Physicochemical study of rock crab lipoproteins

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab (Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL₃ revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL₃. A fourth apolipoprotein was found in both male and female HDL₃. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.     

Instability of crab vitellogenin and its immunological relatedness with mammalian atherogenic lipoproteins

Vitellogenesis is the process of accumulation of vitellogenin (Vg) in rapidly growing oocytes of oviparous animals and its' subsequent transformation into lipovitellin (Lv). Lipovitellin, which forms the major yolk protein, serves as a principal nutrient reserve for the developing embryo. In the present study, Vg and Lv were purified from the hemolymph and ovary, respectively of the crab Scylla serrata by gel filtration followed by preparative gel electrophoresis. It was observed that purified Vg, but not Lv, possessed an intrinsic protease activity with which it underwent autoproteolysis giving rise to several smaller proteins. Furthermore, urea-mediated unfolding studies by UV-spectral analysis revealed clearly that Vg was easily disrupted by urea whereas Lv was resistant. Taken together, these results suggest that although Lv had a stable conformation, its precursor Vg was labile and highly sensitive to degradation. Another aspect that was investigated in the present study was the immunological kinship of crab Vg and Lv to mammalian atherogenic lipoproteins, the low density lipoprotein (LDL), very low density lipoprotein (VLDL), and apolipoprotein B (apoB). By Western blot analysis, it was demonstrated that crab Vg and Lv were immunoreactive to antibodies to human LDL, VLDL, and apoB. These observations suggest the existence of common epitope recognition sites in crab Vg and mammalian lipid transferring proteins. This corroborates well with our earlier study on the recognition of crab Vg receptor by mammalian lipoproteins.     

Properties of serum high-density lipoproteins in the crab, Cancer antennarius Stimpson

Two classes of high-density lipoprotein (HDL) comprise virtually all the lipoprotein mass in female hemolymph. These lipoproteins have hydrated densities of 1.187 g/ml (HDL3) and 1.112 g/ml (HDL2). A third species (HDL1, density 1.080 g/ml) appeared in ovigerous crabs. The mean annual HDL protein concentration was 109 mg/dl of which 67% was HDL3. HDL proteins of both HDL2 and HDL3 were mostly insoluble in tetramethylurea. Three major components with apparent mol. wts of 185,000, 100,000 and 84,000 daltons were identified by gel electrophoresis in SDS. Amino acid compositions are reported. Electron microscopy indicated that the HDL are polymorphic and discoidal. Similarities in shape and differences in size of HDL3 and HDL2 particles were consistent with their lipid and protein composition. Phospholipids, mostly phosphatidylcholine, were the dominant lipid class (74%); no cholesteryl esters were detected. Palmitic and oleic acids were the major fatty acid components of esterified lipids.     

Orthogonal microvillus pattern in the eighth rhabdomere of the rock crab Grapsus

Summary The eighth retinular cell (R 8) of Grapsus lacks cytoplasmic pigment granules and basically resembles those previously known in the ghost crab Ocypode and the mysid Praunus. Distally located, R 8 comprises four lobes inserted between the outer ends of the seven regular retinular cells (R 1–R 7). A thin cytoplasmic bridge connects these lobes. One lobe adjacent to R 1 contains the nucleus of R 8 and gives rise proximally to the cell's axon. The short distal eighth rhabdomere consists of microvilli (mvl) protruding axially from all four lobes. Similar R 8's were found also in two other crab families and in two other genera of mysids.In Grapsus the eighth rhabdomere is extraordinary in possessing mvl oriented in two orthogonal directions parallel to the mvl of R 1–R 7. The distal 20% of the rhabdom consists of mvl originating exclusively from R 8. These appear in somewhat irregular bands and are alternately oriented parallel to the animal's vertical or horizontal axis. More proximally the retinula contains eleven sectors but the rhabdom still comprises bands of alternating mvl with those from R 8 joined respectively by the rhabdomeres of R 1, 4, and 5 (horizontal) and R 2, 3, 6 and 7 (vertical). The rest of the rhabdom shows typical decapod organization with seven interdigitating rhabdomeres.This research has been aided by grants from the United States Public Health Service (5 RO1 EY 00405) and the National Geographic Society. The authors are grateful to Mabelita Campbell for her helpful assistance.     

Platelet lipoproteins. A comparative study with serum lipoproteins.

The nature of human platelet lipoproteins was studied in two series of experiments. In the first series, whole platelets were utilized for extraction of lipoproteins by three different methods: chloroform/methanol/phenol; saline; or sucrose-gradient ultracentrifugation of platelet homogenates. By polyacrylamide gel electrophoresis we were able to demonstrate the existence of lipoprotein in the extracts obtained by the last two methods. These lipoproteins were found not to share antigenic determinants with alpha and beta serum lipoproteins. The second series of experiments utilized platelets solubilized either in sodium deoxycholate or sodium dodecyl sulfate. The solubilized product was characterized by double immunodiffusion and polyacrylamide gel electrophoresis. The nonidentity between plasma and platelet lipoproteins previously demonstrated in the first series of experiments was confirmed. This nonidentity was also supported by a comparison between the apoproteins of purified serum lipoproteins and platelet proteins released after solubilization with sodium dodecyl sulfate. No identical protein fractions were found. Our results suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoproteins are structurally different from plasma lipoproteins.     

Invasion of the Atlantic rock crab (Cancer irroratus) at high latitudes

With the increase in global oceanic trade the establishment of non-indigenous marine organisms has become a major environmental and economic problem worldwide. Recently, the Atlantic rock crab (Cancer irroratus) was reported in Icelandic waters, Eastern North Atlantic. This is the first record of this relatively large crab species outside its natural range, i.e. the east coast of North America. The crab was most likely transferred to Iceland as larvae in ballast water and has successfully established a reproducing population in Icelandic waters. The species is distributed along the southwestern- and western-coast of Iceland. Adult specimens are now common in Faxaflói Bay, Southwest Iceland, but with sporadic occurrences in western and northwestern Icelandic waters. The green crab (Carcinus maenas) and the spider crab (Hyas araneus) are the only native brachyuran decapod species commonly found in its new habitat, but despite its recent colonization the rock crab was the most abundant brachyuran in the areas studied in southwest Iceland. Egg bearing rock crab and green crab females were found from June to October, while egg bearing spider crab females were seen from July to December. In Southwest Iceland both rock crab and green crab larvae were abundant in mid-summer but rare in both spring and autumn, which is opposite of what was observed for the spider crab. The size and abundance of adult crabs, their reproductive conditions, and occurrence of all larval stages, indicate that the Atlantic rock crab has successfully colonized Iceland.     

Gastric processing and evacuation during emersion in the red rock crab, Cancer productus

The passage of a radio-opaque meal was followed through the digestive system of the red rock crab, Cancer productus, using a fluoroscope. When the crabs were maintained in seawater, the food was apparent in the foregut as soon as the animals had fed. Release of food from the foregut was routinely slow and digesta appeared in the midgut only in small amounts at any one time. The foregut was emptied between 24 and 36 h, digesta was cleared from the midgut region at 36 h and by 48 h only a small amount of residual digesta was left in the posterior part of the hindgut. Contractions of the cardiac region of the foregut were somewhat sporadic and ranged between 6 and 11 min^-1. Contractions of the pyloric region were more stable, varying between 45-65 min^-1. In both cases, there was no change in rate during 18 h period in seawater. When crabs were subjected to both short- and long-term aerial exposure, release of food from the foregut was halted for the first 4-6 h of emersion. Although, small amounts of digesta appeared in the midgut and hindgut, there was no significant change in the amount in each region during emersion. There was a trend towards a depression of cardiac stomach contraction rates, but this was only significant in 3 h postprandial crabs during short-term emersion. A pronounced decrease in pyloric stomach contraction rate was maintained for the duration of the aerial exposure. When crabs were returned to seawater, contraction rates took 3-5 h to return to normal, but no significant change in gastric evacuation was observed during this period. During re-immersion, over 65% of the animals regurgitated the stomach contents. This regurgitation may act as a protective mechanism to avoid digestion and the subsequent specific dynamic action. The decrease in gastric processing in C. productus is probably part of an overall metabolic depression occurring during emersion.     

Serum high-density lipoproteins in the crab, Cancer antennarius--III. Density-gradient profiles and lipid composition of subclasses.

1. Prestaining the serum total lipoprotein (LP) fraction and density-gradient ultracentrifugation revealed up to five LPs, all in the high-density class (HDL; 1.187 greater than d greater than 1.080 g/ml). These are group-designated as HDL1, HDL2 and HDL3, lowest to highest buoyant density, respectively. The number and precise density of the LPs varied with individual crab and were not gender specific. 2. The percentage of total mass due to triacylglycerides was progressively higher from HDL1 and HDL3, whereas that of phospholipids, cholesterol and combined fat was progressively lower. The percentage mass of combined fat was higher in females than males. 3. As in hemolymph, the LP of eggs was entirely HDL; instances of each HDL subclass were seen, but HDL3 was predominant. Compared with serum HDL, egg HDL was strikingly low in triacyglyceride mass and relatively high in protein. 4. Hemolymph HDL titers of individual females varied independently with egg size through most of the year, but was positively correlated with the egg size in October to December when 2-3-fold increases in HDL titer occur in the female population.     

海洋活化石——鲎的研究进展

海洋活化石——鲎（hòu）是十分宝贵的自然资源,在科研、经济、生态等方面都有着不可估量的价值。鲎作为古老的海洋底栖动物被过度捕捞,现已到了枯竭的程度,2021年2月,我国《国家重点保护野生动物名录》正式更新和发布,我国海域的中国鲎和圆尾鲎被列为国家二级保护动物。为推动鲎研究和有效制定实施鲎资源的保护措施,本文主要从鲎的生物学研究、资源利用和生态保护三个方面入手,瞄准鲎的分类学问题、鲎的生物学前沿问题包括神经系统、视觉系统、消化系统、循环系统、生殖与发育和共附生微生物等、生态保护现状以及存在的问题等方面国内外的研究进展进行综述,明确研究重点和难点的同时,扩大对鲎的认知。     

Susceptibility and resistance of the rock crab,Cancer irroratus,to natural and experimental bacterial infection

The incidence of naturally occuring bacterial infection in a Connecticut population of rock crab, Cancer irroratus, varied between 10 and 60% of the population. The fluctuation in incidence was correlated significantly with hemocyte concentrations. Twelve percent of the crabs sampled during Jenuary were found to be infected with a previously undescribed Vibrio. The Vibrio was demonstrated as pathogenic over a wide range of temperatures. High mortalities in experimentally infected crabs appear to result from hemocyte clumping and extensive intravascular clotting triggered by an endotoxin. The lobster pathogen, Aerococcus viridans var. homari, was found to be pathogenic at 25°C to C. irroratus, a prey species of the lobster. Results suggest that this species can act as a reservior host for A. viridans var. homari.     

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.     

A fluorescent sterol probe study of human serum low-density lipoproteins

The fluorescent sterol probe, ergosta-5,7,9,(11),22-tetraen-3 beta-ol (dehydroergosterol), was utilized as a cholesterol analog to label human serum low-density lipoproteins (LDL). Quenching of dehydroergosterol fluorescence by KI indicated that most of the fluorophore was either buried within the outer phospholipid monolayer of LDL or within the neutral lipid core of LDL. The steady-state anisotropy of dehydroergosterol in LDL detected the cholesteric core phase transition near 30 degrees C. Fluorescence lifetime decays for dehydroergosterol contained two components, both below and above the cholesteric phase transition, with the major lifetime component near 1 ns. Neither lifetime component underwent a detectable change in duration at the core phase transition temperature. Time-correlated fluorescence anisotropy decays of dehydroergosterol indicated a single rotational correlation time near 1.7 ns, which was unaffected by the core phase transition. Time-correlated anisotropy decays also suggested hindered rotation of dehydroergosterol in LDL. These results indicate that unesterified cholesterol is primarily located in the outer phospholipid monolayer of LDL, with the majority of cholesterol not in direct contact with the aqueous phase.     

Physiochemical properties of 7-aminoacetoxycephalosporanic acid]

Potentiometric titration of the zwitter-ion of 7-aminoacetoxycephalosporanic acid (7-ACA) was performed and the constants of its ionization were estimated. The minimum solubility of the 7-ACA zwitter-ion (20 degrees C, 0.1 M NaCl) was determined and the solubility curve of 7-ACA at wide pH ranges was calculated. Equilibrium of the cationic, zwitter-ionic and anionic forms of 7-ACA was estimated as dependent on pH.     

Physiochemical properties of rat liver mitochondrial ribosomes

In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes. The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined. Sedimentation coefficients were established using the time-derivative algorithm (Philo, J. S. (2000) Anal. Biochem. 279, 151-163). The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively. Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA. The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3). The molecular masses of mitochondrial and E. coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively. The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E. coli monosome. The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E. coli ribosomes, respectively. A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation. These measurements of solvation suggest that ribosomes are highly hydrated structures. They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels.     

Amphipathic helixes and plasma lipoproteins: A computer study

A molecular model for the lipid-associating domains of the A and C plasma apolipoproteins was recently proposed. This model consists of helical regions with special properties termed amphipathic. In the present communication we present a computer analysis of the general occurrence of amphipathic helix patterns in proteins with known amino acid sequences.