Humoral responses of congenital immunity in the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work presents analysis of changes of humoral protective factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The total protein concentration and the titers of hemagglutinins and hemolysins in starfish coelomic fluid, as well as the time of human hemoglobin elimination from circulation were estimated for 6–144 h of the experiment. The hemagglutinin titer was determined in hemagglutination reactions, the hemolysin titer—in hemolysis reaction. Time of human hemoglobin elimination from coelomic fluid was determined in a color enzymatic reaction. The starfish coelomic fluid was revealed to contain soluble factors that are able to interact with antigen— antibody complexes of mammals and have an opsonizing activity. It is established that injection of HE does not change the total protein concentration per 1 ml coelomic fluid, but affects dynamics of changes of the hemagglutinins titer. Time of hemoglobin elimination from circulation does not exceed 24 h. Humoral factors of coelomic fluid of the starfish Asterias rubens play an auxiliary role in congenital immunity reactions.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 23–28.Original Russian Text Copyright © 2005 by Kudryavtsev, Dyachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

ISOLATION OF TWO PANTOTHEINE-CONTAINING ACIDIC PROTEINS FROM MONKEY BRAIN

The isolation of two acid-soluble proteins from Macaca irus brain is reported. The proteins contained 34 and 35.6 per cent polar amino acids, respectively. One protein had a mol. wt. of approx. 37,100 and the other of 23,000 with subunits of 11,500. Each protein contained 4′-phosphopantetheine, which was hydrolytically determined as taurine and β-alanine and microbiologically as pantothenate. The smaller protein is found to be present in the supernatant following homogenization both in 0-32 M-sucrose and in 0-32 M-sucrose with 40 mm -NaCl. The larger protein is also mainly present in the supernatant fluid. The function of these proteins is unknown, but the mol. wt. 23,000 species could be acetylated from glucose and acetate in mouse brain tissue slices and the acetyl groups could be isolated as the hydroxamate.     

Chemical characteristics of chalones isolated from bovine spleen.

Isolation of spleen chalones from the postmicrosomal supernatant is described. Two glycoprotein fractions homogeneous on polyacrylamide-gel electrophoresis were obtained. In the biological test on mice, the preparation of 38 000 mol. wt. inhibited mitosis in spleen cells, and the low-molecular fraction (2100 mol. wt.) in thymus cells, showing respectively the properties of chalones B and T.     

Protease activity of Klebsiella pneumoniae of different virulence

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.     

Characterization of a New Bacillus thuringiensis Isolate Highly Active Against Cochylis hospes

A new bacterial isolate, 00-50-5, from sunflower head extracts was identified as Bacillus thuringiensis (Bt) according to its morphology. Bt isolate 00-50-5 was highly active against the banded sunflower moth (BSM), Cochylis hospes Walsingham. A sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 4–15% gradient gel of whole strain protein of 00-50-5 revealed six proteins with molecular masses (M_r) of 133, 80, 60, 27, 15, and 14 kDa. SDS-PAGE of pH 4.2-precipitated proteins (PP) or activated proteins formed by adding the BSM larval gut protease at 1:50 (wt/wt, protease/PP) showed five bands, including two major proteins of M_r 60 kDa and 27 kDa, and three small peptides of M_r 15, 13, and 7 kDa. The BSM larval gut protease was able to completely digest the proteins when present at a high ratio (10:1, wt/wt, protease/PP). The 60- and 27-kDa proteins could be digested by subtilisin Carlsberg at ratios of 1:50 or 1:1 (wt/wt, protease/PP), but neither BSM larval gut protease nor trypsin was effective at the same ratios. Three small peptides of M_r 15, 13, and 7 kDa were digested by the gut protease at a ratio of 1:1 (wt/wt). The N-terminal sequence of 1–31 amino acid residues for the 27-kDa protein showed 96.7% homology to a 31-amino acid fragment from camelysin, a protease from B. cereus, indicating that the 27-kDa protein may be a camelysin and a novel active protein against BSM. Received: 9 July 2001 / Accepted: 8 August 2001     

Earthworm leukocytes kill HeLa, HEp-2, PC-12 and PA317 cells in vitro

Earthworm coelomic fluid contains biologically active molecules and leukocytes that participate in phagocytosis, encapsulation. Presumably they synthesize and secrete several effector modulators of innate immune responses such as antibacterial molecules, cytotoxic proteins and cytokines. Several lytic molecules have been detected in coelomic fluid previously but it is not yet clear which are actually released from the coelomocytes. Our aim was to analyze the cytotoxic effects of coelomocytes on mammalian target cells and to provide evidence that the lytic factors originate from coelomocytes. Cell-free coelomic fluid, supernatants of short-term cultured coelomocytes, and lysates from coelomocytes--derived by mechanical and detergent extraction--were used in cytotoxicity assays performed on different mammalian standard tumor cell lines and mouse fibroblasts. We used native and denaturized (using proteinase K, and trypsin digestions, or heat-inactivation) coelomocyte lysates (CCL). The viability controls of targeted cells were made by measuring photometrically and analyzing by inverted microscopy. According to our results the coelomic fluid, the supernatant of cultured coelomocytes, and the CCL significantly decreased ratios of living cells compared to controls in a dose-dependent manner. Our experiments performed with CCLs suggest that coelomocytes are responsible for the productions of cytotoxic components presumably proteins.     

Adenosine 3'':5''-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.     

Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid

The major yolk protein (MYP) is localized to the egg and coelomic fluid of the adult sea urchin. While the egg‐localized MYP has been extensively studied, much less is known about the coelomic fluid‐localized protein. Therefore, we have conducted a comparative biochemical analysis of these proteins. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg, 170‐ and 240 kDa, and the coelomic fluid, 180‐ and 250 kDa. Fractionation in polyacrylamide gels revealed that under reducing conditions both species were present in each location. However, in the absence of reducing agent only one species was present in each fraction: 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 peptide mapping indicated that all four species have very similar primary structures. Circular dichroic spectral analysis and endogenous tryptophan measurements of the purified 170‐ and 180 kDa species revealed distinctive secondary and tertiary structural features with notable differences in their responses to calcium: apparent Kds of 245‐ and 475 μmol/L were measured for the 170‐ and 180 kDa species, respectively. Further analysis revealed that both species have differing calcium requirements for binding to membranes as well as protein‐dependent, membrane‐membrane interaction. We discuss the functional implications arising from the structural differences which exist between the egg and coelomic fluid resident MYPs.     

Alkaline phosphatase activities in human amniotic fluid, chromatographic separation of the foetal intestinal component

Hydrophilic gel permeation chromatography of 14-36 wk human amniotic fluid on Fractogel columns divides the total alkaline phosphatase (AP) activity in a higher and a lower mol wt zones. Differential inhibition testing, isoelectric focusing, cellulose acetate, agarose and polyacrylamide gel electrophoreses before and after neuraminidase treatment show the higher mol wt zone to be homogeneous and to be made of the higher mol wt foetal intestinal isoenzyme form whereas the lower mol wt zone represents an unresolved mixture of hepatic, placental and lower mol wt foetal intestinal isoenzymes. In the early stages of pregnancy, the activity associated with the higher mol wt zone outweighs by far that of the lower mol wt zone; however from the 24 th week one notes a steady increase in the relative magnitude of this second zone until at the end of the gestation period both zones assume near equal importance albeit within a lower total AP activity. Satisfactory quantitation of the higher mol wt foetal intestinal isoenzyme form in one ml amniotic fluid can be attained after a 3-h chromatography run using p-nitrophenylphosphate as substrate.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Uridine phosphorylase from Escherichia coli B. Enzymatic and molecular properties

Uridine phosphorylase (EC 2.4.2.3) from Escherichia coli B is an oligomeric protein composed of four identical subunits of 29,000 mol. wt. The enzyme has four half-cystine residues per subunit titrable only in denaturing condition. No disulphide linkages either inter- or intra-chain are present. The isoelectric point is 5.25. The enzyme shows strict specificity toward uridine and 5-methyluridine and is inhibited by thymine, deoxycytidine and heavy metal ions.     

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.     

Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein.

The interaction of mammalian reoviruses with sialylated glycoproteins was studied and found to be highly serotype specific in that attachment of type 3 Dearing reovirus to murine L cell receptors could be strongly inhibited by bovine submaxillary mucin (BSM), fetuin, and alpha 1 acid glycoprotein, albeit at different efficiencies, whereas attachment of type 1 Lang reovirus was inhibited only by fetuin. We subsequently demonstrated, by using reassortants between type 3 and 1 reoviruses, that inhibition of reovirus attachment to cell receptors was specified by the viral attachment protein gene S1. Using a solid-phase binding assay, we further demonstrated that the ability of reovirus type 3 or reassortant 1HA3 and the inability of reovirus type 1 or reassortant 3HA1 to bind avidly to BSM was a property of the viral S1 genome segment and required the presence of sialic acid residues on BSM oligosaccharides. Taken together, these results demonstrated that there is a serotype-specific difference in the ability of the reovirus attachment protein, sigma 1, to interact with sialylated oligosaccharides of glycoproteins. Interaction of reovirus type 3 with sialylated oligosaccharides of BSM is dramatically affected by the degree of O-acetylation of their sialic acid residues, as indicated by the findings that chemical removal of O-acetyl groups stimulated reovirus type 3 attachment to BSM, whereas preferential removal of residues lacking or possessing reduced amounts of O-acetyl groups per sialic acid molecule with Vibrio cholerae sialidase abolished binding. We also demonstrated that BSM was 10 times more potent in inhibiting attachment of infectious reovirus to L cells than was V. cholerae-treated BSM. The results are consistent with the hypothesis that sialylated oligosaccharides on host cells or erythrocytes may act as binding sites or components of binding sites for type 3 reovirus through a specific interaction with the virus attachment protein.     

Comparative effects of soil organic matter fractions on phosphatase activities in wheat roots

Summary Soil organic matter was obtained from two agricultural soils using alkali extraction followed by acidification to produce humic and fulvic acids which were further fractionated by adsorption and gel chromatography.All the products inhibited the activity of phosphatase prepared from wheat roots, but to different extents. Humic acids produced a greater inhibition of enzyme activity than either the fulvic acids or water extracts of soil. Aspergillin, fromAspergillus niger, had a similar C, H and N content to humic acid and produced a similar inhibition of phosphatase activity.The inhibitions produced by corresponding fractions derived from the two soils were slightly different, but the trends between similar fractions from different soils were comparable. The lower mol. wt. components of humic acid inhibited phosphatase activity to a greater extent than higher mot. wt. fractions. Although fulvic acid comprised only low mol. wt. components it was less effective in inhibiting enzyme activity than those components of comparable mol. wt. present in the corresponding humic acid. Synthetic polymaleic acid, produced an inhibition of phosphatase activity similar to that caused by fulvic acid.     

Liproprotein inhibitor of bone marrow cells in tumor-bearing rats.

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon).

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.     

Purification and characterization of a poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl

A poly(A)-binding protein (PABP) with mol wt 72,000 has been purified from chickpea (Cicer arietinum) epicotyls by ammonium sulfate fractionation, Cibacron blue F3-GA and poly(A) agarose chromatography. The binding properties and the specificity of binding show that the purified protein is an analogue of PABPs in other eukaryotes. This PABP is highly susceptible to proteolysis and upon degradation forms a polypeptide fragment of mol wt 21,000 which has an independent poly(A) binding activity.     

Purification and characterization of pancreatic elastase from Atlantic cod (Gadus morhua)

1. A pancreatic elastase from Atlantic cod (Gadus morhua) has been purified and characterized. 2. The enzyme is a very basic protein with an approximate mol. wt of 28,000. 3. The cod elastase has higher elastin specificity than porcine elastase, and it is inhibited by soybean trypsin inhibitor, which has no effect on porcine elastase. 4. The cod elastase expresses a higher turnover number (kcat) and catalytic efficiency (kcat/Km) than porcine elastase, but it is less thermostable.     

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.