Purification and characterization of a secreted protease from Tetrahymena pyriformis

A simple major protease, secreted into the medium during growth of Tetrahymena pyriformis strain W, has been purified about 4000-fold by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and affinity chromatography on organomercurial-Sepharose. The purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22 000-23 000. Amino acid analysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena protease consists of multiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5-8.0 with alpha-N-benzoyl-DL-arginine-p-nitroanilide and with azocasein, while it was in the lower pH range (4.5-5.5) for denatured hemoglobins. The purified enzyme was inhibited by compounds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.     

Oxygen consumption by eggs and larvae of striped mullet, Mugil cephalus, in relation to development, salinity and temperature

Oxygen consumption rates during embryonic and the first 38 days of larval development of the striped mullet were measured at 24° C by differential respirometry. Measurements were obtained at the blastula, gastrula and four embryonic stages, and at the yolk-sac, preflexion, flexion and post-flexion larval stages.
Oxygen uptake rates of eggs increased linearly from 0.024 μl O₂ per egg h^-1 (0·323 μl O₂ mg^-1 dry wt h^-1) by blastulae to 0·177 μlO₂ per egg h^-1 (2·516 μlO₂mg ¹dry wth^-1) by embryos prior to hatching. Respiration rates did not vary significantly among four salinities (20,25, 30, 35%₀).
Larval oxygen consumption increased in a curvilinear manner from 0·243 μl O₂ per larva h^-1 shortly after hatching to 18·880 μl O₂ per larva h^-1 on day 38. Oxygen consumption varied in direct proportion to dry weight. Mass-specific oxygen consumption rates of preflexion, flexion, and postflexion larvae did not change with age (10·838 μl O₂ mg ¹dry wt h^-1).
Larval oxygen consumption rates did not vary significantly among salinities 10–35%. Acute temperature increases elicited significant increases in oxygen consumption, these being relatively greater in yolk-sac larvae ( Q₁₀ = 2·75) than in postflexion larvae ( Q₁₀ = 1·40).     

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.     

Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to beta-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The beta-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.     

Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females

Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.     

Pathway-specific profiling identifies the NF-kappa B-dependent tumor necrosis factor alpha-regulated genes in epidermal keratinocytes

Identification of tumor necrosis factor alpha (TNF alpha) as the key agent in inflammatory disorders led to new therapies specifically targeting TNF alpha and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNF alpha, drugs targeting TNF alpha have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNF alpha therapy. TNF alpha regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNF alpha signals to the nucleus. Hypothesizing that specific TNF alpha-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-kappa B-specific inhibitor, to identify the NF-kappa B-dependent set of the TNF alpha-regulated genes in human epidermal keratinocytes. Expression of approximately 40% of all TNF alpha-regulated genes depends on NF-kappa B; 17% are regulated early (1-4 h post-treatment), and 23% are regulated late (24-48 h). Cytokines and apoptosis-related and cornification proteins belong to the "early" NF-kappa B-dependent group, and antigen presentation proteins belong to the "late" group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-kappa B-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-kappa B pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-kappaB binding sites in their promoter DNA and are, presumably, directly regulated by NF-kappa B, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-kappa B-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNF alpha-dependent signaling pathway may inhibit a specific TNF alpha-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.     

Antisense oligodeoxyribonucleotide as to the growth factor midkine suppresses neointima formation induced by balloon injury

Restenosis is the major clinical problem of angioplasty. We have previously shown that neointima formation is strikingly suppressed in midkine (MK)-deficient mice. Neointima formation is restored if MK protein is administrated to the deficient mice. MK is a heparin-binding growth factor and implicated in the migration of inflammatory cells and vascular smooth muscle cells. Consistently, the suppression of neointima formation in the deficient mice is accompanied by suppression of recruitment of inflammatory cells into the vascular wall. Here, we evaluated the potential of MK antisense oligodeoxyribonucleotide (ODN) for the prevention of restenosis. We cloned the cDNA of rabbit MK, which showed a strongly conserved sequence in mammals. The balloon injury induced MK expression, with the maximum level occurring 7-14 days after angioplasty, in the rabbit carotid artery. Two antisense ODNs suppressed the production of MK in a rabbit kidney cell line, RK13 cells, one of which was then transfected into the arterial wall by means of lipofection immediately after balloon treatment. The antisense ODN suppressed MK induction in vivo and consequently suppressed neointima formation to 60% of the control level. These results suggest that MK is a candidate molecular target for the therapy for vascular restenosis.     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)