Reach Peak Activity Research Articles

Development of thermophilic populations, amylase and cellulase enzyme activities during thermophilic aerobic digestion of model agricultural waste slurry

Thermophilic aerobic digestion (TAD) of a model agricultural waste slurry was carried out in a continuously stirred tank reactor for a total of 156 h at 55 °C. The pH of the digestion was either unregulated or regulated at 6.0, 7.0, 8.0, 9.0 and 9.5 at 0.5 volume of air per volume of waste slurry per minute (vvm) aeration rate. The effect of aeration rate was studied at 0.1, 0.25, 0.5 and 1.0 vvm at pH 7.0. Development of thermophilic populations and amylase and cellulase enzyme activities in the digesting slurry were studied. Thermophiles developed rapidly to reach peak populations in 24 h or earlier. After 96 h, thermophiles were present mostly as spores. They were only slightly affected by aeration rate, but more by digestion pH. Amylase developed rapidly to reach peak activity before declining as rapidly. activity Cellulase was considerably affected by aeration rate but not digestion pH. Amylase profile closely reflected development of microbial activity, and so the progress of TAD. It may be useful as a control tool, either alone or in combination with other process data, for monitoring the TAD of complex waste slurries.

Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation.

Hog barn workers have an increased incidence of respiratory tract symptoms and demonstrate an increase in lung inflammatory mediators, including interleukin (IL)-8 and IL-6. Utilizing direct kinase assays for protein kinase C (PKC) activation, we demonstrated that dust from hog confinement facilities, or hog dust extract (HDE), augments PKC activity of human airway epithelial cells in vitro. A 5% dilution of HDE typically stimulates an approximately twofold increase in human bronchial epithelial cell (HBEC) PKC activity compared with control medium-treated cells. This increase in PKC is observed with 15 min of HDE treatment, and kinase activity reaches peak activity by 1-2 h of HDE treatment before returning to baseline PKC levels between 6 and 24 h. The classic PKC inhibitor, calphostin C, blocks HDE-stimulated PKC activity and associated IL-8 and IL-6 release. Desensitization to HDE stimulation of PKC activation does not appear to occur because subsequent exposures to HDE after an initial exposure result in further augmentation of PKC. Detoxification of HDE with polymyxin B to remove endotoxin did not change PKC activation or IL-8 release, suggesting that endotoxin is not solely responsible for HDE augmentation of PKC. These data support the hypothesis that HDE exposure augments HBEC IL-8 and IL-6 release via a PKC-dependent pathway.

The developing cholinergic system as target for environmental toxicants, nicotine and polychlorinated biphenyls (PCBs): implications for neurotoxicological processes in mice.

During neonatal life, offspring can be affected by toxic agents either by transfer via mother's milk or by direct exposure. In many mammalian species the perinatal period is characterized by a rapid development of the brain - "the brain growth spurt" (BGS). This period in the development of the mammalian brain is associated with numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult. In rodents, the cholinergic transmitter system undergoes a rapid development during the neonatal period, a time when spontaneous motor behaviour also reaches peak activity. We have observed that low-dose exposure to environmental toxicants such as nicotine, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDE, flame retardants) during the "BGS" can lead to irreversible changes in adult brain function in the mouse. The induction of persistent effects on behaviour and cholinergic nicotinic receptors in the adult animal appears to be limited to a short period during neonatal development. Furthermore, the neurotoxic effects were shown to develop over time, indicating a time-response/time-dependent effect. This indicates that environmental toxicants, such as nicotine, PCBs and probably PBDEs, might be involved in the slow, implacable induction of neurodegenerative disorders and/or interfere with normal aging processes.

Cell wall and membrane-associated exo-beta-D-glucanases from developing maize seedlings.

A beta-D-glucan exohydrolase was purified from the cell walls of developing maize (Zea mays L.) shoots. The cell wall enzyme preferentially hydrolyzes the non-reducing terminal glucosyl residue from (1-->3)-beta-D-glucans, but also hydrolyzes (1-->2)-, (1-->6)-, and (1-->4)-beta-D-glucosyl units in decreasing order of activity. Polyclonal antisera raised against the purified exo-beta-D-glucanase (ExGase) were used to select partial-length cDNA clones, and the complete sequence of 622 amino acid residues was deduced from the nucleotide sequences of the cDNA and a full-length genomic clone. Northern gel-blot analysis revealed what appeared to be a single transcript, but three distinct polypeptides were detected in immunogel-blot analyses of the ExGases extracted from growing coleoptiles. Two polypeptides appear in the cell wall, where one polypeptide is constitutive, and the second appears at the time of the maximum rate of elongation and reaches peak activity after elongation has ceased. The appearance of the second polypeptide coincides with the disappearance of the mixed-linkage (1-->3), (1-->4)-beta-D-glucan, whose accumulation is associated with cell elongation in grasses. The third polypeptide of the ExGase is an extrinsic protein associated with the exterior surface of the plasma membrane. Although the activity of the membrane-associated ExGase is highest against (1-->3)-beta-D-glucans, the activity against (1-->4)-beta-D-glucan linkages is severely attenuated and, therefore, the enzyme is unlikely to be involved with turnover of the (1-->3), (1-->4)-beta-D-glucan. We propose three potential functions for this novel ExGase at the membrane-wall interface.

Ethylene production, cluster root formation, and localization of iron(III) reducing capacity in Fe deficient squash roots.

Dicots and non-graminaceous monocots have the ability to increase root iron(III) reducing capacity in response to iron (Fe) deficiency stress. In squash (Cucurbita pepo L.) seedlings, Fe(III) reducing capacity was quantified during early vegetative growth. When plants were grown in Fe-free solution, the Fe(III) reducing capacity was greatly elevated, reached peak activity on day 4, then declined through day 6. Root ethylene production exhibited a temporal pattern that closely matched that of Fe(III) reducing capacity through day 6. On the 7th day of Fe deficiency, cluster root morphology developed, which coincided with a sharp increase in the root Fe(III) reducing capacity, although ethylene production decreased. Localization of Fe(III) reducing capacity activity was observed during the onset of Fe deficiency and through the development of the root clusters. It was noted that localization shifted from an initial pattern which occurred along the main and primary lateral root axes, excluding the apex, to a final localization pattern in which the reductase appeared only on secondary laterals and cluster rootlets.

The Formation of Ceramide-1-phosphate during Neutrophil Phagocytosis and Its Role in Liposome Fusion

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

Contraction of knee flexors and extensors in skiing related to the backward fall mechanism of injury to the anterior cruciate ligament

The purpose of this study was to examine the motion pattern and muscle activity during jumping and landing in downhill skiing to estimate the muscular involvement during the jump-landing-backward fall-anterior cruciate ligament (ACL) injury. A digitized 2-D video motion analysis and a synchronous 8-channel electromyography registration was performed over a jump on a downhill slope during 3 runs by 2 skiers and during 6 control jumps in the laboratory. The knee flexors (lateral gastrocnemius and biceps femoris) were recruited before touchdown and mean 60 ms earlier than the extensors (vastus lateralis and rectus femoris). Extensors and flexors reached peak activity simultaneously at the instant of landing. The knee flexion angle at the instant of landing was mean 36 degrees. As the landing was stabilized, the extensor activity persisted during eccentric work at increasing knee flexion as a corresponding flexor relaxation took place. The control jump in the laboratory showed a similar order of recruitment and timing of muscle contraction. The gastrocnemius was recruited mean 96 ms earlier and the biceps femoris mean 63 ms earlier than the extensors. This recruitment pattern during the landing movement suggest a learned motor program engaging the kinetic chain of the lower extremity to intercept the landing energy. The results also indicate that the knee flexor activity may protect the ACL at the instant of landing but that it is minimal shortly thereafter and probably not able to protect the ACL when a backward fall occurs. The knee was flexed substantially as the extensors became the dominating muscles. Thus, their ability to apply any anterior drawer force able to rupture the ACL is questioned.

Il penogramma radioisotopico dinamico con nitroglicerina sublinguale nella diagnostica non invasiva dell'impotenza

Nuclear medicine techniques may be used to test vasculogenic impotence. Dynamic radioisotope penogram with 0.3 mg sublingual nitroglycerin was performed in 50 impotent patients. The penogram index (Ip) and the time required to reach peak activity (Tp) was calculated. Ip was useful to differentiate vasculogenic and psychogenic impotence. The radioisotope penogram is a simple, less invasive and valuable screening test in the identification of vasculogenic impotence.

Osmoresponsive units in sheep median preoptic nucleus

Single-unit neural activity in the lamina terminalis, a region implicated in osmoregulation, was studied in alpha-chloralose-anesthetized sheep during mild hyperosmotic stimulation (intracarotid infusions of 1.65 M NaCl, 3 M sorbitol in 0.15 M NaCl, or 3 M urea in 0.15 M NaCl, at 1 ml/min). Twelve of 121 units (9.9%) were activated significantly (by 82 +/- 52%) by 2- to 3-min infusions of 1.65 M NaCl. Eleven of these and one untested unit were excited by hypertonic sorbitol (91 +/- 40% increase). Of five units further tested with urea, two were excited (by 19 and 58%). Isotonic or hypotonic NaCl infusions were without effect (eight osmoresponsive units tested). All responsive units were in the median preoptic nucleus (MnPO; nucleus medianus). MnPO units were compared with neurohypophysial fibers (multiunit recordings). Osmotic response profiles were similar; both MnPO units and neurohypophysial fibers responded equally to hypertonic NaCl and sorbitol but less to equiosmolal urea. Both MnPO units and neurohypophysial fibers responded slowly, taking 50 and 30 s of NaCl infusion, respectively, to show significant increases and approximately 2 min to reach peak activity. Their hemodynamic responses differed, however; neurohypophysial fibers were strongly excited by nitroprusside-induced hypotension (three of three animals) but MnPO osmoresponsive units were not (zero of five units). Osmoresponsive MnPO units may contribute osmotic, but not hemodynamic, inputs to control vasopressin secretion and/or osmoregulatory responses.

Developmental and thyroid hormone-dependent regulation of pancreatic genes in Xenopus laevis.

We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.

Neurochemical effects of prenatal treatment with ochratoxin A on fetal and adult mouse brain

Ochratoxin A (OA) is a mycotoxin produced by several storage fungi, such as Aspergillus ochraceus and several Penicillium species. OA (3 mg/kg) was given intraperitoneally to pregnant mice on day 11 of gestation (day 1 = day of insemination), and neurochemical changes in brains of their offspring were examined at fetal and adult stages. OA treatment produced retardation of intrauterine growth as well as microencephaly and reductions in total weight and DNA content of fetal brains. Specific activities of lysosomal enzymes in fetal brains began to increase by the 2nd day after treatment and to reach peak activities by the 3rd or 4th day after injection, indicative of cell death in the developing brains. Examination of brain regions of offspring three months after birth revealed that both tissue weight and DNA content were reduced to 80% of control in cerebral hemispheres (CHs; cerebral cortex and subjacent white matter, hippocampus, and amygdala) and to 90% of control in remainder of the brain (BGDM; basal ganglia, diencephalon, and mesencephalon). Total content of noradrenaline (NA), dopamine (DA) 5-hydroxytryptamine (5-HT) in treated CH showed about 15% reduction, although, expressed on a tissue weight basis, concentrations of these monoamines were increased by about 15%. Total DA content in BGDM was also reduced to 85% of controls, but total content of NA and 5-HT in BGDM and pons-medulla oblongata did not change. These results suggest that synaptogenesis of monoamine neurons in the cerebrum is impaired by prenatal treatment with OA, and that dopaminergic neurons show a slight selective vulnerability to the toxin.

Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol

AbstractThe levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0–1‐h old contain about 2.5 and 35 μ/g of the 2‐ and 26‐phosphates of 26‐hydroxyecdysone, respectively, and 1 μg/g of 26‐hydroxyecdysone. During embryogenesis of 26‐hydroxyedcdysone 26‐phosphate is hydrolyzed to 26‐hydroxyecdysone, which reaches a peak titer in 1–18‐h‐old eggs; the level of 26‐hydroxyecdysone 2‐phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26‐hydroxyecdysone 26‐phosphate decreases there is a progression of other ecdysteroids. C‐20 hydroxylation first appears in 24–40‐h‐old eggs and reaches peak activity in 48–64‐h‐old eggs, where 20‐hydroxyecdysone and 20, 26‐dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 h‐old. C‐3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3‐epi‐26‐hydroxyecdysone and 3‐epi‐20, 26‐dihydroxyecdysone are the major free ecdysteroids in 96‐h‐old eggs. A new ecdysteroid conjugate, 26‐hydroxyecdysone 22‐glucoside, first appears at 24–40h and becomes the major conjugate in 72–80‐h‐old eggs; it represents an apparent end‐product as its peak titer is reached and maintained throughout the final embryonic stages. 20‐Hydroxyecdysonoic acid occurs in 48–64‐h‐old eggs, and along with 3‐epi‐20‐hydroxyecdysonoic and ecdysonoic acids in 72–88‐h‐old eggs. 20‐Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there is a concurrent increase in the level of 3‐epi‐20‐hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0–1‐h‐old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26‐hydroxyecdysone 26‐phosphate serves as source for 26‐hydroxyecdysone and the numerous metabolites; that 26‐hydroxyecdysone and 20,26‐dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26‐phosphate in the developing eggs of m. sexta is presented.

Sexual behaviour and pheromone titre in the tomato looper, Plusia chalcites (Esp.) (Lepidoptera: Noctuidae)

The sexual behaviour of Plusia chalcites (Esp.) was recorded in the laboratory and field. Females reached peak activity on the 4th night between 0.5–2 h into the scotophase. Maximal amounts of pheromone were found in the pheromone glands of 4–5-day old females. Male activity was monitored in a wind tunnel and peak activity was observed with 5–8-day old males, 1–4 h after the onset of the scotophase. Mating occurred 1–2 h after the beginning of the scotophase. In the field, sexual activity of both sexes was synchronized to the first 2 h of night and mating pairs were collected only during the first 3 h, although single females and males were present throughout the night. This timing of sexual behaviour is probably an important factor, in addition to pheromone specificity, in the sexual separation between P. chalcites and other, sympatric, Plusiinae species.

5′-Methylthioadenosine Nucleosidase and 5-Methylthioribose Kinase Activities and Ethylene Production during Tomato Fruit Development and Ripening

5'-Methylthioadenosine (MTA) nucleosidase and 5-methylthioribose (MTR) kinase activities were measured in crude extracts of tomato fruits (Lycopersicon esculentum Mill cv Rutgers) during fruit development and ripening. The highest activity of MTA nucleosidase (1.2 nanomoles per milligram protein per minute) was observed in small green fruits. The activity decreased during ripening; at the overripe stage only 6.5% of the peak activity remained. MTR kinase activity was low at the small green stage and increased thereafter until it reached peak activity at the breaker stage (0.7 nanomoles per milligram protein per minute) followed by a sharp decline at the later stages of fruit ripening. 1-Amino-cyclopropane-1-carboxylic acid (ACC) levels peaked at the red stage, while ethylene reached its highest level at the light-red stage. Several analogs of MTA and MTR were tested as both enzyme and ethylene inhibitors. Of the MTA analogs examined for their ability to inhibit MTA nucleosidase, 5'-chloroformycin reduced enzyme activity 89%, whereas 5'-chloroadenosine, 5'-isobutylthioadenosine, 5'-isopropylthioadenosine, and 5'-ethylthioadenosine inhibited the reaction with MTA by about 40%. 5'-Chloroformycin and 5'-chloroadenosine inhibited ethylene production over a period of 24 hours by about 64 and 42%, respectively. Other analogs of MTA were not effective inhibitors of ethylene production, whereas aminoethoxyvinylglycine showed a 34% inhibition over the same period of time. Of the MTR analogs tested, 5-isobutylthioribose was the most effective inhibitor of both MTR-kinase (41%) and ethylene production (35%).

Induction of tumor-inhibitory macrophages with a novel synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738).

3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.

Ribonucleic Acid Metabolism in Rat Liver During Long-Term Adaptation to Malnutrition

Ornithine decarboxylase (ODC) and nucleolar DNA-dependent RNA polymerase (RNA polymerase I) activities increased in the liver of young adult male rats fed a 6% casein diet (malnourished) for 1 week when compared with rats fed a 25% casein diet (control). ODC activity increased progressively and reached a peak after 3 weeks of malnutrition and then decreased to control values by 5 weeks. RNA polymerase I reached peak activity 1 week after malnutrition was imposed, decreasing thereafter to control values by 3 weeks. At 4 and 5 weeks, RNA polymerase I activity in malnourished animals was lower than control. Nucleoplasmic DNA-dependent RNA polymerase activity remained unchanged in the first 2 weeks of malnutrition and decreased thereafter to values significantly lower than control. The data confirm our previous observations of cyclical changes during prolonged malnutrition and suggests a process of “biochemical adaptation” to malnutrition in which the organism enhances essential metabolic processes to maintain cellular homeostasis to the detriment of less essential functions like systemic growth.RNA polymerase malnutrition RNA metabolism polyamines ornithine decarboxylase

Myocardial kinetics of thallium-201 after dipyridamole infusion in normal canine myocardium and in myocardium distal to a stenosis.

The purpose of the present study was to define myocardial and blood thallium-201 (Tl-201) kinetics after infusion of dipyridamole in normal canine myocardium and in myocardium distal to a coronary artery stenosis. Miniature radiation detector probes were implanted in the left ventricle in 39 open-chest dogs. A balloon constrictor was placed around the proximal left circumflex coronary artery. Electromagnetic flow probes were positioned proximally around both the left circumflex and left anterior descending coronary arteries. In five control dogs (group 1) the balloon occluder was not inflated; in 12 dogs (group 2) a mild stenosis was created such that resting flow was not reduced, yet the hyperemic response after 10 s of total occlusion was partially attenuated; in nine dogs (group 3) a moderate stenosis was created such that resting flow was not reduced, yet the hyperemic response was completely eliminated; and in 13 dogs (group 4) a severe stenosis was created such that resting flow was reduced. After intravenous dipyridamole (0.08 mg/kg . min-1 x 4 min), 1.5 mCi Tl-201 was injected intravenously and probe counts were collected continuously for 4 h. The mean 4-h fractional myocardial Tl-201 clearance for nonstenotic zones was 0.35, 0.27 for group 2 stenotic zones, 0.19 for group 3 stenotic zones, and 0.05 for group 4 stenotic zones (P less than 0.0001). After reaching peak activity, myocardial Tl-201 activity cleared biexponentially with a final decay constant lambda 2 = 0.0017 +/- 0.0001 min-1 (SE) for nonstenotic zones, 0.0011 +/- 0.0001 min-1 for group 2 stenotic zones, and 0.0006 +/- 0.0001 min-1 for group 3 stenotic zones (P less than 0.01). Group 4 stenotic zone Tl-201 clearances were negligible (decay constant essentially zero). Blood Tl-201 activity decayed triexponentially with a final blood lambda 3 = 0.0018 +/- 0.0001 min-1, which was almost identical to the final myocardial lambda 2 decay constant. Thus, the rate of myocardial Tl-201 clearance can distinguish between coronary stenoses of graded hemodynamic severity. These results may be applicable to quantitative techniques for determining myocardial Tl-201 clearance rates on serial clinical images after dipyridamole administration.

Characterization of the platelet response to exogenous arachidonic acid

When aggregation and release are the end points, washed human platelets treated with a range of concentrations of arachidonic acid display a bell-shaped dose-response curve. The peak is at approximately 200 μM, followed by a sharp decrease with minimum reaction at 400 μM. Prostaglandin synthesis, measured by malondialdehyde production, reaches peak activity at 400–500 μM and does not decrease. Platelet lysis, as manifested by lactate dehydrogenase release, increases only in response to arachidonate concentrations above 400 μM. When platelets are treated with 400 μM arachidonate for three minutes, subsequent reaction with thrombin, but not with A23187, is inhibited. This inhibitory activity is not sensitive to aspirin, indomethacin or ETYA and cannot be transferred in the supernatant fraction of the platelet suspension. The inhibitory activity generated from platelets reacted with 400 μM arachidonate is not a product of the cyclooxygenase or lipoxygenase systems.

Proteases and Peptidases of Castor Bean Endosperm

The endosperm of castor bean seeds (Ricinus communis L.) contains two -SH-dependent aminopeptidases, one hydrolyzing l-leucine-beta-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-beta-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an -SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two -SH-dependent enzymes hydrolyzing the model substrate alpha-N-benzoyl-dl-arginine-beta-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6.The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence.

Tissue-factor coagulant activity of cultured human endothelial and smooth muscle cells and fibroblasts

The tissue-factor (thromboplastic) activity of cultured human endothelial cells and fibroblasts is low at time of transfer into fresh medium but increases 3-10 fold. Endothelial cells reach peak activity (400 U/10(5) cells) 5-8 hr after subculture. Activity in fibroblast cultures peaks (3000-12,000 U/10(5) cells) 7-12 hr after subculture. After attaining maximum activity, endothelial and fibroblast tissue-factor content decreases in a time course similar to other cells studied in this laboratory, approaching basal levels by 24-50 hr after subculture. If medium over fibroblasts is changed every 12 hr, activity can be sustained at the peak level for an additional day but cannot be maintained at a high level indefinitely. The kinetics of expression of smooth muscle cell tissue factor are markedly different from other cell types. There is always a pronounced lag (30 hr or more) before the activity increases, and then, in most cases, there is no subsequent decline in activity even though the cells are not refed or restimulated. The activity of each of these cell types is cryptic but becomes available after freeze-thaw disruption of cells.