Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins

Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains.     

Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro

LIM mineralization protein-1 （LMP-1） is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in viva. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 （hLMP-1 [t]）, lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with AdS-hLMP-1 （t）-green fluorescent protein or viral vector control. The expression of hLMP-1 （t） RNA and the truncated protein were examined. The results showed that hLMP-1 （t） blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase （ALP） activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein （BMP）-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP- 1 （t） can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 （t） enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.     

Structural characteristic,pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T _m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T _m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T _m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T _m values of both holo bLF and holo cLF were reduced showing T _m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with ¹²⁵I-labeled Cry1Ac, Cry1Ac mutant ⁵⁰⁹QNR-AAA⁵¹¹ (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both ¹²⁵I-Cry1Ac and ¹²⁵I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots ¹²⁵I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, ¹²⁵I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. ¹²⁵I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured ¹²⁵I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) ¹²⁵I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14

The intracellular chaperone heat‐shock protein 70 (Hsp70) can be secreted from cells, but its extracellular role is unclear, as the protein has been reported to both activate and suppress the innate immune response. Potential immunomodulatory receptors on myelomonocytic lineage cells that bind extracellular Hsp70 are not well defined. Siglecs are Ig‐superfamily lectins on mammalian leukocytes that recognize sialic acid‐bearing glycans and thereby modulate immune responses. Siglec‐5 and Siglec‐14, expressed on monocytes and neutrophils, share identical ligand‐binding domains but have opposing signaling functions. Based on phylogenetic analyses of these receptors, we predicted that endogenous sialic acid‐independent ligands should exist. An unbiased screen revealed Hsp70 as a ligand for Siglec‐5 and Siglec‐14. Hsp70 stimulation through Siglec‐5 delivers an anti‐inflammatory signal, while stimulation through Siglec‐14 is pro‐inflammatory. The functional consequences of this interaction are also addressed in relation to a SIGLEC14 polymorphism found in humans. Our results demonstrate that an endogenous non‐sialic acid‐bearing molecule can be either a danger‐associated or self‐associated signal through paired Siglecs, and may explain seemingly contradictory prior reports on extracellular Hsp70 action.     

Discovery of novel 1,4-dihydropyridine-based PDE4 inhibitors

Substituted 1,4-dihydropyridines were discovered as a novel and potent series of phosphodiesterase 4 (PDE4) inhibitors. Structure–activity relationships within this series have been carried out and studies revealed that the dihydropyridine core, with indole moiety and 3,4-dimethoxybenzyl group, is a potent analogue for PDE4 inhibition. These novel series of compounds were prepared via a 3-component reaction in a single pot. In vitro biological activity, modeling studies and crystallography data are also reported.     

Some ecological observations on two agarophytes from India

The responses of Gracilaria corticata and Gelidiopsis gracilis (agarophytes) to different concentrations of sea water, pH range and lights of different colours have been studied to understand the ecological amplitudes. Both algae had a very narrow range of salinity tolerance from 29.0 to 33.1. With respect to pH also, they could thrive only under pH values from 7.0 to 8.0. Growth of these algae was retarded under all coloured lights. However, under yellow light growth was nearer to that of controls.     

Decolourization of Industrial Effluents – Available Methods and Emerging Technologies – A Review

Water pollution control is presently one of the major thrust areas of scientific research. While coloured organic compounds generally impart only a minor fraction of the organic load to wastewaters, their colour renders them aesthetically unacceptable. Stringent regulating measures are coaxing industries to treat their waste effluents to increasingly high standards. Colour removal, in particular, has recently become an area of major scientific interest as indicated by the multitude of related research reports. During the past two decades, several decolourization techniques have been reported, few of which have been accepted by some industries. There is a need to find alternative treatments that are effective in removing dyes and colourants from large volume of effluents, which are cost-effective, like the biological or integrated systems. This article reviews some of the widely used and most promising industrial wastewater decolourization methods. Data on decolourizing efficiencies of different causative agents, obtained by means of different physical, chemical and biological methods are discussed. Further a critical review is made on the various treatment methodologies and emerging technologies with a note on their advantages and disadvantages.     

Carbohydrate analyses of Manduca sexta aminopeptidase N, co-purifying neutral lipids and their functional interactions with Bacillus thuringiensis Cry1Ac toxin.

Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.