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.     

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     

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.     

Niche differentiation with respect to light utilization among coexisting dwarf shrubs in a subarctic woodland

Summary Canopy structure, shoot design, and photosynthetic light recruitment were used to compare four coexisting dwarf shrub species with respect to light utilization. All four species showed different shoot designs which probably result in different light interception properties. Leaves of Vaccinium uliginosum showed the highest levels of photosynthetic light saturation but in situ the shoots of this species reached their maximum photosynthetic rate at the lowest photon flux densities. No consistent differences with respect to photosynthetic light responses were found between deciduous and evergreen species. At sites dominated by one of the deciduous species (Vaccinium uliginosum or V. myrtillus), the two evergreen species studied (V. vitis-idaea and Empetrum hermaphroditum) occurred in the understory, i.e., with their leaf distribution slightly below that of the deciduous species. Sites dominated by one of the evergreen species showed less vertical differentiation in leaf distribution between species.     

Micro-site performance of evergreen and deciduous dwarf shrubs in a subarctic heath in relation to nitrogen status

The relationship between micro-site nitrogen status (total soil N conc., total vegetation N, and leaf N conc.) and performance (biomass) for two evergreen and two deciduous dwarf shrubs was studied in a subarctic heath. One deciduous species, Vaccinium uliginosum , dominated on sites having the highest vegetation nitrogen content and highest leaf nitrogen concentrations. The evergreen Empetrum hermaphroditum dominated on sites with a low total vegetation nitrogen content and low leaf nitrogen concentrations. Two other species, V. myrtillus (deciduous) and V. vitis-idaea (evergreen), showed intermediate patterns that were not clearly separated from either V. uliginosum or Empetrum . Soil nitrogen content showed no significant differences between micro-sites. Possible physiological reasons for the observed patterns are discussed.     

A new moderately repetitive DNA sequence family of novel organization.

In cloning adenovirus homologous sequences, from a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digests of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (4q31) and 19 (q13.1-q13.5). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.