Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg.

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Zinc increases the longevity of unfertilized sea urchin eggs

Summary We have found that Zn²⁺ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn²⁺ inhibited this proteolysis. Therefore, Zn²⁺ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.     

Involvement of a sperm aminopeptidase in fertilization of the sea urchin

Summary Inhibitory efficiencies of bestatin methyl ester and its nine analogs for sea urchin sperm aminopeptidase activity were similar to the efficiency of the same compounds as inhibitors either of sperm respiration or of fertilization. This suggests that a sperm aminopeptidase plays a role in fertilization in the sea urchin, possibly through its involvement in sperm respiration.Acknowledgment. We are grateful to Dr Wataru Tanaka of Nippon kayaku co. for his gifts of bestatin and its analogs. We are also indebted to Mrs Akiko Tsuchida-Watanabe for her technical assitance, and to the staff of Asamushi Marine Biological Station, Tohoku University, where part of this work was carried out. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Antibacterial activity in the egg mass of a sea hare

Summary The eggs of a sea hare,Aplysia kurodai, contained antibacterial factors which probably play a role in the defense of eggs against bacterial infection. The active factors were composed of several heat-labile proteins, unrelated to lysozyme, and were produced in the albumen gland.     

Trypsin and trypsin-like proteases in the brain: Proteolysis and cellular functions

Several serine proteases including thrombin, tissue-type plasminogen activator and urokinase-type plasminogen activator have been well characterized in the brain. In this article, we review the brain-related trypsin and trypsin-like serine proteases. Accumulating evidence demonstrates that trypsin and trypsin-like serine proteases play very important roles in neural development, plasticity, neurodegeneration and neuroregeneration in the brain. Neuropsin is able to hydrolyze the extracellular matrix components by its active site serine, and regulates learning and memory in normal brain. The mutant neurotrypsin contributes to mental retardation in children. Neurosin seems to be involved in the pathogenesis of neurodegenerative disorders, like Alzheimer’s disease, Parkinson’s disease or multiple sclerosis. Although mesotrypsin/trypsin IV is also implicated in neurodegeneration, its functional significance still remains largely unknown. Particularly, mesotrypsin/trypsin IV, P22 and neurosin exert their physiological and pathological functions through activation of certain protease-activated receptors (PARs). In the brain, the presence of serpins controls the activity of serine proteases. Therefore, understanding the interaction among brain trypsin, serpins and PARs will provide invaluable tools for regulating normal brain functions and for the clinical treatment of neural disorders. Y. Wang, W. Luo: These authors made equal contributions. Received 26 June 2007; received after revision 13 August 2007; accepted 12 September 2007     

Effect of 2 inhibitors of glycoprotein synthesis, tunicamycin and 2-deoxyglucose, on development of the sea urchin (Paracentrotus lividus) egg]

Tunicamycin, an inhibitor of glycoprotein synthesis, inhibits the development of the sea Urchin egg. Gastrulation is very sensitive to the Tunicamycin. Fertilization is not inhibited. 2-deoxy-D-glucose inhibits gastrulation and induces the hyperdevelopment of ectodermic structures.     

Incorporation of S35-Methionine in the microsomes and soluble proteins during the early development of the sea urchin egg

Riassunto Nelle uova diParacentrotus lividus già pochi minuti dopo la fecondazione si inizia una attiva incorporazione di metionina-S35 nei microsomi e nelle proteine solubili. Si avanza l'ipotesi che cio possa indicare l'iniziarsi molto precoce della sintesi di una o più proteine.     

Chymotrypsin gene expression during the intermolt cycle in the shrimpPenaeus vannamei (Crustacea; Decapoda)

InPenaeus vannamei, chymotrypsin is present as two isoenzymes in the hepatopancreas. The enzyme has been localized in F-cells by immunocytochemistry using a specific antibody. By in situ hybridization, with a 510 pb cDNA probe encoding for the first 170 amino acids of the shrimp chymotrypsin, mRNA was localized in the same cells. Gene expression was followed during the intermolt cycle by measuring changes in specific activity in crude extracts, and by the estimation of mRNA levels by Northern blots using the same probe. The increase in specific activity in premolt is preceded in early premolt by an increase in the amount of chymotrypsin mRNA. A second increase is observed in postmolt, suggesting a different mode of regulation of gene expression.     

A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet

Summary A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.     

Peroxidase and gamma-glutamyl transpeptidase activities duringEimeria nieschulzi (Apicomplexa) and/orNippostrongylus brasiliensis (Nematoda) infections in the rat

Summary Results suggest that malabsorption of amino acids which occurs duringEimeria nieschulzi andNippostrongylus brasiliensis infections in rats is not due to impairment by intestinal inflammation of gamma-glutamyl transpeptidase activity.Supported by NIH MBRS grant RR08012-13.     

Histochemical localization of alkaline and acid phosphatase activities in the skin ofMystus (Mystus) vittatus Bl. (Siluriformes)

Summary An attempt has been made to localize alkaline and acid phosphatase activities in the skin ofMystus vittatus by using histochemical techniques. The alkaline phosphatase activity is found in metabolically active cells such as basal columnar cells, mucous cells and polygonal support cells. The acid phosphatase activity is intense in the outermost squamous support cells and in the basal columnar cells. These activities have been correlated with some physiological functions of the epidermis.Acknowledgment. We are thankful to P. Vishwanatham, Government College, Mhow, and Dr R.S. Shrivastava, Holkar Science College, Indore, for providing laboratory facilities and to the Council of Scientific and Industrial Research, New Delhi, for a fellowship for M.S.     

Peroxidase and gamma-glutamyl transpeptidase activities during Eimeria nieschulzi (Apicomplexa) and/or Nippostrongylus brasiliensis (Nematoda) infections in the rat

Results suggest that malabsorption of amino acids which occurs during Eimeria nieschulzi and Nippostrongylus brasiliensis infections in rats is not due to impairment by intestinal inflammation of gamma-glutamyl transpeptidase activity.     

Common antigenic properties of a g-type (goose) and a c-type (duck) egg white lysozyme: Antibody responses in rabbits and mice

Embden goose (GEWL) and Barbary duck (DEWL) egg white lysozymes possess different amino acid sequences corresponding to the g-type and c-type, respectively. GEWL was shown to be a better immunogen than DEWL in both rabbits and mice. The antigenicity of the two lysozymes was tested using different technique (i.e. indirect ELISA, inhibition tests and immunoabsorption experiments). Injection of either GEWL or DEWL into rabbits and mice induced both specific antibodies and cross-reacting antibodies. Moreover, anti-GEWL antibodies, in contrast to anti-DEWL antibodies, did not cross-react with hen egg white lysozyme (HEWL), a c-type lysozyme. While the structure of GEWL was not modified after binding to plastic, DEWL was denaturated, but it did keep some native epitopes. It was concluded that g-type and c-type lysozymes, which have different amino acid sequences, exhibit strong common antigenic properties.     

Variations in DNA synthesis and the activities of acid and alkaline deoxyribonucleases during regeneration of planaria (Polycelis tenuis)

Variations of DNA synthesis were established for the first time during planarian regeneration by 32P phosphoric acid incorporation. A first peak of DNA synthesis occurred between the 10th and the 18th hour after sectioning (maximum at 12 hrs.). Subsequently, DNA synthesis increased again progressively after the 24th hour to a plateau between 48 and 72 hrs. after regeneration. Simultaneously, variations of alkaline and acid DNases were determined.     

Studies on some enzymes of the toad (Bufo melanostictus) testis and their probable role at the time of fertilization

Summary Glycosidases like sialidase,-galactosidase, -L-fucosidase, N-acetyl hexosaminidase and proteases were detected in toad testis. Neuraminic acid aldolase activity was also detected. The enzyme activities were found to vary as production of spermatozoa varied. All enzymes, except N-acetyl glucosaminidase, were shown to decrease after injection of toad pituitary extract and they were also found to be absent from testis containing no spermatozoa. The glycosidases were found to act on toad oviduct jelly and they may therefore be involved in the degradation of the jelly after fertilization, into smaller bits, which may be utilized as nutrients by the fertilized zygote.Acknowledgment. We thank Prof. T.R. Ramaiah, Head of the Department of Biochemistry, University of Mysore, for his help. We also acknowledge the financial assistance of University Grants Commission to one of us (MS) and CSIR through a grant No. 9 (165)83/EMR-II to HSS. Please address all correspondence to H.S. Seshadri.