Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

Immunohistochemical distribution of basic fibroblast growth factor in experimental retinal ischaemia and reperfusion in the rat

Summary It has been proposed that basic fibroblast growth factor (basic FGF) mediates the neovascular response in a variety of conditions, including diabetic retinopathy and branch retinal vein occlusion. To test the hypothesis that basic FGF was released from retinal stores as a result of retinal ischaemia, transient retinal ischaemia was induced, followed by 48 h of reperfusion, in the rat by combined central retinal vasculature and optic nerve ligation. The immunolocalization of basic FGF was studied in the retina. We found that basic FGF in the normal retina is present around the deeper retinal vessels and in the neuronal tissue of the outer plexiform layer. In the eyes that had ischaemia followed by reperfusion, there was moderate cellular oedema with retinal swelling, and mitoses in the inner nuclear and plexiform layers. There were no changes evident at the immunohistochemical level either in the intensity or distribution of stores of basic FGF. We conclude from these data that stores of basic FGF are not altered dramatically under the conditions of transient experimental ischaemia and reperfusion in the rat, despite the presence of cellular proliferation.     

The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2–3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle orl-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

The Structure and Origin of the Plasmodium of Rhopalura ophiocomae(Phylum Orthonectida)

Abstract Adult orthonectids develop from germinal cells within a cytoplasmic matrix called a plasmodium. This is generally assumed to be formed by the parasite. In the case of Rhopalura ophiocomae, which lives in the brittle star Amphipholis squamata, the plasmodia occupying the perivisceral coelom are closely associated with the walls of the genital bursae or the gut, and they are covered by peritoneum. They have been reported to contain scattered small nuclei distinct from those within germinal cells, embryos, and adults, but the results of the present study indicate that such nuclei probably do not exist. Furthermore, electron micrographs show that some plasmodia are in continuity with the cytoplasm of contractile cells that lie beneath the peritoneum of a genital bursa or the gut of the host. The matrix of a plasmodium of R. ophiocomaeappears, therefore, to consist of cytoplasm of a contractile cell. It is proposed that after a contractile cell has been entered by an infective cell of the parasite, it hypertrophies, bulging progressively farther into the perivisceral coelom and lifting up the peritoneum, which remains in intimate contact with it.