Recombinant expression,characterization and expressional analysis of clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> cathepsin B,an enzyme involved in nutrient digestion

Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K _m, V_max and k _cat are 6.11 μM, 0.0174 μM min⁻¹ and 277.57 s⁻¹, respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix.     

Inflated protoconchs and internally dissolved, coiled protoconchs of nudibranch larvae: different developmental trajectories achieve the same morphological result

Abstract. Light and scanning electron microscopy were used to examine protoconch form in eight species of planktotrophic heterobranch larvae, including four nudibranch species with a coiled (type 1) protoconch, two nudibranch species with an inflated (type 2) protoconch, and two cephalaspid species with a coiled protoconch. The coiled protoconchs of the cephalaspids and nudibranchs have a similar form at hatching, and shell growth up to metamorphic competence is hyperstrophic. Shell added to coiled protoconchs during the larval stage overgrows all but the left wall of the initial protoconch that exists at hatching. The entire protoconch of cephalaspids, including overgrown areas, is retained through metamorphosis. However, during later larval development in nudibranchs with a coiled protoconch, overgrown shell is completely removed by dissolution. As a result, regardless of whether nudibranch larvae hatch with an inflated or coiled protoconch type, the protoconch is a large, hollow cup at metamorphic competence. The protoconch of nudibranchs is shed at metamorphosis and absence of a post-metamorphic shell is correlated with absence of visceral coiling in this gastropod group. Internal dissolution of the coiled protoconch in nudibranchs allows the left digestive gland to uncoil prior to metamorphic shell loss. Retention of overgrown protoconch whorls in cephalaspids allows the attachment plaque of the pedal muscle to migrate onto the parietal lip of the post-metamorphic shell. Release from this constraint in nudibranchs, in which the larval pedal muscles and the entire protoconch are lost at metamorphosis, may have permitted internal protoconch dissolution and precocious uncoiling of the visceral mass, as well as evolutionary emergence of the inflated larval shell type.     

Observations on development,larval growth and metamorphosis of four species of aplysiidae (gastropoda: Opisthobranchia) in laboratory culture

The development of simple, reliable techniques for the laboratory culture of aplysiid gastropods through their complete life cycle, has enabled us to study the larval biology, metamorphosis, and early juvenile development of these animals. Egg masses, duration of the embryonic phase, veligers, and larval growth and development are described for four species of Hawaiian Aplysiidae, namely, Aplysia dactylomela Rang, Aplysia Juliana Quoy and Gaimard, Dolabella auricularia (Lightfoot) and Stylocheilus longicauda (Quoy and Gaimard). Metamorphosis and early juvenile development of A. Juliana are described in detail with additional comments on these processes in the other three species. Length of the embryonic phase and size of the veliger at hatching are a function of the size of the uncleaved egg. All four species develop planktotrophically and have ≈ 30-day larval phases. In each species the larval phase includes a period of rapid shell growth to a species-specific size followed by a non-growth period during which other morphological developments occur to culminate in metamorphic competence. The larvae of each species metamorphose preferentially on a particular species of benthic algae. The events of metamorphosis require 2 to 4 days for completion and transform the planktonic filter-feeding larva into a benthic, radular-feeding juvenile. Postlarval development includes growth of the shell, parapodia, oral tentacles, rhinophores, anal siphon, and structures of the mantle cavity.     

Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation.

Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblast-myoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.     

CA-074, but not its methyl ester CA-074Me,is a selective inhibitor of cathepsin B within living cells

Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA-074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.     

Development of digestive secretions in white sturgeon juveniles (Acipenser transmontanus)

Development of the digestive enzyme complement corresponded with this species' carnivorous habits and a natural dietary shift at metamorphosis from larval pelagic zooplanktivory to benthic carnivory. During the first developmental stanza when the digestive tract was differentiating and the larvae were dependent on endogenous nutritional reserves, digestive enzyme concentrations were low. Initiation of gastric secretion (pepsin and acid) was concomitant with onset of exogenous feeding. Highest amylolytic and lipolytic activities were recorded from feeding larvae prior to their metamorphosis to juveniles.     

Effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum

The effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum at the temperature of 19.6–21.6 °C, the salinity of 34‰ and pH of 8.0 were investigated from May 18 to July 18, 2006. In this study, the early, middle and late umbo-veliger larvae with the shell lengths of 100, 140, and 190 μm were subject to temporary food deprivation for up to 4.5, 20, and 25d at 0.5, 4, 5d intervals, followed by refeeding for the remaining of a 24, 20, 25d period, respectively. The results suggested that the larvae should have shown considerable tolerance to starvation due to their endogenous and exterior nutrition material, for larvae and time to the point-of-no-return (PNR: the threshold point during starvation after which larvae could no longer metamorphose even if food is provided) were calculated to be 4.25, 17.54, and 22.17d. As the starvation period prolonged, the mean shell length of larvae starved got close to constants at 1.5, 4, and 15d after starvation, which were different for larvae at different stages when starvation began, survival of larvae decreased, and was lower in treatments starved earlier in development than those starved later, for the early, middle and late umbo-veliger larvae, after 4.5, 20 and 25d of starvation period, few larvaes were alive. After starvation period, the alive larvaes were able to metamorphose and had a capability of compensatory growth when refeeding was given. Starvation not only affected metamorphosis rate, but also caused the delay in the time to metamorphosis and the decrease in the metamorphosed sizes. For example, for the continuously-fed larvae, duration to metamorphosis was 20.7d, for larvae with a size of 100-μm starved for up to 4d, larvae with a size of 140-μm starved for up to 16d, larvae with a size of 190-μm starved for up to 20d, duration to metamorphosis were 29.7, 31.7, and 37.7d, the delay in duration to metamorphosis were 9, 11, and 17d, respectively. Furthermore, importance of nutrition material for maintaining larval survival during starvation and the compensatory growth on larvae at the same feeding time were discussed.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.