Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Alcohol dehydrogenase polymorphism in Apis mellifera

A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1 ¹, Adh-1², and Adh-1 ³. The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1 ¹ and Adh-1², respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1 ³ was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.This work was supported by the Brazilian Research Council (CNPq) and S~ao Paulo State Research Foundation (FAPESP). This study was performed by the senior author in partial fulfillment of the requirements for a master's degree and was directed by Dr. M. A. Mestriner, Genetics Department, University of São Paulo at Ribeirão Preto.     

Evidence and properties of digestive proteases in young mugilidae during the first year of life]

Proteolytic activity was observed in extracts from the digestive tract of Mugil auratus and Mugil capito; its maximum occurs at about pH 2 and 9. Activity in the acid range is mainly found in stomach extracts and the enzyme may be considered as similar to pepsin and cathepsins. In the alkaline range, the main activity is recorded in the region of the pyloric caeca. Activities of the trypsin, chymotrypsin, A and B carboxypeptidases and elastase type are found. The effects of pH and temperature on some activities are studied: optimal pH are about 2.6 and 3,4 with pepsin-like activity, between 8,1 and 8,5 with trypsin-like activity and between 7,8 and 8,5 with chymotrypsin-like activity; incubation optimal temperature (pH 2,2) is found between 35 degrees and 40 degrees.     

转Btc-ry1 Ah基因玉米花粉对龟纹瓢虫解毒酶和中肠蛋白酶活性的影响

研究了取食转Bt-cry1Ah基因玉米花粉对龟纹瓢虫Propylaea japonica（Thunberg）体内解毒酶和中肠蛋白酶活性的影响。利用饲喂结合比色方法,比较龟纹瓢虫取食转Bt-cry1Ah基因玉米花粉和非转基因玉米花粉后体内α-乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽-S-转移酶、中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的酶活性。结果发现：在解毒酶方面,取食Bt玉米花粉的龟纹瓢虫4龄幼虫和蛹的α-乙酸萘酯酶活性显著低于取食非Bt玉米花粉的龟纹瓢虫（对照组）,取食Bt玉米花粉的龟纹瓢虫的乙酰胆碱酯酶和谷胱甘肽-S-转移酶活性在各个发育时期与对照相比均无显著差异。在中肠蛋白酶方面,与对照组相比,取食Bt玉米花粉的龟纹瓢虫的总蛋白酶和强碱性类胰蛋白酶活性在各个发育时期均无显著差异;但取食Bt玉米花粉的龟纹瓢虫的弱碱性类胰凝蛋白酶和类胰凝乳蛋白酶活性在蛹期显著低于取食非Bt玉米花粉的龟纹瓢虫。由此可见,龟纹瓢虫取食含有Cry1Ah杀虫蛋白的玉米花粉后,体内代谢解毒酶和中肠蛋白酶与Cry1Ah杀虫蛋白相互作用,可能会引起某些酶活性的变化。因此,转cry1Ah基因玉米花粉对龟纹瓢虫的潜在影响还需要进一步的研究。     

Enzymatic properties of the proteasome purified from starfish oocytes and its catalytic subunits involved in oocyte maturation

The 20S proteasome was purified from oocytes of the starfish Asterina pectinifera and its enzymatic properties were investigated. The chymotrypsin-like activities were potently inhibited by PSI as well as MG115, whereas the trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were not or only weakly inhibited by PSI and MG115. The inhibitory ability of MG115 toward germinal vesicle breakdown (GVBD) coincided with those toward the trypsin-like and PGPH activities, and PSI showed no inhibitory effect on GVBD. We have previously reported that the inhibition pattern toward GVBD of peptidyl-argininals, which potently inhibited the proteasomal trypsin-like activity rather than the chymotrypsin-like activity, correlated with the inhibition pattern toward the chymotrypsin-like activity of the proteasome. These results, together with the peptidyl-argininals scarcely inhibiting the PGPH activity at concentrations sufficient for the inhibition toward GVBD, indicate that both the chymotrypsin-like and trypsin-like activities, but not the PGPH activity, of the proteasome are responsible for degradation of the physiological substrate during starfish oocyte maturation. It was also suggested that the inhibition of a single catalytic site of the proteasome is not sufficient for prevention of the proteasomal function.     

Distribution of haemolymph in the honeybee (Apis mellifica) in relation to season,age and temperature

[¹⁴C]Polyethylene glycol was proved to be a suitable marker for the determination of the extracellular space, which is identical with the volume of the haemolymph in insects. Bees kept at temperatures above 22°C show a fast circulation of haemolymph independent of the season. In winter at 5–25°C, immobilized isolated bees have a thoracic temperature which differs very little from that of the ambient. Under these conditions the time of mixing and the amount of haemolymph in head, thorax and abdomen depends on the temperature. At 5°C bees have much more haemolymph in their heads than at 25°C. At low temperatures the mixing time was increased and finally the mixing process was nearly stopped. Although bees are motionless at 5°C, limited activity of the circulatory system could be shown.The correlation between these distribution studies and the functional status of the bees within their colony is discussed.     

The ontogeny of immunity: development of innate immune strength in the honey bee (Apis mellifera)

Honey bees (Apis mellifera) are of vital economic and ecological importance. These eusocial animals display temporal polyethism, which is an age-driven division of labor. Younger adult bees remain in the hive and tend to developing brood, while older adult bees forage for pollen and nectar to feed the colony. As honey bees mature, the types of pathogens they experience also change. As such, pathogen pressure may affect bees differently throughout their lifespan. We provide the first direct tests of honey bee innate immune strength across developmental stages. We investigated immune strength across four developmental stages: larvae, pupae, nurses (1-day-old adults), and foragers (22-30 days old adults). The immune strength of honey bees was quantified using standard immunocompetence assays: total hemocyte count, encapsulation response, fat body quantification, and phenoloxidase activity. Larvae and pupae had the highest total hemocyte counts, while there was no difference in encapsulation response between developmental stages. Nurses had more fat body mass than foragers, while phenoloxidase activity increased directly with honey bee development. Immune strength was most vigorous in older, foraging bees and weakest in young bees. Importantly, we found that adult honey bees do not abandon cellular immunocompetence as has recently been proposed. Induced shifts in behavioral roles may increase a colony's susceptibility to disease if nurses begin foraging activity prematurely.     

Cry2Ab及Cry1Ac杀虫蛋白对棉铃虫中肠蛋白酶活性的影响

为明确Cry2Ab和Cry1Ac2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera（Htibner）中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异。结果发现：Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6．67ug／gCry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6．43倍。Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理。Cru2Ab和Cry1Ac2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2．22ug／g混用时,处理12h后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照。     

Significance of microflora in proteolysis in the colon

Protease activities in human ileal effluent and feces were compared by using a variety of native and diazotized protein substrates. In many cases the diazotized proteins had altered susceptibilities to hydrolysis compared with the native proteins. Proteolytic activity was significantly greater than (P less than 0.001) in small intestinal effluent than in feces (319 +/- 45 and 11 +/- 6 mg of azocasein hydrolyzed per h per g, respectively). Moreover, fecal proteolysis was qualitatively different in that ileal effluent did not hydrolyze the highly globular protein bovine serum albumin, whereas all fecal samples tested degraded this substrate. Inhibition experiments provided further evidence that fecal protease activity differed from that in the small intestine. Physical disruption of fecal bacteria released large quantities of proteases, indicating that the lysis of bacteria in the colon may contribute to the extracellular proteolytic activity in feces. Protease inhibition studies with washed fecal bacteria showed that they produced serine, cystine, and metalloproteases, and experiments with synthetic p-nitroanilide substrates indicated that low levels of trypsin- and chymotrypsin-like activities were associated with whole cells. An elastase-like enzyme was bound to the outer membranes of some fecal bacteria.     

Significance of microflora in proteolysis in the colon.

Protease activities in human ileal effluent and feces were compared by using a variety of native and diazotized protein substrates. In many cases the diazotized proteins had altered susceptibilities to hydrolysis compared with the native proteins. Proteolytic activity was significantly greater than (P less than 0.001) in small intestinal effluent than in feces (319 +/- 45 and 11 +/- 6 mg of azocasein hydrolyzed per h per g, respectively). Moreover, fecal proteolysis was qualitatively different in that ileal effluent did not hydrolyze the highly globular protein bovine serum albumin, whereas all fecal samples tested degraded this substrate. Inhibition experiments provided further evidence that fecal protease activity differed from that in the small intestine. Physical disruption of fecal bacteria released large quantities of proteases, indicating that the lysis of bacteria in the colon may contribute to the extracellular proteolytic activity in feces. Protease inhibition studies with washed fecal bacteria showed that they produced serine, cystine, and metalloproteases, and experiments with synthetic p-nitroanilide substrates indicated that low levels of trypsin- and chymotrypsin-like activities were associated with whole cells. An elastase-like enzyme was bound to the outer membranes of some fecal bacteria.     

The colony environment, but not direct contact with conspecifics, influences the development of circadian rhythms in honey bees

Honey bee (Apis mellifera) workers emerge from the pupae with no circadian rhythms in behavior or brain clock gene expression but show strong rhythms later in life. This postembryonic development of circadian rhythms is reminiscent of that of infants of humans and other primates but contrasts with most insects, which typically emerge from the pupae with strong circadian rhythms. Very little is known about the internal and external factors regulating the ontogeny of circadian rhythms in bees or in other animals. We tested the hypothesis that the environment during early life influences the later expression of circadian rhythms in locomotor activity in young honey bees. We reared newly emerged bees in various social environments, transferred them to individual cages in constant laboratory conditions, and monitored their locomotor activity. We found that the percentage of rhythmic individuals among bees that experienced the colony environment for their first 48 h of adult life was similar to that of older sister foragers, but their rhythms were weaker. Sister bees isolated individually in the laboratory for the same period were significantly less likely to show circadian rhythms in locomotor activity. Bees experiencing the colony environment for only 24 h, or staying for 48 h with 30 same-age sister bees in the laboratory, were similar to bees individually isolated in the laboratory. By contrast, bees that were caged individually or in groups in single- or double-mesh enclosures inside a field colony were as likely to exhibit circadian rhythms as their sisters that were freely moving in the same colony. These findings suggest that the development of the circadian system in young adult honey bees is faster in the colony than in isolation. Direct contact with the queen, workers, or the brood, contact pheromones, and trophallaxis, which are all important means of communication in honey bees, cannot account for the influence of the colony environment, since they were all withheld from the bees in the double-mesh enclosures. Our results suggest that volatile pheromones, the colony microenvironment, or both influence the ontogeny of circadian rhythms in honey bees.     

Proteolytic activity in Plagiodera versicolora Laicharting (Coleoptera: Chrysomelidae): Characterization of digestive proteases and effect of host plants

Proteolytic profiles in the midgut of Plagiodera versicolora were studied using biochemical approaches, and the effects of host plants on possible changes in their activity were determined. Morphology of the alimentary canal revealed several areas of sections, namely bucca, pharynx, esophagus, crop, midgut, ileum, rectum and anus. A pH of 6 and 11 was found to be optimal for soluble and membrane-bound fractions, by using azocasein 2% as a substrate. Determination of specific proteases demonstrates the presence of trypsin-like, chymotrypsin-like, elastase, cathepsin B, cathepsin L and cathepsin D, as well as two exopeptidases. Regarding site of activity for each specific protease, it was found that the major activity of cathepsin B and cathepsin L was in the soluble fraction, chymotrypsin, cathepsin D and two exopeptidases in membrane-bound fraction. Additionally, trypsin-like and elastase activities had no significant differences between fractions. The presence of the above mentioned specific proteases was verified using the specific inhibitors PMSF, TLCK, TPCK, cystatin, phenanthroline and DTT. Feeding of the beetle on four host plants: including Salix aegyptica, S. alba, Populus alba and P. caspica, from the 1st larval instar to adult, revealed the highest trypsin-, chymotrypsin-like and elastase activities in the individuals fed on S. aegyptica and S. alba, respectively. Regarding cathepsins B and L, the highest activities were observed on S. alba and S. aegypticum but cathepsin D was higher in S. Alba and P. alba. Feeding on S. alba and S. aegypticum showed the highest activities of amino- and carboxy-peptidases, respectively.     

虫蛹活性多肽的制备、分离和生物活性研究进展

我国拥有悠久的蜂、蚕养殖历史,其虫蛹富含各种营养物质,其中蛋白质的含量最高。现代营养学研究发现,通过酶解等生物手段从虫蛹蛋白中分离出的生物活性肽,具有多种生物活性,如抗氧化、抗肿瘤、降血压和增强免疫等,同时可有效地提高虫蛹的利用率。基于此,就近年来国内外对虫蛹多肽的制备、分离纯化的方法以及生物活性的研究进展作一综述,以期为虫蛹多肽的深入研究与开发提供参考。     

Modulation of the chymotrypsin-like activity of the 20S proteasome by intracellular redox status: effects of glutathione peroxidase-1 overexpression and antioxidant drugs

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.     

The effects of zinc deficiency on pancreatic carboxypeptidase activity and protein digestion and absorption in the rat

1. Proteolytic enzyme activities were examined in the pancreas of zinc-deficient and control rats. 2. No change was detected in trypsin-plus-chymotrypsin activity. 3. Carboxypeptidase activity was appreciably lowered in zinc deficiency and returned rapidly to normal on zinc therapy. 4. In experiments in which U-¹⁴C-labelled Chlorella protein was fed no evidence was obtained which suggested that the reduction in carboxypeptidase activity had limited the rate of protein digestion or absorption. 5. The specific activity of pancreatic protein synthesized during these experiments was appreciably lower in zinc-deficient than in control rats. 6. A higher proportion of the total activity present, in each organ examined, was in the non-protein fraction in zinc-deficient rats.     

Acetylcholinesterase in Apis mellifera head during post-embryonic development. Existence of a glycoinositol-anchored membrane form at eary pupal stages

• 1.1. Properties of acetylcholinesterase (AChE, EC 3.1.1.7) from Apis mellifera head were studied during pupal development and at the adult stage.
• 2.2. During post-embryonic development, tissue and specific activities were closely related and increased to reach a maximum value at emergence and at last pupal stage, respectively.
• 3.3. In adults, AChE activity was weaker in foragers than in emerging bees.
• 4.4. The membrane form occurred in adult bees as well as in pupae whereas the soluble enzyme only appeared from Pd pupal stage.
• 5.5. The proportion of soluble and membrane forms fluctuated during late development but, in all cases, the percentage of the soluble form remained less than 10% of total AChE activity.
• 6.6. At all post-embryonic stages, the membrane form was sensitive to the action of phosphatidylinositol-specific phospholipase C (PI-PLC) and was converted into a hydrophilic enzyme.
• 7.7. In adult bees, the sensitivity to PI-PLC depended on the season. In summer, about 60% of the membrane activity could be solubilized by PI-PLC vs only 5% in winter.
• 8.8. The sensitivity of AChE to pirimicarb varied with the developmental stage.
• 9.9. In foraging bees, AChE was more susceptible to pirimicarb than in emerging bees. This difference of sensitivity to carbamate was abolished after removal of the membrane anchor either by mild trypsin digestion of PI-PLC treatment.

     

Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor

The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 10³ or 10⁵ fold the levels of their uninfested brood. In contrast, POL (10² or 10⁴ fold) and RHB (10² or l0⁴ fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism.     

蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响

为明确蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响,采用室内人工接虫和生化测定的方法,研究了在离体条件和饲喂条件下4种蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶的抑制作用,并测定了绿豆象幼虫取食不同含量的绿豆胰蛋白酶抑制剂(MBTI)的人工绿豆后,其中肠内总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的变化.结果表明:在离体条件下,供试4种蛋白酶抑制剂对绿豆象幼虫总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性均有明显的抑制作用,且浓度越大,抑制效果越显著,其中以20μg·mL^-1的MBTI对3种酶活性的抑制效果最强,3种酶活性分别比对照降低了62.5%、41.2%和38.7%,而卵粘蛋白抑制剂(OI)抑制效果最弱.绿豆象幼虫取食含不同抑制剂的人工绿豆后,中肠内3种酶活性也均受到一定的抑制作用,取食后随龄期的延长,3种酶活性有所升高但仍显著低于对照,且以MBTI的抑制作用最强.当绿豆象幼虫取食不同含量MBTI的人工绿豆后,随MBTI含量的增加,对总蛋白酶活性和类胰蛋白酶活性的抑制作用均逐渐增强,但对类胰凝乳蛋白酶活性的抑制作用并不显著,只有当MBTI含量达20%时,对类胰凝乳蛋白酶活性才表现出明显的抑制作用.