Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

PROTEINS OF AXONAL TRANSPORT: INTERACTION OF RAPIDLY TRANSPORTED PROTEINS WITH LECTINS

Rapidly transported fucose-labelled glycoproteins from the optic system of the rabbit were solubilised with the non-ionic detergent Berol 172. The major labelled components were bound to wheat germ agglutinin or Concanavalin A coupled to Sepharose but not to other lectins or glycoproteins. It was concluded that rapidly transported proteins contain exposed N-acetyl-D-glucosamine     

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

Rapid Migration of Inositol Phospholipids with Axonally Transported Substances in the Rabbit Optic Pathway

Following an intraocular injection of myo-[2-3H]inositol, the axonal transport of labelled water-soluble substances and inositol phospholipids was investigated. Evidence was obtained for a rapid axonal transport of a relatively small amount of labelled inositol phospholipids. In contrast to other axonally transported phospholipids, there was no significant accumulation of labelled, rapidly transported inositol phospholipids in the nerve terminal region at later time intervals following the isotope administration.     

Thyroxine,methimazole, and thyroid microsomal autoantibody titres in hypothyroid Hashimoto's thyroiditis.

Ten hypothyroid patients with Hashimoto''s thyroiditis were treated with methimazole 30 mg in addition to thyroxine 0.15 mg daily. Another 10 hypothyroid patients with Hashimoto''s thyroiditis were given thyroxine 0.15 mg alone. After 22 weeks of treatment significant decreases in thyroid microsomal autoantibody titres were observed in both groups (p less than 0.01). There was no difference in the mean change in titre between the two groups. When the patients treated with methimazole were subsequently given thyroxine 0.15 mg alone for a further 22 weeks no additional change in titre was observed. The data suggest that thyroxine, by normalising serum thyroid stimulating hormone concentrations, may reduce the autoantigenic properties of the thyrocytes with a subsequent decrease in autoantibody titres.     

Separation of enantiomeric amines and acids using chiral ion-pair chromatography on porous graphitic carbon

The application of porous graphitic carbon as adsorbing phase for direct separation of enantiomeric acids and amines using chiral ion-pair chromatography is described. The enantiomeric amines were separated as diastereomeric ion pairs with N-benzyloxycarbonylglycyl-L -proline, N-benzyloxycarbonylglycylglycyl-L -proline, or captopril as the chiral counterion. High enantioselectivities were obtained for amines having a hydrogen bonding function in the vicinity of the asymmetrical carbon atom. Quinine was the chiral counterion used to separate the enantiomeric acids. The strongly UV-absorbing quinine improved detection of solutes having low UV-absorbing properties, e.g., (R,S)-2-chloropropionic acid, by “indirect detection.” Retention and stereoselectivity of enanticmeric acids were regulated by the quinine concentration and by the addition of carboxylic acids as well as polar modifiers, e.g., methanol and 2-propanol, to the mobile phase. © 1992 Wiley-Liss, Inc.     

Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1

The binding of Shiga toxin isolated from the bacterium Shigella dysenteriae type 1 to a series of glycolipids and to cells or cell homogenates has been studied. Bound toxin was detected using either 125I-labeled toxin or specific monoclonal antibody and 125I-labeled anti-antibody. Overlay of toxin on thin-layer chromatograms with separated glycolipids and binding to glycolipids coated in microtiter wells established that the toxin specifically bound to Gal alpha 1-4Gal beta (galabiose) placed terminally or internally in the oligosaccharide chain. No glycolipid shown to lack this sequence binds the toxin. Most of the glycolipids with internally placed galabiose were not active, indicating a sterical hindrance for toxin access to the binding epitope. Binding of toxin to HeLa cells in monolayers could be inhibited by preincubation of the toxin with galabiose covalently linked to bovine serum albumin (BSA), but not with free oligosaccharides containing galabiose or with lactose coupled to BSA. This demonstrated that the inhibition is specifically dependent on galabiose and requires multivalency of the disaccharide to be efficient. The inhibitory effect was successively enhanced by increasing the substitution on BSA (7, 18, and 25 mol of galabiose/mol of BSA). The BSA-coupled galabiose could also prevent the cytotoxic effect on HeLa cells (detachment of killed cells). There are cell lines with a dense number of receptor sites, but which are resistant to toxin action (uptake and inhibition of protein synthesis) which may suggest two types of receptor substances which are functionally different and unevenly expressed. In analogy with the mechanism earlier formulated for cholera toxin, we propose glycolipid-bound, bilayer-close galabiose as the functional receptor for membrane penetration of the toxin, while galabiose bound in glycoproteins affords binding sites but is not able to mediate penetration.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.