Zymogen granules in enzyme liberation and activation in pea seeds.

Pea seeds have zymogen-like granules that contain an inactive form of the enzyme amylopectin-1,6-glucosidase. The enzyme can be liberated from the particles in the inactive form and can then be activated by limited proteolysis with trypsin.

Permeation of dry seeds with chemicals: use of dichloromethane.

Dichloromethane can be used to introduce chemicals into dry seeds in the absence of water. The solvent has no effect on germination or respiration. The chemicals become effective when the seeds are placed in water under normal conditions for germination. The potential of the new method is discussed.

Murine sarcoma virus pseudotypes used as immunogens against viral and chemical oncogenesis.

The purpose of this study was to develop an animal system of protective immunity against oncornaviruses and to test whether such immunization had an inhibitory effect upon chemical sarcomagenesis. Several murine sarcoma virus (MSV) pseudotypes were used as immunogens and tested against themselves, against other pseudotypes, against leukemogenesis by their helper viruses, and against sarcomagenesis by 3-methylcholanthrene. Five MSV pseudotypes were obtained by rescuing complete MSV from MSV-genome carrier, nonproducer hamster tumor cells, using five different leukemia viruses as helpers. The immunogenic properties of these pseudotypes could be specified on the basis of the following observations. 1) They all induced sarcomas in newborn mice and regressing sarcoma nodules in young adult mice. After regression, most mice remained free of neoplastic disease, but some developed sarcoma or leukemia relapses. 2) They had an individual host range pattern, usually determined by the helper virus, as tested by inoculation of a constant virus dose in BALB/c, C57BL/Ka, and Swiss mice. 3) They were all immunogenic, in the sense that the first virus inoculation prevented sarcoma induction by a second challenge, either viral or cellular. 4) They were cross-reactive in vivo, one pseudotype immunizing against another, in the combinations tested. 5) They were able to immunize against leukemogenesis induced by their helper viruses. This was shown by prevention of leukemic deaths by Rauscher and Friend viruses, by a slight prolongation of survival after challenge with the Precerutti-Law leukemia virus, and by inhibition of splenomegaly by Moloney leukemia virus. In a second stage of the study, we investigated whether immunization with any of the MSV psuedotypes had an inhibitory effect upon sarcomagenesis induced by near-threshold doses of 3-methylcholanthrene. The incidence of these sarcomas was essentially the same in virus-immunized and control mice. It was concluded that immunizing procedures able to prevent sarcomagenesis when the inducer is a virus did not have any consistent preventive effect when the inducer was a chemical.

Functional characterization of peripheral circulating and liver recruited neutrophils in endotoxic rats.

Neutrophil accumulation in tissue is a hallmark of inflammation and is associated with a variety of pathological conditions. In bacterial infection neutrophils are selectively attracted in large numbers to phagocytose and kill invading microorganisms. However, activated neutrophils can also cause injury to tissues. To investigate functional alterations in liver recruited neutrophils (PMNs), we studied the functional characteristics of circulating blood and liver sequestered PMNs in terms of host defense mechanisms, such as nitric oxide (NO) and superoxide (SO) generation, beta 2 integrin expression, phagocytosis, and eicosanoid profile. Cells were isolated from rats infused with a nonlethal dose (320 micrograms/kg) of E. coli endotoxin (ET) or pyrogen-free saline for 90 min. Liver PMNs produced significantly more NO both in the absence and in the presence of an in vitro endotoxin challenge than did blood PMNs. No significant difference was observed in phorbol myristate acetate-stimulated SO generation. Endotoxin infusion significantly up-regulated the expression of CD11b/c in circulating and even more so in liver PMNs. Phagocytosis was significantly enhanced by in vivo ET treatment in blood PMNs, and liver PMNs showed even greater phagocytic activity than blood PMNs or Kupffer cells. The percent distribution of prostaglandins D2 and E2 of total 14C-eicosanoids was significantly higher and that of thromboxane B2 and 5-, 12-, and 15-HETEs was significantly lower in liver than in blood PMNs. Our study demonstrates several functional differences between liver-recruited and circulating PMNs in an acute endotoxic model. The implications of altered neutrophil function may extend to mechanisms of host defense and hepatotoxicity associated with sepsis and endotoxemia.

Diabetes complications and mortality among Alaska Natives: 8 years of observation.

OBJECTIVE: To determine the prevalence and incidence of diabetes in Alaska Natives and the incidence of cerebrovascular accidents (stroke), myocardial infarction (MI), end-stage renal disease (ESRD), lower-extremity amputations (LEA), and mortality over a 6- to 8-year period. RESEARCH DESIGN AND METHODS: The data derive from a registry of diagnosed diabetes (World Health Organization [WHO] criteria) of the Alaska Area Native Health Service (AANHS), from medical records, and from the Alaska Bureau of Vital Statistics. RESULTS: From 1986 to 1993, the prevalence of diabetes in Alaska Natives increased by 22% from 15.7 to 19.2 per 1,000 people. Of these cases, nearly all were diagnosed with type II diabetes. During the same period, 614 new cases were diagnosed. The incidence was 1.5 per 1,000 Alaska Natives per year. The incidence of confirmed MI was 8.0 per 1,000 person-years of diabetes. Aleuts had the highest rate, followed by Indians and Eskimos. The incidence of confirmed stroke was 10.6 per 1,000 person-years of diabetes. Eskimos had a significantly higher rate than Indians (P = 0.002), and women had a higher rate than men. The incidence of LEA was 5.0 per 1,000 person-years of diabetes. The incidence rate dropped significantly after instituting a foot care program. The incidence for ESRD was 3.3 per 1,000 and also showed a decline with time. The all-cause mortality rate of 43.2 per 1,000 person-years of diabetes was nearly equal between men and women. Among Alaska Natives with diabetes, cardiovascular disease (CVD) was the most common cause of death, followed by cancer and diabetes, per se. CONCLUSIONS: We conclude that diabetes is increasing in Alaska Natives, who are experiencing both the microvascular and macrovascular complications of diabetes. The incidence of LEA and ESRD show some evidence of a decrease after intervention efforts.

Diabetes and impaired glucose tolerance in three Alaskan Eskimo populations. The Alaska-Siberia Project.

OBJECTIVE: The objectives of this study were to determine the prevalence of diabetes and impaired glucose tolerance (IGT) in three Alaskan Eskimo populations, using standardized diagnostic criteria, and to evaluate family history and obesity as risk factors. RESEARCH DESIGN AND METHODS: This cross-sectional study involved men and women > or = 25 years of age from three Eskimo ethnic groups (Siberian Yupik, Central Yupik, and Inupiat) residing in northwestern Alaska. Glucose tolerance status was defined by World Health Organization criteria and was based on a 75-g oral glucose tolerance test. Data on age, family history of diabetes, and degree of Eskimo ancestry were obtained from a personal interview. Obesity was assessed using BMI. RESULTS: A total of 454 of 899 (50.5%) eligible participants were examined for diabetic status (239 Siberian Yupik, 106 Central Yupik, and 109 Inupiat participants). The prevalence of diabetes was more than twice as high among the Siberian Yupik (9.6%) as among the Central Yupik (2.8%) and Inupiat participants (3.7%). Diabetes was more prevalent in women than men (8.8 vs. 4.2%). IGT was found in an additional 11.7% of the women and 4.7% of the men. The combined prevalence of diabetes and IGT in the population > or = 55 years of age was 30.4% (diabetes 12.0%, IGT 18.4%). Of the people identified with diabetes, 47% had not been previously diagnosed. Age-specific prevalences were similar to those found in U.S. whites in the National Health and Nutrition Examination Survey II. After adjustment for age, family history of diabetes was associated with diabetes in study participants with an odds ratio of 4.4, while obesity was associated with diabetes with an odds ratio of 2.6. CONCLUSIONS: These prevalences of diabetes are the highest yet reported among Eskimo populations. Obesity and family history of diabetes are associated with increased odds of developing diabetes. These data underscore the need to further examine risk factors and to design effective interventions.

Increased surface expression of CD11b/c and CD18 appears to be dissociated from anti-CD11b/c monoclonal antibody stimulated O2- anion generation in in vivo Escherichia coli lipopolysaccharide and tumor necrosis factor-alpha-treated rat neutrophils.

The purpose of this investigation was to determine the effect of anti-rat CD11b/c monoclonal antibody (MAb) on in vitro superoxide anion (O2-) generation in in vivo Escherichia coli lipopolysaccharide (LPS) and tumor necrosis-alpha (TNF)-treated rat polymorphonuclear leukocytes (PMN). After a continuous infusion of a nonlethal dose of E. coli LPS (.5 mg/kg) or TNF (6.0 x 10(5) units) into rats, PMN were recovered by centrifugal elutriation and discontinuous density gradient centrifugation from the liver (LPS-treated) and whole blood (LPS- and TNF-treated) and compared for CD11b/c, CD11a, and CD18 upregulation and their capacity for basal and agonist-stimulated O2- production. Immunofluorescence flow cytometry studies of rat whole blood PMN demonstrated that, upon LPS infusion, there was a time-dependent upregulation of CD11b/c and CD18, but not of CD11a. Similarly, TNF infusion upregulated CD11b/c although to a lesser degree. Stimulation of LPS- and TNF-treated PMN with phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OPZ), and anti-rat CD11b/c MAb triggered O2- generation. Although total O2- generated by OPZ and anti-rat CD11b/c MAb was less than that generated by PMA stimulation, the in vivo LPS- and TNF-induced beta 2 integrin upregulation did not result in a statistically significant enhancement of O2- generation with respect to normal saline-treated PMN. Our results do not appear to support the hypothesis that enhanced expression of CD11b/c or CD18 might be associated with enhanced in vitro anti-CD11b/c MAb-triggered O2- generation in LPS- and TNF-treated PMN in vivo.

Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation: correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-alpha, thromboxane B2, and metalloprotease release.

The effects of lipopolysaccharide (LPS) on the central nervous system, one of the first organs to be affected by sepsis, are still incompletely understood. Rat microglia (BMphi) constitute the main leukocyte-dependent source of reactive oxygen species in the central nervous system. The in vitro effect of LPS on agonist-stimulated superoxide (O2-) generation from BMphi appears controversial. Our purpose was to determine the time- and concentration-dependent effect of Escherichia coil LPS on phorbol-12 myristate 13-acetate-stimulated O2- generation from BMphi. Our results demonstrate that BMphi O2- generation in vitro peaked 17 h after stimulation of with .3 ng/mL LPS. Furthermore, stimulation of BMphi with LPS for 17 h resulted in the following concentration-dependent responses: .1-1 ng/mL LPS induced no prior mediator generation but potently enhanced subsequent phorbol-12 myristate 13-acetate-stimulated O2- generation; 3-10 ng/mL LPS caused nitric oxide, tumor necrosis factor-alpha (TNF-alpha), thromboxane B2 and matrix metalloproteinase-9 release although partially inhibiting ensuing phorbol-12 myristate 13-acetate-stimulated O2- generation; 30-100 ng/mL LPS, maximized nitric oxide, TNF-alpha, thromboxane B2, matrix metalloproteinase-9 generation with concomitant lactic dehydrogenase release although strongly deactivating successive phorbol-12 myristate 13-acetate-stimulated O2 production. Our in vitro studies suggest that enhanced release of these four mediators (nitric oxide, TNF-alpha, thromboxane B2, and matrix metalloproteinase-9) during stimulation of BMphi with LPS might play a critical role in the subsequent ability of BMphi to generate O2- in vivo. Potential clinical implications of our findings are suggested by the fact that LPS levels similar to the ones used in this study have been observed in cerebrospinal fluid both in Gram-negative meningitis and sepsis.

The possible function of the glucan sheath of Botrytis cinerea: effects on the distribution of enzyme activities.

The effect of a beta-1,3-glucanase (Glucanex) on cultures of Botrytis cinerea was examined. The enzyme released reducing sugars from the mycelium and from the glucan secreted into the culture medium. The morphology of the mycelium was changed in the presence of Glucanex. The measured activity of guaiacol peroxidase, laccase, and catalase was increased when the mycelium was treated with Glucanex. Culture of the mycelium in the presence of Glucanex resulted in an increase in catalase activity. We suggest that the glucan plays a role in protecting the fungus from host response and may assist in the initial stages of host infection.

Enzymes of Botrytis cinerea capable of breaking down hydrogen peroxide.

The amounts of intra- and extracellular guaiacol peroxidase, ascorbic peroxidase, glutathione peroxidase, superoxide dismutase, laccase, and catalase present in Botrytis cinerea, cultured in three different media: Kovac synthetic medium, Sabouraud fluid medium, and a medium containing malt extract, were determined. The activity of two enzymes, ascorbic peroxidase and glutathione peroxidase, has not been previously described in B. cinerea. The detected amount of the enzymes showed considerable variability in the three different culture media. The presence of an array of enzymes capable of metabolizing hydrogen peroxide, whose levels are determined by the conditions under which the fungus grows, shows that B. cinerea is well equipped to contend with the occurrence of host-produced active oxygen species.

Prohormone convertases PC2 and PC3 in rat neutrophils and macrophages. Parallel changes with proenkephalin-derived peptides induced by LPS in vivo.

Prohormone- or proneuropeptide-converting enzymes PC2 and PC3 have been observed exclusively in nervous and endocrine tissues. In this work the presence of these enzymes in cells of the immune system was demonstrated. PC2 was detected in peripheral and liver-infiltrating polymorphonuclear leukocytes (PMN) but not in alveolar macrophages (AM) or spleen mononuclear cells (SMC). PC2 proteins corresponded to 75, 71 and 56 kDa forms. PC3 appeared in AM and SMC but not in PMN, and a 66 kDa protein was the only PC3 form detected. Proenkephalin-derived peptides (PENKp) were observed in PMN and AM, showing peptides of 35, 28, 21, 18 and 14 kDa in the former cells and a doublet of 35 and 32 kDa in the latter. PC2 proteins and PENKp decreased in liver PMN and peripheral PMN 90 min after intravenous (i.v.) infusion of LPS, suggesting an increased release. However, in vitro assays showed that the chemotactic peptide FMLP but not LPS increased the basal secretion of PC2 proteins and PENKp in PMN. These results indicate that PC2 proteins are released from PMN, together with PENKp, and suggest that LPS in vivo may act through an indirect mechanism. Low levels of PC3 and PENK were detected in the AM of rats treated for 90 min with SAL or LPS. However, a significant increase of PC3 and PENKp appeared 30 h after LPS infusion. These results show for the first time that PC2 and PC3 are differentially expressed in PMN and AM, respectively, which were paralleled by the presence of different post-translational products of PENK. In addition, the in vivo effect of LPS on PC2, PC3 and PENKp levels in PMN and AM resembles the effect of LPS on prohormone levels in endocrine tissues, suggesting that similar mechanisms may control the turnover of PENK in endocrine and in these immune cells.

Inhibitors of polygalacturonase in calli of Orobanche aegyptiaca.

The presence of at least three distinct polygalacturonases (PGase) in callus of Orobanche was demonstrated. The PGase activity is labile and at pH 4.5 does not require activation by cations. It can be partially purified on Biogel P 100 columns and can be resolved by PAGE into several bands. Broomrape callus tissue also contains inhibitors of PGase activity. One of these is a low M(r) compound, stable to boiling and removable by dialysis. An additional inhibitor precipitable by ammonium sulphate is also present.

Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response.

The hypersensitive response (HR), elicited when resistant hosts are infected by incompatible races of biotrophic fungi, has been researched extensively. New studies on host responses to necrotrophic fungi are beginning to show that when the HR occurs in hosts colonized by necrotrophs, fungal growth is accelerated rather than retarded. We review current knowledge about how necrotrophs survive in host plants in which the HR is expressed. We discuss how necrotrophs cope with the environmental factors formed as a result of the HR. Necrotrophs contain an array of enzymes, which can help in exploiting the hostile environment in order to colonize the host and to remove or inactivate active oxygen species (AOS). Among this array of enzymes are superoxide dismutase (SOD), peroxidases, catalase, and perhaps laccases and polyphenol oxidases. Of these, only SOD and catalase have been studied in any detail. The precise significance of SOD and catalase in host invasion and fungal resistance is still not adequately known. The importance of different peroxidases is also still far from clear. We speculate that AOS species may trigger the response of necrotrophs to the host environment.

Biotransformation of constituents of essential oils by germinating wheat seed.

Wheat seeds, when exposed to essential oils, are able to metabolise certain monoterpenes. The actual amounts of the compounds and their derivatives in the endosperm and embryo of wheat seeds, after exposure to the monoterpenes were determined. Neral and geranial, which are the constituents of citral, are reduced and oxidised to the corresponding alcohols and acids. Similarly citronellal, pulegone and carvacrol are converted partly to the corresponding reduction and oxidation products. The aromatic compound vanillin is partly reduced to vanillyl alcohol or oxidised to vanillic acid. In all cases it seems that part of the compounds applied are degraded, as indicated by the inability to account for all the compounds, which were supplied to the germinated seeds. In most cases the derivatives of the essential oil applied were less toxic than the parent compound. The possible role of non-specific enzymes by which the compounds are oxidised or reduced is discussed.

Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea.

The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds. RNA was isolated from the cultures and probed with specific DNA probes for laccase. As an internal control, the RNA was probed for Botrytis beta-tubulin mRNA. From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase. This could account for the previously observed repression of laccase formation by cucurbitacins.

The marine toxin okadaic acid reduces O2- generation and tyrosine phosphorylation in LPS-primed rat neutrophils.

Contrasting effects of okadaic acid (OKA) on neutrophil (PMN) superoxide anion (O2-) generation have been reported. In this study, we examined the effect of OKA on phorbol myristate acetate (PMA)-stimulated O2- generation in rat PMNs primed with LPS in vivo (LPS-PMN) and saline-treated rat PMNs (SAL-PMN). The following results were observed: (1) OKA, but neither genistein nor vanadate, markedly reduced O2- generation in a dose and time-dependent manner; (2) genistein, a tyrosine kinase inhibitor, as well as OKA, reduced tyrosine phosphorylation; (3) sodium orthovanadate, a tyrosine phosphatase inhibitor, potently enhanced tyrosine phosphorylation. Our studies suggest that OKA might reduce tyrosine phosphorylation by affecting the activity of tyrosine phosphatases regulated by serine-threonine phosphorylation.

Pharmacological characterization of the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae.

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.

Marine pharmacology in 1999: antitumor and cytotoxic compounds.

During 1999 marine antitumor pharmacology research involved researchers in Austria, Australia, England, France, Germany, Greece, Holland, Italy, Japan, Spain, Taiwan and the United States. Thirty six papers were published in peer-reviewed journals describing the antitumor and cytotoxic properties of 30 marine natural products belonging to four structural types, namely polyketides, terpenes, nitrogen-containing compounds and polysaccharides. The organisms yielding these bioactive marine compounds comprised a diverse group of marine animals, algae, fungi and bacteria. A variety of antitumor pharmacological studies were conducted with 17 marine natural products with established mechanisms of action in a number of experimental and clinical models. Didemnin B, a tunicate-derived depsipeptide with potent antitumor effects, completed a Phase II anticancer clinical trial which resulted indeterminate in respect to activity against human melanoma due to anaphylactoid reactions. In vitro cytotoxicity data with murine and human cell lines were reported for 14 novel marine chemicals with as yet undetermined mechanisms of action. This 1999 literature overview thus highlights the fact that the multinational effort aimed at the discovery of novel marine antitumor agents remained at the same level of research activity as during 1998.

Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.

BACKGROUND: The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-). RESULTS: In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release. CONCLUSIONS: To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.