Defective Expression of β1-Integrins in Cells with Constitutively Active αLβ2-Integrins

We have investigated a potential relationship between expression of β1-integrins and adhesiveness of the β2-integrin LFA-1 (αLβ2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-β1 cell surface expression. Thirty-seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-β1 expression. Conversely, 7 of 21 clones selected for lack of β1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between β1 expression and LFA-1 activity, we further analyzed at which level β1 expression was blocked. We focused on one clone, HAP4, with activated LFA-1 and no detectable β1 cell surface expression and found, surprisingly, that it expressed wild-type levels of β1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of β1 protein. However, in addition to β1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-β1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-β1 antibody 4B4 was hidden or lost. The α4-chain was found in its precursor form but it did not associate with any β-chain, and it was not processed to its mature form. Instead α4-chains were eventually degraded. Taken together this showed that β1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper β1 folding and for repression of LFA-1 adhesiveness.     

Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.     

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.     

Seasonal variation in catabolic enzyme activities in breast muscle of some migratory birds

Summary Five bird species were examined in order to ascertain if any changes in flight muscle catabolism take place between breeding season and migration. Two different patterns were discovered. The first consists of a high oxidative capacity and a low glycolytic and anaerobic capacity during migration. The converse occurs during the breeding season, i.e. low oxidative, high glycolytic and anaerobic capacity. The pattern was found in those species that deposit large amounts of fat prior to migration. The second pattern was similar to the first, but there was no change in fatty acid oxidation capacity between breeding season and migration. The pattern was found in those species that do not deposit much fat towards migration. These changes are believed to reflect differences in migration strategy and differences in locomotory activity during different seasons. Deviations from these patterns are discussed.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques

Remsen, C. C. (Swiss Federation Institute of Technology, Zurich, Switzerland), and D. G. Lundgren. Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques. J. Bacteriol. 92:1765-1771. 1966.-A comparison was made of the fine structure of the cell envelope of the gram-negative bacterium Ferrobacillus ferrooxidans when cells were prepared for microscopy by freeze-etching and chemical fixation techniques. Cell envelopes of chemically fixed cells appeared as five separate layers distinguishable by their location and electron density. Frozen-etched cells showed a three-layered complex with each layer measuring approximately 100 A in thickness. The latter technique is considered to be "artifact-free" and, as a technique, yields purely morphological information on the natural state. The three layers revealed by freeze-etching are: the outer layer, a lipoprotein-lipopolysaccharide layer; the middle layer, a layer composed of globular protein attached to fibrillar mucopeptide; and the innermost layer, the cytoplasmic membrane. The latter was covered with 100 to 120 A particles. The relationship of the aforementioned layers to those seen in chemically fixed cells is discussed.     

Polymorphism in the interferon-alpha gene family.

A pronounced genetic polymorphism of the interferon type I gene family has been assumed on the basis of RFLP analysis of the genomic region as well as the large number of sequences published compared to the number of loci. However, IFNA2 is the only locus that has been carefully analyzed concerning gene frequency, and only naturally occurring rare alleles have been found. We have extended the studies on a variation of expressed sequences by studying the IFNA1, IFNA2, IFNA10, IFNA13, IFNA14, and IFNA17 genes. Genomic white-blood-cell DNA from a population sample of blood donors and from a family material were screened by single-nucleotide primer extension (allele-specific primer extension) of PCR fragments. Because of sequence similarities, in some cases "nested" PCR was used, and, when applicable, restriction analysis or control sequencing was performed. All individuals carried the interferon-alpha 1 and interferon-alpha 13 variants but not the LeIF D variant. At the IFNA2 and IFNA14 loci only one sequence variant was found, while in the IFNA10 and IFNA17 groups two alleles were detected in each group. The IFNA10 and IFNA17 alleles segregated in families and showed a close fit to the Hardy-Weinberg equilibrium. There was a significant linkage disequilibrium between IFNA10 and IFNA17 alleles. The fact that the extent of genetic polymorphism was lower than expected suggests that a majority of the previously described gene sequences represent nonpolymorphic rare mutants that may have arisen in tumor cell lines.     

Intracellular calcium oscillations induced in a T-cell line by a weak 50 Hz magnetic field

Applied weak magnetic fields have been shown to affect cellular activity on several levels, but the mechanisms involved remain elusive. We have decided to study an early signal transduction event in the human T cell line Jurkat; oscillations of free [Ca²⁺]i, of the type seen by crosslinking the CD3 complex. Cells were exposed to a 50 Hz, 0.1 mT, sinusoidal magnetic field while intracellular free calcium was measured in individual cells, using fura-2 as a probe. An acute response was observed with oscillatory increases in [Ca²⁺]i, which subsided when the field was turned off. The effect of the magnetic field on [Ca²⁺]i was comparable to that achieved by an anti-CD3 monoclonal antibody. © 1993 Wiley-Liss, Inc.     

Cellular Localization of Transforming Growth Factor-α mRNA in Rat Forebrain

Abstract: The cellular localization of transforming growth factor-α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a ³⁵S-labeled cRNA probe. TGFα cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam-pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.