Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K_m was 0.17 mM for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K_i of 2–3 mM. Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO₂ elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Trypanosoma rangeli: Differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation

In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.     

Lactobacillus coryniformis subsp. coryniformis Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound

The antifungal activity spectrum of Lactobacillus coryniformis subsp. coryniformis strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds Aspergillus fumigatus, A. nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, F. graminearum, F. culmorum, and F. sporotrichoides. A weaker activity was observed against the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. The yeasts Rhodotorula glutinis, Sporobolomyces roseus, and Pichia anomala were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121°C for 15 min. Maximum activity was observed at pH values of between 3.0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH₄)₂SO₄ precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from L. coryniformis subsp. coryniformis.     

Biomphalaria glabrata: lysozyme activities in the hemolymph, digestive gland, and headfoot of the intermediate host of Schistosoma mansoni.

Lysozyme (mucopeptide N-acetylmuramylhydrolase EC 3.2.1.17) activity has been found in the hemolymph, digestive gland, and headfoot extracts of Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. Partial purification of the bacteriolytic enzyme was attained by gel chromatography on Sephacryl S-200 and active lytic fractions were concentrated by Amicon filtration. The properties of the lytic enzymes from the three tissue extracts were identical. Enzyme activity was determined by the rate of lysis of cell wall suspension of Micrococcus lysodeikticus. Lysis of the cell walls was accompanied by a release of reducing sugar groups and N-acetylhexosamines. The enzyme was stable to heating at 100 C for 2 min and had an optimum activity at pH 4.5 to 5.0 in 0.066 M glycylglycine buffer. Low concentrations (5 mM) of NaCl, KCl, and LiCl increased the activity of the enzyme, whereas high concentrations (25 mM) of the same ions caused about 50% inhibition of the enzyme activity. MgCl₂ and CaCl₂ also inhibited the enzyme activity. Addition of 1 mM EDTA or EGTA resulted in about a twofold increase in enzyme activity. Double reciprocal plots of enzyme velocities and substrate concentrations yielded an apparent Michaelis-Menten constant (K_m) of 0.05 ± 0.01 mg/ml of M. lysodeikticus.     

Inhibition of muscle phosphorylase a by natural components of the sarcoplasm

Using a reconstituted glycolytic enzyme system from muscle tissue, it was shown that phosphorylase activity was regulated by some process to provide only the required amount of glucose 1-phosphate, regardless of the percentage of phosphorylase in the a form. By carrying out phosphorylase a assays at high enzyme concentration (2 mg ml^?1), the same concentration as in the reconstituted system and comparable with in vivo, it was shown that (a) the K_m for phosphate was higher and V lower than at low enzyme concentration (2 μg ml^?1), (b) the presence of other glycolytic enzymes at 40 mg ml^?1 suppressed the activity a further threefold, and (c) phosphocreatine inhibited the enzyme. Taken together, these three effects were sufficient to explain the relative lack of activity of phosphorylase a in the reconstituted system. The inhibition by phosphocreatine is seen as a mode of feedback control on phosphorylase activity in vivo.     

Do tigers hunt during the day? Diel activity of the Asian tiger mosquito,Aedes albopictus (Diptera: Culicidae), in urban and suburban habitats of North America

BackgroundAedes (Stegomyia) albopictus (Skuse) impacts human outdoor activity because of its aggressive biting behavior, and as a major vector of mosquito-borne diseases, it is also of public health importance. Although most mosquito species exhibit crepuscular activity by primarily host seeking at dawn and dusk, Ae. albopictus has been traditionally characterized as a diurnal or day-biting mosquito. With the global expansion and increased involvement of Ae. albopictus in mosquito-borne diseases, it is imperative to elucidate the diel activity of this species, particularly in newly invaded areas.Methodology and principal findingsHuman sweep netting and carbon dioxide-baited rotator traps were used to evaluate the diel activity of Ae. albopictus in two study sites. Both trapping methods were used in New Jersey’s Mercer County, USA (temperate/urban), while only human sweep netting was used in Florida’s Volusia County, USA (subtropical/suburban). Human sweep netting was performed to determine adult mosquito activity at Sunrise, Solar Noon, Sunset, and Lunar Midnight. Because New Jersey is in a temperate area, diel activity was investigated during the early season (3–19 July), peak season (25 July-19 September), and late season (22 September- 22 October). Aedes albopictus showed the highest activity during peak and late seasons at Solar Noon (P < 0.05). At Sunrise and Sunset during the peak season, Ae. albopictus activity was similar. Lunar Midnight activity was significantly lower than Sunrise and Solar Noon (P < 0.05) but was similar to that of Sunset. In the late season, the highest activity was observed during Solar Noon while the least activity was observed during Sunrise and Lunar Midnight (P<0.05). Bottle rotator traps used in conjunction with the human sweep net technique exhibited similar results. Seasonal activity was not differentiated in Florida due to the consistent subtropical climate. The highest adult activity was observed at Sunrise using human sweep netting, but it was not significantly different from Solar Noon and Sunset. The lowest adult activity was observed at Lunar Midnight; however, it was not significantly different from Solar Noon and Sunset. These results provide evidence that the diel activity of Ae. albopictus, contrary to the common perception of its diurnal activity, is much more varied.Conclusion/SignificanceInvolvement of Ae. albopictus in the transmission of debilitating mosquito-borne pathogens such as chikungunya, dengue, and Zika virus, coupled with its affinity to thrive in human peridomestic environments, substantiates that our findings have global implications in areas where Ae. albopictus populations established. It also highlights the importance of behavioral studies of vector species which will not only help mosquito control professionals plan the timing of their control efforts but also provides empirical evidence against conventional wisdoms that may unjustly persist within public health stewards.     

Production of peroxidase with horseradish hairy root cells in a two step culture system

Horseradish hairy root cells transformed by a soil bacterium Agrobacterium rhizogenes had peroxidase (POD) activity comparable to that of the original plant root tubers. To enhance POD production by the hairy root culture, various additives to the medium were tested including casein hydrolysates and plant extracts. Polypepton addition had a significant effect on the growth and POD production; at low concentrations (below 1 g/l) the growth was stimulated, while at high concentrations (3–10 g/l), POD activity in the cells was enhanced in spite of a low growth rate. Therefore, the hairy roots were at first cultured in the medium with 1 g/l Polypepton, and then 5 g/l Polypepton was added to enhance intracellular POD activity. POD activity in this two step culture system was 5.4 times higher than that in conventional culture in Polypepton-free medium.     

Characterization of liver flavin-containing monooxygenase of the dogfish shark (Squalus acanthias) and partial purification of liver flavin-containing monooxygenase of the silky shark (Carcharhinus falciformis)

Flavin-containing monooxygenase (FMO) activity as N,N-dimethylaniline (DMA) N-oxygenation was characterized in microsomes from the smooth dogfish shark (Squalus acathias). DMA N-oxygenase activity from the liver of the dogfish shark was linear with increasing protein content and over 60 min. The optimal temperature for catalysis was 25°C with a 76 percent reduction in activity when incubated at 15°C and 99 percent loss of activity at 45°C. Optimal pH was approximately 9.6. The maximum velocity for DMA N-oxygenase activity was calculated to be 1.3 nmol min⁻¹ mg⁻¹ with an apparent Michaelis constant of 44 μM. Methimazole oxidase activity was also observed in dogfish liver microsomes which was inhibited by trimethylamine (TMA). Inhibition of DMA N-oxygenase activity by TMA and thiobenzamide was competitive, while inhibition by methimazole was not competitive. Western blot analysis indicated a single liver protein from both Squalus and Carcharhinus of approximately 50 kDa that bound to antibodies raised against FMO 2. An attempt was made to purify FMO as methimazole oxidase from the liver of the silky shark. A single peak of about 10-fold purity was observed following passage through two chromatographic media (CM-Sepharose and HA-Agarose). However, no activity was recoverable after the FMO-containing fractions were applied to a 2′5′ ADP-Sepharose column.     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Activated macrophages mediate interferon-independent inhibition of herpes simplex virus

Activated macrophages exhibit extrinsic antiviral activity (inhibition of virus replication in other cells) which may involve mechanisms similar to macrophage antitumor activity or macrophage-mediated immunosuppression. Peritoneal macrophages elicited in mice by Corynebacterium parvum vaccine suppressed the growth of herpes simplex virus (HSV) in infected cells by an interferon-independent mechanism. This was demonstrated by expression of activity against HSV-infected xenogeneic (Vero) cells. Culture supernatant fluids also did not mediate antiviral activity, and did not contain detectable levels of interferon (< 3 IU/ml). Moreover, antiviral activity was not affected by the presence of anti-mouse interferon IgG. Antiviral activity was expressed at 12–16 hr after infection, at the end of the first cycle of virus replication. Cell contact was required for optimal activity. No enhanced adsorption or phagocytosis of HSV by C. parvum macrophages could be detected nor was macrophage cytotoxicity responsible for the activity. Cytotoxicity (⁵¹Cr release) by macrophages for virus infected cells was low (< 6% specific cytotoxicity), and was not significantly higher with C. parvum macrophages than with resident macrophage controls. Although C. parvum macrophages were not cytotoxic at the macrophage-host cell ratio employed, they did significantly inhibit uptake of [³H]leucine by the host Vero cells. This suggests that inhibition of host cell metabolism by the macrophage, similar to macrophage immunosuppression, may be responsible for the antiviral activity in this system.     

Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation

Influence of water activity (a_w) on biosynthesis of polygalacturonase, d-xylanase and β-glucosidase in solid culture system of Trichoderma viride TS was studied. It was found that the production of enzymes was strongly affected by water activity of substrate and nature of a_w depressor used. The polygalacturonase and d-xylanase production were maximized at a_w = 0.995 whereas β-glucosidase formation was favored at a_w = 0.96–0.98. The influence of water activity on catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as a_w depressor was also investigated. It was observed that sorbitol improved the thermal stability of polygalacturonase and d-xylanase.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

In situ comparison of activity in two deep-sea scavenging fishes occupying different depth zones

The activity of two scavenging deep-sea fishes occupying the same niche in overlapping depth zones were compared by in situ measurements of swimming speeds, tail-beat frequencies and by arrival time at baits. At 4800 m on the Porcupine Abyssal Plain, the grenadier Coryphaenoides (Nematonurus) armatus was the dominant scavenger, arriving at baits after 30 min, and swimming at relatively slow speeds of 0.17 body lengths (BL) sec^-1. At 2500 m in the relatively food rich Porcupine Seabight both C. (N.) armatus and the blue-hake, Antimora rostrata, were attracted to bait, but A. rostrata was always the first to arrive and most of the bait was consumed before the C. (N.) armatus arrived. A. rostrata swam at mean speeds of 0.39 BL sec^-1, similar to related shallow water species at equivalent temperatures. Observations on tail-beat frequency from video sequences confirmed the greater activity of A. rostrata. The data indicate that, given sufficient food supply, high pressure and low temperature do not limit activity levels of demersal deep-sea fishes. Low activity of C. (N.) armatus is an adaptation to poor food supply in the abyss, where these fishes dominate, but prevents it competing with the more active A. rostrata in shallower depths.     

Biochemical Characterization of C-type Lectin in the Diamondback Moth,Plutella xylostella (Yponomeutidae: Lepidoptera)

Lectins due to their affinity to carbohydrate moiety are involved in diverse functions like cell attachment in embryogenesis, organogenesis and cellular trafficking as well as nonself recognition in immune responses. Agglutinating activity was detectable in Plutella xylostella (Yponomeutidae: Lepidoptera) against 14 different species including bacterial and yeast cells, among which the whole body homogenate of P. xylostella agglutinated Providencia vermicola, Flavobacterium sp., and Saccharomyces cerevisiae with high titers. On analysis of physico-chemical properties, this putative agglutinating factor (s) was specifically dependent on the presence of Ca⁺⁺ for its activity and was reversibly sensitive to EDTA. The agglutinating activity was stable at pH 6–8, but was heat-labile. The agglutinating factor (s) was proteinaceous in nature as it was completely precipitable by ammonium sulphate. Its carbohydrate binding activity was demonstrated by inhibition assay, which revealed that methyl α-D-mannopyranoside inhibited agglutination against P. vermicola. In contrast, P. xylostella parasitized by an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera), also showed the agglutination properties with somewhat higher activity than the nonparasitized. Carbohydrate inhibition assay with methyl α-D-mannopyranoside was detectable at one-fold higher concentration in the homogenate of the parasitized larvae, suggesting that the agglutinating factor (s) is inducible or due to de novo parasitism-specific synthesis. These results suggest the presence of calcium-dependent lectin in P. xylostella and an alteration in the agglutinating property by C. plutellae parasitization.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Esterase activity in soy sauce Moromi as a factor hydrolyzing flavor esters

In order to elucidate the reason for the meager occurrence of volatile esters in soy sauce, the ester-decomposing activities of microorganisms concerned in soy sauce fermentation were examined. Soy yeasts showed at least 10 times higher esterase activity than the other yeasts used for fermented beverages. The yeast esterase was not greatly affected by the pH or NaCl concentration. Soy koji cultured with Aspergillus sojae or A. oryzae showed very high ester-splitting activity. By gel-filtration of koji esterase, the i-amylacetate (i-AmAc) decomposing fraction was obtained. This fraction showed a decrease of activity at lower pH or higher NaCl concentration. Koji esterase decreased its activity in moromi but remained over the entire moromi period. Koji esterase exhibited a higher activity than yeast esterase in fermenting moromi. These strong esterase activities are thought to be one of the causes of the low concentration of ester flavor in soy sauce.     

Proteinase Enzyme System of Lactic Streptococci II. Role of Membrane Proteinase in Cellular Function

The effect of several environmental conditions on the structure and activity of a membrane-associated proteinase from Streptococcus lactis was investigated. The activity of the enzyme varied with pH. Before storage at 3 C, maximal activity occurred at pH 6.0, but was minimal at this pH after storage. At all pH values tested, the enzyme was inactivated after storage. After storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicated that inactivation of the enzyme was the result of aggregation to higher molecular weight forms. p-Hydroxymercuribenzoate prevented inactivation of the enzyme during storage by preventing aggregation. Activity was correlated with disaggregation of polymer forms of the enzyme to an active monomer. The storage-inactivated enzyme could be reactivated by treatment of the enzyme with cysteine, glutathione, or ferrous ion. Glutathione enabled stored cells to produce acid at their original rate when subcultured in milk. This was attributed to the effect of glutathione on the membrane proteinase. The data suggested that the biological activity of stored cells may be dependent upon the activity of the membrane proteinase.