Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

Substitution of heat from exercise and digestion by ducks diving for mussels at varying depths and temperatures

Diving birds can lose significant body heat to cold water, but costs can be reduced if heat from exercising muscles or the heat increment of feeding (HIF) can substitute for thermogenesis. Potential for substitution depends jointly on the rate of heat loss, the rate of heat produced by exercise, and the level of HIF. To explore these interactions, we measured oxygen consumption by lesser scaup ducks (Aythya affinis) diving to depths of 1.2 and 2 m at thermoneutral (23°C) and sub-thermoneutral (18 and 8°C) temperatures. Birds dove while fasted and when feeding on blue mussels (Mytilus edulis). Substitution occurred if HIF or costs of diving above resting metabolic rate (RMR) were lower at 18 or 8°C than at 23°C, indicating reduction in the thermoregulatory part of RMR. For fasted scaup diving to 1.2 m, substitution from exercise heat was not apparent at either 18 or 8°C. At 2 m depth, dive costs above RMR were reduced by 5% at 18°C and by 40% at 8°C, indicating substitution. At 1.2 m depth (with voluntary intake of only 14–17% of maintenance requirements), HIF did not differ between temperatures, indicating no substitution. However, at 2 m (intake 13–25% of maintenance), substitution from HIF was 23% of metabolizable energy intake at 18°C and 22% at 8°C. These results show that even with low HIF due to low intake rates, substitution from HIF can add to substitution from the heat of exercise.     

构建丝状真菌大片段基因组文库载体DNA的制备

载体DNA的制备是构建大片段基因组文库的关键步骤之一,高质量载体DNA受到酶切、脱磷等因素的影响,以载体pBHYG为材料,优化了限制性内切酶胁HindⅢ酶切和小牛肠碱性磷酸酶(CLAP)脱磷的作用条件,并在T4连接酶作用下自连,通过胶回收纯化制备了可用于进一步构建大片段基因组文库的线性载体DNA。     

Comparisons of digestive function between the European hare (Lepus europaeus) and the European rabbit (Oryctolagus cuniculus): Mastication, gut passage, and digestibility

The European hare Lepus europaeus and the European rabbit Oryctolagus cuniculus are sympatric in many areas of the world. They are medium-sized herbage-feeding lagomorphs and trophic competitors. Both species feed on twigs under extreme and perhaps limiting conditions. To ascertain whether fine niche separation mechanisms occur, several comparative tests of digestive function were undertaken on samples of animals drawn from sympatric populations. The weights of the organs constituting the abdominal alimentary canal, the rates of passage and the extent of trituration of dietary markers intended to mimic twigs, and the digestibility of fibre, protein, and fat were compared. Both the stomach and the caecum of the hare were significantly smaller as a proportion of body weight, and this would result in a higher power–weight ratio. Both species rapidly passed the digestive marker, but passage was significantly faster in the hare. The rabbit chewed twig-like material with a scissor cutting and crushing action, whereas the action of the hare included a stripping action that would more efficiently access soluble carbohydrates stored in vascular rays. Both species were poor digesters of fibre, but digestibility of hemicelluloses was significantly greater in the rabbit. The faeces of both species of lagomorphs contain nutrients that can be attractive to more efficient fermenters of plant fibre, and consumption of those faeces may confound lagomorph population surveys that rely on dung counts.     

Gastric processing in the Dungeness crab, Cancer magister, during hypoxia

The gastric physiology of the Dungeness crab, Cancer magister, was investigated over a range of oxygen tensions. Postprandial crabs reacted differently to hypoxia compared with unfed animals. The bradycardic response in postprandial animals was reduced, suggesting a summation of responses with feeding. A similar pattern was observed for ventilation rate. In unfed animals ventilation rate increased slightly as oxygen levels declined, but dropped significantly in oxygen tensions below 3.2 kPa, whereas in postprandial crabs it increased significantly in the lower oxygen regimes. Gastric processing of the meal was followed using a fluoroscope. Pyloric contraction rates were maintained during mild hypoxia, but decreased in 5.3 kPa oxygen tension and below. This led to an increase in clearance times of digesta from the foregut, midgut and hindgut regions. The slowing of gastric processing in the lower oxygen tensions suggested that the animals were unable to maintain their internal oxygen concentration. A significant reduction in efficiency of assimilation only occurred in the lowest oxygen regime tested (1.6 kPa). The range of hypoxia where gastric processing was affected corresponded closely to the levels of oxygen that modulate the foraging behaviour of C. magister. By using both physiological and behavioural mechanisms C. magister can maintain digestive processes, even in severely oxygen depleted environments.     

磷酸化对人肝癌细胞株SMMC-7721转醛醇酶活性的影响

目的:研究人肝癌细胞株SMMC-7721中转醛醇酶(Transaldolase,TAL)活性变化与磷酸化的关系。方法:SMMC-7721细胞培养,细胞周期同步化后分组加不同浓度(10%、1%)胎牛血清孵育,分别测定不同孵育时间(1、2、4、8h)TAL酶活性;Western blot检测TAL蛋白表达水平。磷酸酶消化法检测TAL活性变化与磷酸化的关系。结果:①人肝癌细胞株SMMC-7721中TAL活性在10%胎牛血清培养时明显高于1%胎牛血清,2、4、8h显著性差异(P<0.05)。②不同浓度(10%.1%)胎牛血清孵育2、4、8hTAL蛋白表达水平无显著性差异。③磷酸化消化后TAL活性,与消化前相比呈显著性下降。结论:人肝癌细胞株SMMC-7721中增加血清浓度明显提高TAL活性,酶活性变化与TAL蛋白表达水平无关,提示活性调节发生在蛋白翻译后修饰,其中磷酸化起重要作用。     

Changes in the digestive gland of the loliginid squid Sepioteuthis lessoniana (Lesson 1830) associated with feeding

Changes associated with feeding in the histological and cytological structure of the digestive gland of the loliginid squid Sepioteuthis lessoniana were examined, along with the nature of both the intracellular and extracellular enzymes produced by the gland. The timing of the release of the extracellular enzymes during the digestive cycle was also determined using a quantitative experimental program. Like that of all coleoid cephalopods, the digestive gland was characterised by one type of cell with several functional stages. As is the case for other loliginid squids, however, the digestive cells did not contain the large enzyme-carrying boules that characterise the digestive glands of most cephalopods. Instead, smaller secretory granules were found in the digestive cells and these may be the enzyme carriers. The prominent rough endoplasmic reticulum, large mitochondria and active Golgi complexes present in the digestive cells are characteristic of cephalopods and indicate a high metabolic activity. Like that of other cephalopods, endocytotic absorption of nutrients and intracellular digestion occurs in the digestive gland of this squid. From quantitative and qualitative examinations of structural changes in the digestive gland of S. lessoniana after feeding, a schedule of its function during the course of digestion was proposed. This indicated that digestion was very rapid, being completed in as little as 4 h in S. lessoniana. Extracellular digestive enzymes were only released after the first hour following feeding, which implies that they are stored in the stomach between meals to increase digestive efficiency.     

Ultrasound-assisted digestion: a useful alternative in sample preparation

Ultrasound-assisted digestion is a promising alternative in the analysis of solid samples when either simple dissolution or direct analysis is not applicable. However, the field of application of ultrasonic sample digestion is still small in comparison with classical digestion alternatives and, particularly, with microwave-assisted digestion. This fact can be justified by the scant knowledge analytical chemists have about the advantages ultrasonic energy provides to digestion. Among these, the strict control at low temperatures of ultrasound applications allows the implementation of ultrasonic-assisted steps in biochemical analyses. In this connection, two specific biological applications, ultrasonic enzymatic digestion and assistance of ultrasound for cell disruption, are also reviewed.     

黑腹绒鼠消化道长度和重量的季节变化

能量获得与分配的效率和速率是决定动物能量收支的主要因子,对于动物的生存和繁殖是至关重要的（Karasov, 1986;Weiner,1992）.动物的消化道容纳和处理食物的能力,以及消化和吸收营养物质的能力是限制其能量收支的重要因素,也是理解生活史进化和最优资源分配理论的关键（Derting and Bogue,1993;Derting and Noakes,1995）.