Algal phospholipids by 31P NMR: Comparing isopropanol pretreatment with simple chloroform/methanol extraction

• 1.1. A modified Folch procedure [potassium (ethylenedinitrilo)-tetraacetic acid at pH 6 substituted for KC1] is suitable for the extraction of marine algae.
• 2.2. The quantitative ³¹P nuclear magnetic resonance phospholipid profiles of four marine algae, Gracilaria verrucosa, Bryothamnion triquetrum, Padina gymnospora, and Caulerpa sertularioides, were obtained from Folch and Nichols extractions of both fresh and dried algae, and essentially identical results were obtained using either extraction procedure.
• 3.3. Extracts of air-dried algae are statistically different when compared to extracts of living algae, suggesting that tissue handling is a critical factor in phospholipid extractions.

     

Lipid composition of the red and green forms of Actina equinia from the black sea

• 1.1. The fatty-acid composition of the red and green forms of Actinia equina from the Black Sea have been determined by methods involving silver-ion HPLC and GC-MS.
• 2.2. The fatty acid compositions of both forms of A. equina resemble those of most marine invertebrates. Substantial amounts of C₂₀ and C₂₂ polyunsaturated fatty acids were found.
• 3.3. Balck Sea Actenia equina contains a large amount of plasmalogens, mainly phospotidylcholine and phosphatidylserine plasmalogens.
• 4.4. The red form of A. equina contains more arachidonic acid in glycolipid fraction and more phosphatidylethanolamine and its plasmalogen, while the green from contains more sphingomyelin. These differences are an indication that the species A. equina can be divided into two subspecies—green and red.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

A comparative study of seafish erythrocytes and agglutinins from seaweeds

• 1.1. To date, only a few authors have assayed the agglutinic activity of marine algae against fish erythrocytes, and in these cases, mainly against freshwater fish.
• 2.2. For the first time, the hemagglutinic activity of 70 seaweeds (29 brown, 37 red and four green algae) against erythrocytes of 16 seaflsh species is reported.
• 3.3. The presence of agglutinins was demonstrated in 100% of algae assayed, against at least one of the different types of erythrocytes tested.
• 4.4. The results obtained confirm the presence of receptors for algae agglutinins on the surface of the erythrocytes of the fish studied.
• 5.5. This could be useful in establishing the origins of fish populations, as these serological differences could distinguish between populations of cultivated and wild fish.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Esterases in quail (Coturnix coturnix). Physicochemical and developmental aspects

• 1.1. Soluble esterases of digestive system organs of various developmental stages in the quail (Coturnix coturnix) were resolved by polyacrylamide gel electrophoresis into several molecular forms which were characterized as carboxylesterases, acetylesterases, cholinesterases and esterases sensitive to eserine.
• 2.2. The pI of the majority of esterasic activity in several quail and chicken tissues was observed in the range of 5.1–5.6, while the apparent molecular weight in liver extracts was 60,000.
• 3.3. The expression of the esterase multiple molecular forms was found to be both tissue- and developmental stage-specific, with electrophoretic patterns becoming more complex in number and/or staining intensity upon hatching and thereafter, especially in liver and intestine.

     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Biochemical and functional characterization of Rapana thomasiana hemocyanin

• 1.1. The hemocyanin (Hc) of the marine gastropod mollusc Rapana thomasiana was collected from animals living on the west coast of the Black Sea and characterized for its biochemical and functional properties.
• 2.2. This Hc is very similar to other gastropod Hcs as far as amino acid composition, general structure and reactivity of the binuclear copper active site are concerned.
• 3.3. Some peculiarities in the dissociation-reassociation pattern are observed in comparison to other gastropod Hcs, in particular with respect to the ability to form sopramolecular aggregates.
• 4.4. Changes in pH disclose a strong reverse Bohr effect. Different R and T states are required to describe the oxygen binding curves at the different pHs.

     

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

• 1.1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence.
• 2.2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata.
• 3.3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated.
• 4.4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

     

Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts

• 1.1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).
• 2.2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.
• 3.3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions.
• 4.4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Presynaptic modulation of sensory afferents in the invertebrate and vertebrate nervous system

• 1.1. Ultrastructural examination of the central terminals of sensory afferent neurons in both invertebrates and vertebrates demonstrates that the synapses that form the substrate for presynaptic inhibition and facilitation are almost universally present.
• 2.2. Presynaptic modulation of afferent input acts in many ways which tailor the inflow of sensory information to the behaviour of the animal, effectively providing a means of turning this on and off, or of combining information of the same or different modalities to refine responsiveness or clarify ambiguity.
• 3.3. Presynaptic modulation may act in several different roles on the same afferent.
• 4.4. A comparison of the mechanisms of presynaptic inhibition in different animals demonstrates the likelihood of a variety of common mechanisms,several of which may act simultaneously on the same terminal.These include changes in the conductance of the afferent membrane to Cl^-, K⁺and Ca²⁺ions, in addition to less well understood mechanisms that directly affect transmitter release.
• 5.5.A single transmitter can produce several effects on a terminal through the same or different receptors.
• 6.6. Ultrastructural studies of afferent terminals reveal that only a proportion of boutons on a given afferent may receive presynaptic input and that this may depend on the region of the nervous system in which these are found or on the identity of the postsynaptic neurons contacted.
• 7.7. The synaptic relationships of afferent terminals can be complex. In invertebrates different types of presynaptic neuron may interact synaptically,as may postsynaptic dendrites in vertebrates.
• 8.8. Axons presynaptic to afferent terminals in vertebrates frequently synapse also with dendrites postsynaptic to the afferents.
• 9.9. In both invertebrates and vertebrates reciprocal interactions between afferents and postsynaptic neurons are seen.
• 10.10. Ultrastructural immunocytochemistry reveals the likely dominance of GABA as an agent of presynaptic inhibition but also demonstrates the possible presence of other transmitters some of whose roles are less completely understood.

     

Novel tetraamines,pentaamines and hexaamines in sea urchin,sea cucumber,sea squirt and bivalves

• 1.1. Polyamines were extracted from the guts and ovaries of the sea urchin Anthocidoris crassispina, and the guts and flesh of the sea cucumber Stichopus japonicus and the sea squirt Halocynthia roretzi, the oyster Crassostrea gigas and the short-necked clam Tapes philippinarum, and analyzed by ion-exchange high-performance liquid chromatography and gas chromatography-mass spectrometry.
• 2.2. Norspermidine and norspermine as well as putrescine, cadaverine, spermidine, spermine and agmatine were the ubiquitous polyamines in these invertebrates. These results suggest the widespread distribution of norspermidine and norspermine in invertebrates.
• 3.3. Thermopentamine, thermohexamine and homothermohexamine were found in the sea urchin. This in the first report on the occurence of thermopentamine and hexaamine in invertebrates.
• 4.4. Homospermidine, canavalmine, aminopropylhomospermidine, homospermine, caldopentamine, homocaldopentamine and aminopropylcanavalmine were found in the sea cucumber. Homospermidine, aminopropylhomospermidine and homospermine were found in the squirt. This is the first report on the occurence of canavalmine, aminopropylhomospermidine, homospermine, homocaldopentamine and aminopropylcanavalmine in invertebrates.

     

Human spleen cathepsin D: Its characterization and localization in human spleen

• 1.1. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography.
• 2.2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins.
• 3.3. Inhibition with thiol compounds was found to be a specific phenomenon of the cathepsin D from the human spleen.
• 4.4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel.
• 5.5. In an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen.
• 6.6. The role of this enzyme in human spleen cell was discussed.

     

Structural features of carbohydrate chains in human salivary mucins

• 1.1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated.
• 2.2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units.
• 3.3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants.
• 4.4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine.
• 5.5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Proteolysis of skeletal muscle in yellowfin tuna (Thunnus albacares): Evidence of calpain activation

• 1.1. White muscle of yellowfin tuna is subject to a form of deterioration known as “burnt tuna”.
• 2.2. TEM and SDS-PAGE were used to quantify cellular differences in deteriorated white muscle of yellowfin tuna.
• 3.3. Electron micrographs showed a significant loss of Z-disc integrity and an increase in intracellular edema in burnt tuna.
• 4.4. Electrophoresis established that a specific doublet of proteins, 42 kD and 46 kD was lost.
• 5.5. Proteolysis of isolated myofibrils incubated in calpain (EC 3.4.22.17) was greatest at pH 7.5 and was selective for intermediate molecular weight proteins.
• 6.6. This evidence suggests that burnt tuna is a specific and limited proteolysis of myofibrillar structural proteins characteristic of calpain proteolysis.