Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays.

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

TEMPO-mediated glycoconjugation: a scheme for the controlled synthesis of polysaccharide conjugates

A method for the controlled conjugation of polysaccharides (PSs) to carrier proteins based on the stoichiometric oxidation of PSs with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is described. In this scheme, a PS can be stoichiometrically activated, by the formation of minimal amounts of carboxylic acids with TEMPO, and subsequently coupled to a carrier protein through amide bond formation. Our exploratory studies included a number of readily available glucans, which were oxidized with several combinations of TEMPO-sodium hypochlorite preparations followed by conjugation to BSA. Following these investigations, more structurally complex bacterial capsular PSs were stoichiometrically oxidized with TEMPO-sodium hypochlorite and conjugated to BSA. Hypochlorite was deemed to be the reaction component responsible for the extent of oxidation. Collectively, the data showed this approach to be effective in designing PS-conjugates with no disruption to the structural integrity and immunochemical properties of the PS, and thus has the potential to become a reliable method for glycoconjugate vaccine synthesis.     

Quality control of polyvalent pneumococcal polysaccharide-protein conjugate vaccine by nephelometry.

A nephelometric method was used for quantitative analysis of individual polysaccharides (PSs) in a polyvalent pneumococcal conjugate vaccine using CRM(197) as carrier protein. Using this method, the individual types 4, 6B, 9V, 14, 18C, 19F and 23F PSs were found to range between 82.3 to 119% of the manufacturer's indicated values.During conjugation using reductive amination, pneumococcal PS was first oxidized to introduce aldehyde groups. Higher or lower levels of antigen-antibody reaction were observed in periodate activated and then reduced PS of some serotypes compared to non-treated PS. Use of oxidized and reduced PS may provide an early indication of change in conjugation process. Furthermore, since the final monovalent and polyvalent conjugate vaccines gradually change during the storage period, the nephelometry provides an useful analytical method for stability study of these vaccines.     

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.     

Binding diversity of monoclonal antibodies to α(2 → 8) polysialic acid conjugated to outer membrane vesicle via adipic acid dihydrazide

Abstract Murine monoclonal antibodies (mAbs) were generated using group B Neisseria meningitidis and Escherichia coli K1 polysaccharides (PSs) conjugated to outer membrane vesicle (OMV) via adipic acid dihydrazide, and were used to identify the immunodeterminants expressed on these capsular PSs. Ten mAbs representative of IgM and all subclasses of IgG were obtained which recognized diverse immunodeterminants on α(2 → 8) polysialic acid (PSA). The specificity of mAbs to different antigenic determinants was assessed by their differential binding to PSA attached to a solid phase by different methods and confirmed by absorption studies. Two mAbs from the E. coli K1 fusion were directed to the O -acetyl epitope and the rest reacted with both the PSs only when attached to a solid phase by certain means. The methods by which PSA was coated on the solid phase had an impact on the epitope expression and binding pattern. At the concentrations used, the O -acetyl-specific mAbs, IgG1 and IgG3 mAbs were not bactericidal against group B N. meningitidis , whereas other mAbs were. The conjugates B and K1 PSs present to the murine immune system different antigenic determinants, some of which elicit bactericidal antibodies.     

Cellulases of Penicillium verruculosum

Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-β-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated.     

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.     

Purification and characterization of bacteriophage receptor on Veillonella rodentium cells

Veillonellophage N2 adsorbed to polysaccharides (PSs) on Veillonella rodentium ATCC 17743 cell wall, and the bacteriophage receptor contained only glucosamine. D(+)-glucosamine hydrochloride (Sigma) also adsorbed the veillonellophage N2. These results therefore indicate that the receptor to the veillonellophage N2 is cell wall PSs. The PSs of the host cells as receptor have been characterized. Glucosamine accounted for approximately 100% of the weight of the PSs. The PSs which were partially resolved by Sephadex G-75 chromatography comprised approximately four glucosamine units. Their primary structure was determined by 400 MHz n.m.r. spectroscopy. One- and two-dimensional ¹H-nmr experiments showed the PS to be a branched polymer. Glucosamine linkage was detected in one of the branches.     

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.     

Structural and actin-binding properties of the trypsin-produced HMM and S1 from gizzard smooth muscle myosin

The reaction of trypsin on the heavy chain of gizzard myosin and chymotryptic HMM was investigated under restricted fragmentation conditions. The three fragments of the head part with 29 kDa, 50 kDa and 26 kDa were isolated and identified. The 66 K heavy chain segment containing the S1-S2 junction was slowly but extensively degraded liberating a S1-like entity which lacked an intact COOH-terminal 26 kDa region; this isolated species displayed full intrinsic ATPase activities but little actin-binding ability. Tryptic HMM was also formed bearing a fragmented heavy chain and lacking the 20 kDa light chain. Its actin-activated ATPase was derepressed upon cleavage of the 66 kDa segment by papain. We propose that the integral 66 kDa heavy chain component is directly involved in the regulation of the gizzard actomyosin ATPase.     

Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha-subunit of the (Na(+)+K(+))-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by (3)H release.     

Melting of myosin rod as revealed by electron microscopy. II. Effects of temperature and pH on length and stability of myosin rod and its fragments

Effects of temperature and pH on intact rabbit and chicken myosin, isolated myosin rods, rabbit subfragment-2 (61 kDa, 53 kDa, and 34 kDa) and chicken light meromyosin (LMM) fragments were tested to induce a phase transition from alpha-helix to coil conformation, within the hinge region. The influence of temperature and pH were studied directly with length determination by electron microscopy. An increase of temperature to 50 degrees C yielded a shortening of 16 nm, 8 to 9 nm and 7 to 11 nm for intact myosin, isolated rods and long S-2 fragments, respectively. The length of the 34 kDa short S-2 and LMM fragments were unchanged. An increase of pH from neutral to pH 8.0 yielded values that were somewhat smaller, e.g. 12 nm, 6 nm and 6 to 8 nm for intact myosin, isolated rods and long S-2 fragments, respectively, whereas the 34 kDa short S-2 LMM fragments were also unaffected. Thus, melting and subsequent shortening is confined to the region between LMM and short S-2 segment, that is the hinge region. Alteration of temperature had a stronger shortening effect than alteration of pH, and shortening of long S-2 was more pronounced under physiological salt conditions as compared with high (0.3 M) salt. The shortening of rods in intact myosin amounted to twice the value observed with isolated rods. The amount of contraction was somewhat smaller in rods than in the 61 kDa and 53 kDa long S-2 fragments.     

Paenibacillus campinasensis BL11: a wood material-utilizing bacterial strain isolated from black liquor

In order to search for new thermophilic microorganisms and their enzymes, bacterial strains from black liquor of brownstock at washing stage of kraft pulping process were screened. Therein a multiple glycosyl hydrolase-producing strain, BL11, was isolated as a dominant species in the xylan-degrading bacterial population and identified as Paenibacillus campinasensis. The bacterial strain used all kinds of saccharides and polysaccharides, except lignin as carbon source and produced multiple extracellular polysaccharide-degrading enzymes, including one xylanase (41 kDa), three cellulases (42, 57 and 86 kDa), one pectinase (28 kDa) and one cyclodextrin glucanotransferase (38 kDa). P. campinasensis BL11 lacked lipase and protease activities and was able to grow over a wide range of pH, but it particularly grew well around neutral pH at 55 degrees C. Based on its physiological characteristics, it has strong potential for industrial application and bioresource utilization.     

Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a gamma-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na(+)-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose at equilibrium were altered by radiation. Thus Na(+)-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.     

Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities

Hermetical microwave was used to degrade Porphyridium cruentum polysaccharides from 2918 to 256.2, 60.66 and 6.55 kDa. The antioxidant properties of different molecular weight polysaccharides were evaluated by determining the scavenging ability of free radicals, inhibitory effects on lipid peroxidation in liver homogenates and hemolysis of mouse erythrocytes. Analysis of physicochemical properties confirmed that microwave degradation might not markedly change the chemical components of the polysaccharides. High-molecular-weight polysaccharides from P. cruentum had no obvious antioxidant activity, but low-molecular-weight fragments after degradation exerted an inhibitory effect on oxidative damage. The 6.55-kDa fragment had stronger antioxidant activity than the 60.66 and 256-kDa fragments.     

On the serological specificity of the Escherichia coli O8 and O9 antigens.

The O8 and O9-specific lipopolysaccharides of Escherichia coli lost their serological activity during liberation of the polysaccharide moieties (alpha-mannans) by mild acid hydrolysis, as tested by passive haemagglutination and haemagglutination inhibition. The serological activities and specificities were restored by substitution of the polysaccharides with 1 to 2 stearoyl groups per polysaccharide chain. The mannans obtained by biosynthesis in vitro were serologically active only when bound to the membrane-associated hydrophobic carrier molecule. Liberation of the polysaccharides from the carrier by treatment with aqueous phenol resulted in loss of the serological activity. The O8- and O9-specific mannans of E. coli are thus serologically active when they are part of an amphiphilic molecule and not as free polysaccharides.     

Fibronectin fragments in human seminal plasma

The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.     

Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling procedure was also performed to detect the breakage of 3'-OH ends of a DNA strand. Furthermore, we found that nuclear lamins were degraded from 88 kDa and 66 kDa to 37 kDa and 47 kDa fragments. The DNA fragmentation could be inhibited by AC-DEVD-CHO and AC-YVAD-CHO. The results indicate that the apoptosis in plant cells may share some similar pathways to apoptosis in animal cells.