A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Nature and distribution of copper in green lagoon crab Scylla serrata (Forskal)

In Scylla serrata (Forskal), copper is present in detectable quantities in all tissues except the central nervous system. 75% of animal's total copper is found in the haemolymph, hepatopancreas, and cuticle. Both the haemolymph and the hepatopancreas share equally ≈ 60% of the animal's total copper. Copper is found in the TCA-soluble and insoluble fractions of water-soluble tissue extracts and in the chloroform : methanol-soluble and insoluble fractions of water-insoluble residue from the hepatopancreas. No other tissue has all the fractions of copper. In haemolymph copper is present only in the TCA-insoluble fraction. The TCA-soluble fraction is also absent in muscles. The absence of any lipid-bound copper in other tissues and the presence of copper in all fractions from the hepatopancreas indicates that this tissue may play a major role in the mobilization, conservation and detoxication of copper.     

An Evaluation of the Metachromasia of Bromophenol Blue

The heterogeneity of bromophenol blue from different commercial sources was revealed by paper chromatography. Isopropanol:ammonia:water (20:1:2) as the solvent system gave the best separation. A variety of impurities: violet, pink, light blue and yellow coloured ones were observed. Two of the yellow fractions showed a spectral shift to red in the presence of ammonia vapour. The respone of the main dye component with the anionic chromotropes such as heparin and hyaluronate was found to be metachromatic similar to that exhibited by the dye solution and not due to a polychromatic effect. The metachromatic effect was blocked by FeCl₃ as in the case of cationic dye metachromasy. The observed metachromatic colour is not one of the colours which characterize those resulting from changes caused by pH.     

Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

Comprehending renin inhibitor’s binding affinity using structure-based approaches

The performance of several structure-based design (SBD) approaches in predicting the binding affinity of diverse small molecule inhibitors co-crystallized to human renin was assessed to ascertain the modeling tool and method of choice required when dealing with structure-based lead optimization projects. Most of the SBD approaches investigated here were able to provide qualitative guidance, but quantitative accuracy as well as decisive discrimination between [in]actives is still not within reach. Such an outcome suggests that the current methods need improvement to capture the overall physics of the binding phenomenon for consistent applications in a lead optimization setting.     

Synthesis and glycosidase inhibitory activities of chain-modified analogues of the glycosidase inhibitors salacinol and blintol

The synthesis of chain-modified analogues of the naturally-occurring glycosidase inhibitor, salacinol, and its selenium analogue, blintol is described. The modification consists of a frame shift of the sulfate moiety by one carbon atom in the zwitterionic structures as well as an extension of the acyclic chain to five carbons. The target molecules were synthesized by alkylation of 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio (or seleno)-D-arabinitol at the ring heteroatom by 2,3,5-tri-O-p-methoxybenzyl D- or L-xylitol-1,4-cyclic sulfate, followed by deprotection with trifluoroacetic acid. Two of the four compounds inhibit recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose oligosaccharides in the small intestine, with Ki values of 20+/-4 and 53+/-5 microM.     

Interleukin-2 binds to ganglioside GD(1b)

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.     

Structural features in the model of a thermostable and stress-resistant protein, SP1 from aspen

A three dimensional theoretical model of SP1 (stable protein 1), which is resistant to high temperature and biotic-stresses, is presented here. The model was generated by the application of homology modeling technique. The conformational rigidity imparted to the fold by the presence of hydrogen-bonded, C5, C7, C10 and C13 structures in the loop regions, multiple aromatic--aromatic interactions at the protein interior and on the surface, in addition to salt-links and hydrogen-bonds are primarily the major factors, responsible for the increased stability of protein. The putative protein family is characterized by motifs, E-x(0,1)-L-x-[AEGQS] and V-x(2,3)-L-x-[ADEGST] and the active site in the tertiary structure is formed by conserved aromatic and isoleucine clusters.