Calcium-activated neutral proteinase in gametes of the sea urchinStrongylocentrotus droebachiensis

By Chromatographic separation of cytosol fraction of homogenates from gonads of females and males of green sea urchinStrongylocentrotus droebachiensis using an AcA-34 ultragel, protein fractions with mol. mass of 123 and 81 kDa are revealed, which have activity of calciumactivated proteinases. Dynamics of activity of the calcium-activated proteinases is studied during annual sexual cycles of the sea urchin. It is shown that the maximal activity of the studied enzyme in females and males of the sea urchin is present at the IV stage of gonad maturation, at the period of their trophic growth which is characterized by formation of stores of nutritient substances necessary for the subsequent development of embryos.     

Lipid peroxidation in embryos and larvae of the sea urchinStrongylocentrotus intermedius

Lipid peroxidation (LP) and glutathione content were studied at different developmental stages of the sea urchinStrongylocentrotus intermedius: egg cell, fertilization, 4 blastomers, blastula, hatching, gastrula, prism, pluteus. A high rate of LP in the total membrane fraction of sea urchin embryos and larvae at the stages from the egg cell to hatching was observed at enzymatic and nonenzymatic activation of LP. The LP rate was significantly reduced at the gastrula stage and at subsequent stages, there was practically no further development of the process. The glutathione concentration remained unchanged at different stages. The alterations in LP seem to reflect participation of free radicals in regulation of individual development.     

Calcium-activated neutral protease activities in brain trauma

This paper investigates the level of cytosolic and synaptosomal forms of calcium activated neutral protease activities in the normal brain and their changes following a freezing lesion in the rabbit. From 1 to 24 hours post lesion we observe a progressive disappearing of the enzyme activities from the cytosolic compartment and concurrently their increase in the membranal fraction. These changes are likely to be due to a rise in intracellular calcium concentration, a well documented consequence of many cellular insults. The specific role of the activation of calpain activities in the pathophysiology of trauma is discussed, an enhancement of excitotoxic mechanisms is proposed.     

The effect of temperature on the rate of oxygen consumption in the sea urchinStrongylocentrotus intermedius

InStrongylocentrotus intermedius acclimated to certain temperatures within the range of 5–20°C, the rate of oxygen consumption increases regularly as the temperature rises, the mean value of theQ ₁₀ coefficient being about 1.9.     

Inhibition of in-vitro fertilization of mouse gametes by proteinase inhibitors

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.     

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchinStrongylocentrotus purpuratus

Summary The general organization and primary amino acid sequences of theS. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26–27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage.The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

Polyfluorochrome marking slows growth only during the marking month in the green sea urchin Strongylocentrotus droebachiensis

Abstract. Used singly, the fluorochrome tags tetracycline and calcein have yielded important insights into sea urchin biology, especially regarding growth. We present a new method of tagging using sequential fluorochrome markers, as well as a more precise method of quantifying growth. Such polyfluorochromes enable repeated markings that allow measurement of multiple growth points and unique identification of individuals or groups. We marked sea urchins, Strongylocentrotus droebachiensis , with four fluorochromes: alizarin complexone, calcein, calcein blue, and tetracycline. All fluorochromes marked both by injection and immersion. We examined the clarity of the mark produced with low, metabolically scaled doses, and higher doses similar to those that have been previously used. We tested the effect of fluorochromes on survival, growth, jaw size, and gonad size by marking a size range (3.9–44.3 mm in diameter) of urchins with either one or all four fluorochromes. We quantified growth using a nominal diameter, that is a fitted constant, times the cube root of weight, which increased the precision of measurements by a factor of six relative to measured diameter. Growth rate was a decreasing function of diameter except for a growth lag in the smallest urchins. Growth rate data for all sizes were fitted using: gamma distributions; Tanaka functions; and, for larger sizes, straight lines (von Bertalanffy model). All treatments produced clear marks, with higher doses producing more reliably clear marks. Tetracycline marking did not affect growth; other treatments produced only transient slowing of growth in the marking month. Growth rate, survival, gonad production, and jaw weight did not differ between control and treatments during the following 6 months. Thus, polyfluorochromes produce reliable marks that do not significantly affect growth or gonad production.     

Larval settlement of the green sea urchin, Strongylocentrotus droebachiensis, in the southern Gulf of Maine

Abstract. Settlement timing and differential settlement for the larval stage of the green sea urchin, Strongylocentrotus droebachiensis , in the southern Gulf of Maine was studied during the summer of 1996. Settlement densities on astroturf panels were highest in June and early July (13 to 37 m ⁻² d⁻¹), and peaked in mid-June (199 m⁻² d⁻¹). Settlement was low to nonexistent from mid-July through August (0 to 2 m⁻² d⁻¹). During the peak in settlement, no selection for substrate type was observed. In the remainder of the settlement period, differential settlement occurred, with a preference for substrate covered with live coralline algae. Test diameter of newly settled urchins varied among the substrates, with urchins settling on live coralline algae having the largest test diameter (0.43 ± 0.01 mm). There were no differences in test diameter among the different weeks in which sampling was done. Sustained onshore winds occurred only during peak settlement, suggesting that wind drift currents may concentrate larvae and influence patterns of larval settlement.     

Calcium-activated neutral protease and its endogenous inhibitor in tissues of dystrophic and normal mice

Calcium-activated neutral protease (milli-CANP) and its endogenous inhibitor are elevated in muscle tissues, primarily the skeletal muscle and heart, of dystrophic mice (C57BL/6J dy/dy) as compared to the control strain (C57BL/10J). Tissues showing relative increase of CANP also show significant loss of enzymes such as CK, LDH in comparison to plasma, where these enzymes register a significant increase. PK is lost minimally from these tissues, probably showing a "sparing effect." Absence of any significant change in CANP activity in the liver points to a specific role of CANP in the dystrophic process. In the skeletal muscle the endogenous CANP inhibitor registers a concomitant increase with CANP without altering the enzyme/inhibitor ratio.     

Histopathology of the disease causing mass mortality of sea urchins (Strongylocentrotus droebachiensis) in Nova Scotia

The disease causing mass mortalities of Strongylocentrotus droebachiensis off Nova Scotia, Canada, from 1980 to 1983 is described. Diseased urchins were characterized by loss of preipheral muscle function in tube feet, spines, and mouth. Signs occurred primarily in the body wall and associated tissues (water vascular system, nerves, spine bases) and coelomic fluid. These symptoms were diffuse and included a general infiltration of tissues with amoebocytes. The coelomic fluid often contained reduced numbers of red and white spherule cells, and clotting was incomplete. Progressive breakdown and fragmentation of muscle cells in tube feet and spine bases resulted in destruction of coherent muscle layers and their replacement by numerous spindle-shaped fibrillar muscle remnants. Coelomic lining cells in the tube feet sloughed off into the lumen, but remained in clumps and phagocytosed muscle remnants.