Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.     

A genome-wide departure from the standard neutral model in natural populations of Drosophila

We analyze nucleotide polymorphism data for a large number of loci in areas of normal to high recombination in Drosophila melanogaster and D. simulans (24 and 16 loci, respectively). We find a genome-wide, systematic departure from the neutral expectation for a panmictic population at equilibrium in natural populations of both species. The distribution of sequence-based estimates of 2Nc across loci is inconsistent with the assumptions of the standard neutral theory, given the observed levels of nucleotide diversity and accepted values for recombination and mutation rates. Under these assumptions, most estimates of 2Nc are severalfold too low; in other words, both species exhibit greater intralocus linkage disequilibrium than expected. Variation in recombination or mutation rates is not sufficient to account for the excess of linkage disequilibrium. While an equilibrium island model does not seem to account for the data, more complicated forms of population structure may. A proper test of alternative demographic models will require loci to be sampled in a more consistent fashion.     

Genetic controls over melanocyte differentiation: interaction of agouti-locus and albino-locus genetic defects

Tyrosinase activities and dopachrome conversion activity were evaluated in extracts made from skins of 6-day-old mice that were mutant at the agouti and albino loci. Dopa oxidase (DO) activity of tyrosinase in fully pigmented (C/C) mice is reduced in extracts made from skins of yellow 6-day-old mice as compared to those of black mice. Dopachrome conversion (DC) activity is absent from skin extracts of normal yellow mice and is present in normal black mice. DC activity is a characteristic of a separate enzyme which has been called dopachrome conversion factor or dopachrome oxidoreductase. We measured the dopa oxidase activity and dopachrome conversion activity in skin extracts of yellow mice and black mice that were mutant at the albino (C) locus. Extracts made from extreme-dilution (ce/ce) mice do not have DO activity. Those from yellow extreme-dilution mice do not have DC activity, while those from black, extreme-dilution mice do. The DO and DC activities that characterize skin extracts made from platinum (cp/cp) yellow mice are similar to those of platinum black mice. These observations suggest possible mechanisms by which the functions controlled by the agouti and albino loci interact to control melanogenesis.     

Synthesis of a metallothionein-like protein in cultured human skin fibroblasts: Relation to abnormal copper distribution in Menkes' disease

A metallothionein-like protein (MTP) is synthesized in normal diploid human skin fibroblasts cultured in Zn- or Cu-supplemented medium. Synthesis of MTP is not detected in cells cultured without metal supplementation of complete tissue-culture medium. Cultured fibroblasts from patients with Menkes' disease accumulate excess Cu which chromatographs both with highmolecular-weight protein(s) and with a Cu-MTP. Under normal culture conditions, the Menkes' MTP incorporates [³⁵S]-cystine, but not appreciable amounts of ⁶⁵Zn. However, Menkes fibroblasts retain the ability to incorporate ⁶⁵Zn into MTP in response to Zn supplementation of the medium. The results do not support the idea that Menkes' disease results from a failure of Cu to bind to MTP, but rather that an elevated intracellular Cu concentration in Menkes' disease fibroblasts leads to association of excess Cu with high-molecular-weight protein, stimulating synthesis of a Cu-binding MTP.     

Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization

Gangliosides are shed by tumor cells and can bind to normal cells in the tumor microenvironment and affect their function. Exposure of fibroblasts to exogenous gangliosides increases epidermal growth factor (EGF)-induced fibroblast proliferation and enhances EGF receptor (EGFR)-mediated activation of the mitogen-activated protein kinase signaling pathway (Li, R., Liu, Y., and Ladisch, S. (2001) J. Biol. Chem. 276, 42782-42792). Here we report that the EGFR itself is the target of this ganglioside effect: Preincubation of normal human dermal fibroblasts with G(D1a) ganglioside enhanced both EGF-induced EGFR autophosphorylation and receptor-tyrosine kinase activity. The enhancement was rapid (within 30 min), not due to alteration of time kinetics of the EGFR response to EGF, and reproduced in purified G(D1a)-enriched cell membranes isolated from ganglioside-preincubated fibroblasts. Evaluating the initial steps underlying activation, EGF binding, and EGFR dimerization, we found that G(D1a) enrichment of the cell membrane increased EGFR dimerization and the effective number of high affinity EGFR without increasing total receptor protein. Unexpectedly, G(D1a) enrichment also triggered increased EGFR dimerization in the absence of growth factor. This resulted in enhanced activation of the EGFR signal transduction cascade when EGF was added. We conclude that membrane ganglioside enrichment of normal fibroblasts (such as by tumor cell ganglioside shedding) facilitates receptor-receptor interactions (possibly by altering membrane topology), causing ligand-independent EGFR dimerization and, in turn, enhanced EGF signaling.     

Sphingolipid-induced enhancement of receptor-mediated uptake of low density lipoproteins in normal and receptor-deficient human skin fibroblasts

(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides G_M1 or G_D1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced supression of [¹⁴C]acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of α₂-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.     

Null alleles for gliadin blocks in bread and durum wheat cultivars

Summary Wheat gliadin proteins are coded by clusters of genes (complex loci) located on the short arms of chromosomes of homoeologous groups 1 and 6 in bread (6x) and durum (4x) wheats. The proteins expressed by the various complex loci have been designated gliadin blocks. In a survey of accessions from the Germplasm Institute (C.N.R., Bari, Italy) collection, several different accessions have been found that lack particular blocks of proteins (null alleles). In some bread wheat accessions, seeds do not express gliadins that are coded by chromosomes 1D and 6A in normal cultivars. Similarly, some durum wheat accessions lack -gliadin components coded for by genes on chromosomes 1A and 1B. The missing proteins do not result from the absence of whole chromosomes, but may be the consequence of partial deletion of these genes at a complex locus or result from their silencing.     

Effect of thryoidectomy on the properties of rat liver mitochondria during liver regeneration]

The properties of regenerating rat liver mitochondria are not changed by the thyroidectomy. Thus, hepatectomy induces, in thyroidectomized rats as in normal ones, a decrease in the activity of the outer membrane enzymes, a stimulation of oxidative phosphorylations and an increase of the turn over rate of mitochondrial proteins. Thyroid hormones do not seem to have a major effect upon the liver regeneration mechanism.     

Deficient regulation of DNA double-strand break repair in Fanconi anemia fibroblasts

Fibroblasts from patients with Fanconi anemia (FA) display genomic instability, hypersensitivity to DNA cross-linking agents, and deficient DNA end joining. Fibroblasts from two FA patients of unidentified complementation group also had significantly increased cellular homologous recombination (HR) activity. Results described herein show that HR activity levels in patient-derived FA fibroblasts of groups A, C, and G were 10-fold greater than those seen in normal fibroblasts. In contrast, HR activity in group D2 fibroblasts was identical to that in normal cells. Western blot analysis revealed that the RAD51 protein was elevated 10-fold above normal levels in group A, C, and G fibroblasts, but was not altered in group D2 fibroblasts. HR activity levels in these former cells could be restored to near-normal levels by electroporation with anti-RAD51 antibody, whereas similar treatment of normal and complementation group D2 fibroblasts had no effect. These findings are consistent with a model in which FA proteins function to coordinate DNA double-strand break repair activity by regulating both recombinational and non-recombinational DNA repair. Interestingly, whereas positive regulation of DNA end joining requires the combined presence of all FA proteins thus far tested, suppression of HR, which is minimally dependent on the FANCA, FANCC, and FANCG proteins, does not require FANCD2.     

Submicroscopic duplication of chromosome 21 and trisomy 21 phenotype (Down syndrome)

Summary A patient with the phenotype of trisomy 21 (Down syndrome) was found to have a normal karyotype in blood lymphocytes and fibroblasts. Assessment of the chromosome 21 markers SOD1, CBS, ETS2, D21S11, and BCEI showed partial trisomy by duplication of a chromosome segment carrying the SOD1, CBS, and ETS2 loci and flanked by the BCEI and D21S11 loci, which are not duplicated. This submicroscopic duplication at the interface of 21q21 and 21q22.1 reduces to about 2000–3000kb the critical segment the trisomy of which is responsible for the phenotype of trisomy 21.     

Phosphatidylcholine mobility in liver microsomal membranes

Analysis of the 35SO4-labelled macromolecules synthesized by cultures of normal )NIL8) and transformed (NIL8-HSV) hamster fibroblasts has revealed the following differences between the two cell lines: (1) The proportion of sulfate incorporated into cell-associated macromolecules is three times higher in normal than in transformed cells. In addition, normal fibroblasts incorporate more sulfate into extracellular, middle and low molecular weight species than do transformed cells. Transformed cells, however, incorporate more sulfate into extracellular, very high molecular weight species than do normal cells. (2) Normal fibroblasts, which synthesize much more extracellular dermatan sulfate than do transformed cells, produce a class of extracellular heterogeneous sulfated proteoglycans absent from transformed cultures. This macromolecular species consists largely of dermatan sulfate. The transformed cells instead release a lower molecular weight class of proteoglycans which consist of chondroitin sulfates A and C. (3) The large, external, transformation-sensitive glycoprotein is sulfated in NIL8 cultures. This macromolecular species is present on the surface membrane of normal cells, but absent from transformed cells. Sulfated large, external transformation-sensitive protein is also present in the conditioned medium from normal cultures. A similar species is present in the conditioned medium from transformed cultures, but has a slightly higher apparent molecular weight and differs in other properties from the large, external, transformation-sensitive protein of normal cells.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Infection of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex virus: study of capacity and Weigle reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.     

Endogenously labeled low density lipoprotein triglyceride and apoprotein B kinetics.

The kinetics of endogenously labeled low density lipoprotein (LDL) triglycerides (TG) and apoprotein B (apoB) have been studied in four normal and in four hyperlipemic subjects using double tracers. Analysis of the data suggests that most LDL triglycerides turn over about 10 times faster than apoB (0.003/min vs. 0.0003/min) and that about 10% of the LDL particles contain most of the TG found with LDL. It is not possible to determine from the analysis whether each new LDL particle arrives with the excess TG or whether only a subpopulation of particles contains most of the TG. The kinetic analysis further suggests that triglyceride-rich LDL particles do not exchange with an extraplasma compartment as most LDL particles do, and thus, they behave more like very low density lipoprotein particles. A compartmental model accounting for both the LDL-TG and LDL-apoB kinetics is proposed.     

Molecular population genetics of inducible antibacterial peptide genes in Drosophila melanogaster

Insects respond to septic infection in part by producing a suite of antimicrobial peptides that may be subject to host-pathogen coevolutionary dynamics. In order to infer population genetic forces acting on Drosophila antibacterial peptide genes, we examine global properties of polymorphism and divergence in the Drosophila melanogaster defensin, drosocin, metchnikowin, attacin C, diptericin A, and cecropin A, B, and C genes. As a functional class, antibacterial peptides exhibit low levels of interspecific amino acid divergence. There are multiple amino acid polymorphisms segregating within D. melanogaster, however, a high proportion of which change the charge or polarity of the variable residue. These polymorphisms are particularly prevalent in processed signal and propeptide domains. We find that models of coevolutionary "arms races" and selectively maintained hypervariability do not adequately describe the population dynamics of mature antibacterial peptides in D. melanogaster, but that a highly significant excess of high-frequency derived polymorphisms coupled with substantial intralocus linkage disequilibrium suggests that positive selection may act on antibacterial peptide genes. Some attributes of the data may be consistent with a simple demographic model of population founding followed by expansion, but departures from the equilibrium null tend to be more pronounced in the peptide genes than at other loci around the genome.     

Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.     

Microinjection of human cell extracts corrects xeroderma pigmentosum defect

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.     

Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus.

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.     

Uptake and degradation of high density lipoprotein: comparison of fibroblasts from normal subjects and from homozygous familial hypercholesterolemic subjects.

Fibroblasts cultured from the skin of subjects with homozygous familial hyperlipoproteinemia (HFH) internalize and degrade low density lipoproteins at a much lower rate than do fibroblasts from normal subjects. Evidence has been presented that this reflects the absence from such mutant cells of specialized binding sites with high affinity for low density lipoproteins. The specificity of this membrane defect in familial hypercholesterolemia is further supported by the present studies comparing the metabolism of low density lipoproteins (LDL) and high density lipoproteins (HDL) in normal fibroblasts and in fibroblasts from HFH patients. The surface binding (trypsin-releasable (125)I) of (125)I-labeled LDL by HFH cells was approximately 30% of that by normal cells at a concentration of 5 micro g LDL protein per ml. At the same concentration the internalization (cell-associated (125)I after trypsinization) and degradation (trichloroacetic acid-soluble non-iodide (125)I) of (125)I-labeled LDL were less than 10% of the values obtained with normal cells. In contrast, the binding of (125)I-labeled HDL to HFH cells was actually somewhat greater than that to normal cells. Despite this, the internalization and degradation of (125)I-labeled HDL by HFH cells averaged only 70% of that by normal cells. [(3)H]- or [(14)C]Sucrose uptake, a measure of fluid uptake by pinocytosis, was similar in normal and HFH fibroblasts. These findings are consistent with the proposal that fibroblasts from subjects with HFH lack high-affinity receptors for LDL. These receptors do not play a significant role in HDL binding and uptake. Instead, as previously proposed, HDL appears to bind randomly on the cell surface and its internalization is not facilitated by the specific mechanism that internalizes LDL. The small but significant abnormalities in HDL binding and internalization, however, suggest that there may be additional primary or secondary abnormalities of membrane structure and function in HFH cells. Finally, the observed overall rate of uptake of LDL (that internalized plus that degraded) by HFH fibroblasts was considerably greater than that expected from fluid endocytosis alone. This implies that adsorptive endocytosis, associated with binding to low-affinity sites on the cell surface, may play a significant role in LDL degradation by HFH cells, even though it does not regulate endogenous cholesterol synthesis in these cells.