Identification of a putative vacuolar serine protease gene in the rice blast fungus,Magnaporthe grisea

We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.     

Treatment of Combined Injuries of the Axillary and Suprascapular Nerves with Scapulothoracic Dissociation

A 20-year-old man suffered the combined axillary and suprascapular nerve palsies associated with scapulothoracic dissociation by motorcycle accident. The dislocated shoulder girdle was reduced and stabilized with osteosynthesis of the fractured clavicle and reattachment of the trapezius avulsed from the scapular spine for removal of continuous traction force to these damaged nerves. Because of no evidence of recovery on manual muscle test and electromyogram, exploration for these nerves was administered 6 weeks after injury. Although neurolysis of both nerves revealed neural continuity, excessive tension still existed on the suprascapular nerve. It was thought that previous operation in which the shoulder girdle had been reduced and stabilized as much as possible could not achieve complete anatomical reduction of the scapula. As an additional treatment, medial walls of the suprascapular and spinoglenoid notches were shaven to relax the suprascapular nerve. After a year, complete recovery of both the axillary and suprascapular nerve was identified. Although scapulothoracic dissociation is commonly recognized as massive injury of the shoulder girdle with poor prognosis because of existence of accompanied severe neurovascular injuries, there are more than a few cases in which partial damage on the infraclavicular brachial plexus is only accompanied. In case of them, there is the possibility of lesions in continuity of the nerves in which good prognosis might be expected with surgical intervention including early reduction of the shoulder girdle for removal of excessive tension to the damaged nerve.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

Production of low temperature active lipase from the pink snow mold, Microdochium nivale (syn. Fusarium nivale)

Summary The pink snow mold, Microdochium nivale (syn. Fusarium nivale) SUF 1377 strain produced an extracellular low temperature active lipase during growth at 4°C. The lipase had the highest activity at 20°C, and retained 19% of its maximum activity at 0°C.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

Androgen-induced growth factor and its receptor: Demonstration of the androgen-induced autocrine loop in mouse mammary carcinoma cells

SC-3 cells derived from mouse mammary carcinoma (Shinogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18–24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matris. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant from of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant from of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.