

The systemic treatment effects of OP-1206 alpha-CD (17S-20-dimethyl-trans-delta 2-PGE1 alpha-cyclodextrin clathrate), a prostaglandin E1 (PGE1) analogue, on walking dysfunction, spinal cord blood flow (SCBF) and skin blood flow (SKBF) were assessed in the rat neuropathic intermittent claudication (IC) model in comparison with nifedipine (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate), ticlopidine (5-[(2-chlorophenyl)methyl]-4,5,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride) and cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)-butoxy]-3,4-dihydro-2(1H)-quinolinone). Two pieces of silicone rubber strips were placed in the lumbar (L4 and L6) epidural space in rats. After surgery, walking function was measured using a treadmill apparatus. SCBF and SKBF were measured using a laser-Doppler flow meter. Drugs were administered orally twice a day for 11 days from day 3 post-surgery. Treatment with OP-1206 alpha-CD significantly improved walking dysfunction on days 5, 7 and 14, and improved SCBF on day 14 post-surgery. SKBF remained unaffected. Treatment with nifedipine, ticlopidine or cilostazol had no significant effects on any of the parameters measured in this model. These data suggest that the therapeutic effect of OP-1206 alpha-CD is primarily mediated by the improved local SCBF at the territory of spinal stenosis and not due to improvement of peripheral perfusion and/or antiplatelet activity.