Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.     

Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

Two micromorphic bivalves from the Silurian of Gotland

Two new micromorphic bivalve species from the Silurian of Gotland are described and assigned to the new genusKenzieana, viz.Kenzieana lata andKenzieana angusta. Kenzieana exhibits morphological features characteristic of some unrelated extant species; above all a conspicuous antero-posterior flattening of the shell. Probably it was a siphonate suspension feeder with a reclining mode of life.     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.     

Nonrecombinant Meiosis I Nondisjunction in Saccharomyces Cerevisiae Induced by Trna Ochre Suppressors

The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.     

Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions.

A physical map of the D. melanogaster genome is being constructed, in the form of overlapping cosmid clones that are assigned to specific polytene chromosome sites. A master library of ca. 20,000 cosmids is screened with probes that correspond to numbered chromosomal divisions (ca. 1% of the genome); these probes are prepared by microdissection and PCR-amplification of individual chromosomes. The 120 to 250 cosmids selected by each probe are fingerprinted by Hinfl digestion and gel electrophoresis, and overlaps are detected by computer analysis of the fingerprints, permitting us to assemble sets of contiguous clones (contigs). Selected cosmids, both from contigs and unattached, are then localized by in situ hybridization to polytene chromosomes. Crosshybridization analysis using end probes links some contigs, and hybridization to previously cloned genes relates the physical to the genetic map. This approach has been used to construct a physical map of the 3.8 megabase DNA in the three distal divisions of the x chromosome. The map is represented by 181 canonical cosmids, of which 108 clones in contigs and 32 unattached clones have been mapped individually by in situ hybridization to chromosomes. Our current database of in situ hybridization results also includes the beginning of a physical map for the rest of the genome: 162 cosmids have been assigned by in situ hybridization to 129 chromosomal subdivisions elsewhere in the genome, representing 5 to 6 megabases of additional mapped DNA.