Biomedical subjects

S Michel

Publications and source records attributed to S Michel.

At least 127 records · Page 7Linked to original sources

PubMed1989

Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes.

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross-linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus-40 (SV-40)-transformed human foreskin keratinocyte line SV-K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane-associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane-associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.

Butyrates↗

PubMed1989

A stopped-flow mixer device for a batch microcalorimeter application to NAD-NADase reaction.

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.

Calorimetry↗

PubMed1989

Atypical human rotaviruses in the G.D.R.

Following the aetiologic agent of diarrhoea in children aged one to three years we found in the stools a large number of rotaviruses by electron microscopy (EM), although ELISA was negative; the sensitivity of ELISA had been confirmed in previous investigations. In 141 children with diarrhoea 10 conventional (group A) and 21 antigenically distinct rotaviruses were detected. The presence of atypical rotaviruses could be confirmed by electrophoretic analysis of their RNA migration profiles. The electropherotype pattern corresponded to the group C rotaviruses reported by others.

Antigens, Viral↗

PubMed1988

Plasma membrane transglutaminase and cytosolic transglutaminase form distinct envelope-like structures in transformed human keratinocytes.

Cross-linked envelope formation in the transformed human keratinocyte line SV-K14 requires treatment of the cells with a Ca2+ ionophore. Depending on the culture conditions, different extracellular Ca2+ concentrations are necessary to trigger the process which is catalyzed by the enzyme transglutaminase. Confluent cells grown in the presence of serum express only the cytosoluble form of the enzyme and need 5 mM Ca2+ for optimum protein cross-linking, whereas serum-starved cells which additionally contain the plasma membrane associated form of the enzyme require only 1 mM Ca2+. The envelope-like structures thus synthesized are morphologically and biochemically distinct.

Cell Line↗

PubMed1988

Complete nucleotide sequence of the neuraminidase gene of the human influenza virus A/Chile/1/83 (H1N1). Brief report.

The complete nucleotide sequence of the neuraminidase (NA) gene of influenza virus A/Chile/1/83 (H1N1) has been determined after reverse transcription and cloning into the plasmid pAT 153/PvuII/8. The gene is 1461 nucleotides long and codes for a protein of 470 amino acids. The overall nucleotide and predicted amino acid sequence of the A/Chile/1/83 NA exhibits a high homology with other N1 neuraminidases. Hyper-variable regions concerning A to G exchanges are discussed.

Amino Acid Sequence↗

PubMed1988

Evolution of influenza polymerase: nucleotide sequence of the PB2 gene of A/Chile/1/83 (H1 N1).

The complete nucleotide sequence of the PB2 gene of influenza virus A/Chile/1/83 (H1 N1) is presented. Sequence comparison between A/Chile PB2 protein and the known PB2 sequences of the influenza strains A/WSN/33 (H1 N1), A/PR/8/34 (H1 N1), A/NT/60/68 (H3 N2), A/Kiev/59/79 (H1 N1), A/FPV/Rostock/34 (H7 N1), and B/Ann Arbor/1/66 indicates extensive amino acid homology for the influenza A virus PB2 proteins. Small clusters of basic amino acids are conserved in all PB2 proteins including the influenza B PB2 protein which has only 39% sequence homology overall to the PB2 polypeptides of type A influenza viruses. The evolutionary rate of 5.7 x 10(-3) nucleotide substitutions per site per year and 0.25% amino acid changes per year between the A/Chile/1/83 and A/NT/60/68 PB2 appears to be higher than that calculated earlier for A/NT, A/PR/8 and A/WSN. An unusually high degree of sequence change between A/Chile/1/83 and A/Kiev/59/79 PB2 polymerase was revealed and this is discussed in terms of its probable origin.

Amino Acid Sequence↗

PubMed1988

Localization and in vivo activity of epidermal transglutaminase.

The plasma membrane-associated transglutaminase is responsible for the formation of a cornified envelope during terminal differentiation of epidermal keratinocytes. We have studied the epidermal distribution of this enzyme ("epidermal transglutaminase") and its activity by fluorescence microscopy in histological sections of normal human skin and human skin grafted onto nude mice. Three different techniques were employed: (i) incubation of skin sections with a monoclonal antibody raised against a purified preparation of the enzyme; (ii) incubation of skin sections with dansylcadaverine, a fluorescent substrate of the enzyme, in the presence of Ca++ ions; and (iii) subcutaneous injection of dansylcadaverine into mice, at least 2 months after grafting. The first technique is supposed to detect all enzyme molecules carrying the epitope that is recognized by the antibody, even when the enzyme is catalytically not active; the second should decorate all sites in which membrane-bound transglutaminase activity is located, and the third detects only sites in which transglutaminase is active in vivo. With the first two techniques a broad band of plasma membrane associated fluorescence, reaching from the middle of the spinous layer to the stratum corneum, was detected in both normal and grafted skin. In vivo enzyme activity, however, was found to be restricted to one, or at most two, cell layers at the interface of the stratum granulosum and stratum corneum and to coincide with the layer in which the antigenicity of involucrin, a natural substrate of epidermal transglutaminase, disappeared.

Calcium↗

PubMed1988

Transglutaminases in normal and transformed human keratinocytes in culture.

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.

Cell Line, Transformed↗

PubMed1988

Morphological and biochemical characterization of the cornified envelopes from human epidermal keratinocytes of different origin.

The formation of a cornified envelope (CE) is a major event in the terminal differentiation of epidermal cells. Nomarski contrast microscopy of the envelopes purified from different sources reveals the existence of two major, but morphologically distinct classes: the very irregularily shaped fragile type CEf, and the polygonal rigid type CEr. Human keratinocytes in submerged culture are only able to produce type CEf. Specimens from healthy human epidermis contain largely type CEr. Psoriatic scales from different patients show both types in varying proportions. Tape stripping of normal epidermis reveals that type CEf is present in the lowermost layers of the stratum corneum and type CEr is present in the upper layers, indicating that the two types represent a different stage of maturation. Cyanogen bromide peptide mapping of electrophoretically purified envelopes reveals striking differences between cultured keratinocytes, normal epidermis, and psoriatic scales but also slight interindividual variations. This variability supports the view that the molecular CE composition is not strictly determined. On the other hand, no difference could be detected in the peptide maps of CEf and CEr obtained after tape stripping from the same healthy volunteer indicating that CE maturation within the stratum corneum does not involve the provision of qualitatively new proteins.

Cell Differentiation↗

PubMed1988

Functional and structural analysis of the ribonucleoprotein complexes of different human influenza virus strains.

Ribonucleoprotein (RNP) cores were prepared from various strains of human influenza virus by treating the purified or spikeless virus particles with non-ionic detergents such as Nonidet P-40 and centrifugation in continuous linear glycerol gradients. In addition to RNA, the purified complexes contained viral nucleoprotein (NP) and the three P proteins (PA, PB1, PB2) as determined by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. Contaminations with other viral polypeptides, especially HA1, HA2 and M were below 1%. All RNP complexes were transcriptionally active in vitro. Comparison of the polymerase activity of purified complexes revealed considerable differences depending not only on the content of polymerase proteins. The activity of RNP complexes was enhanced for all strains tested by adding of ApG.

DNA-Directed RNA Polymerases↗

PubMed1988

Antibodies to polymerase proteins of influenza virus A/PR/8/34 (H1N1): comparison on the immunoreactivity with polymerase proteins of other influenza A and B virus strains.

The polymerase proteins (PB1, PB2, PA) of the influenza virus strain A/PR/8/34 (H1N1) were isolated from whole virion or ribonucleoprotein (RNP) fractions by electrophoresis on polyacrylamide gel and electroelution or Sepharose CL-6B chromatography in the presence of SDS. Antisera to polymerase proteins (P proteins) were raised in rabbits; the immunoglobulins (Ig) were purified by affinity chromatography. Characterization of the antibody fraction by Western blot analysis showed a highly monospecific reaction with the three polymerase proteins. Spot immunobinding assay was used to compare the immunoreactivity of the monospecific polymerase antibodies with the P proteins of other influenza A subtypes and influenza B strains, revealing high immunoreactivity with the components of all influenza A strains and only insignificant reactivity with the components of the influenza B strains tested.

Animals↗

PubMed1988

Interaction between anti-influenza viral polymerase antibodies and RNP particles using the in vitro transcription process and an immunogold labelling technique.

Immunogold labelling and in vitro transcription of influenza virus vRNA have been used to analyse the interaction of anti-influenza polymerase antibodies with influenza-ribonucleoprotein (RNP) complexes. The polymerase proteins (P proteins) were localized exclusively at one end of the RNP segments. In the course of transcription the amount of P protein decreased significantly. The in vitro transcriptase activity y of influenza A virus RNP complexes in the presence of anti-polymerase antibodies to the strain A/PR/8/34 was inhibited by 60%. In contrast, RNP transcriptase activity of influenza B virus was not inhibited by these antibodies.

Animals↗

PubMed1987

Identification and subcellular distribution of cornified envelope precursor proteins in the transformed human keratinocyte line SV-K14.

SV-40 transformed human foreskin keratinocytes (line SV-K14) develop under conditions of serum starvation the competence to form cornified envelopes that are characteristic of terminally differentiating epidermal cells. In this cell line, the final assembly of the envelope does not occur spontaneously but must be induced using a calcium ionophore. Five potential precursor proteins with molecular weights of 140K, 90K, 61K, 53K, and 36K, respectively, could be detected in the extracts of envelope competent and noncompetent cells. The 61 kD and the 36 kD precursors were specifically decorated in immunoblots when using an antiserum directed against the purified cornified envelope of SV-K14 cells. The 140 kD protein was identified as involucrin by means of a commercial anti-involucrin antibody. Part of the 61 kD protein was found to be inserted into the plasma membrane after the cells gained envelope competence. The set of precursor proteins used by SV-K14 cells differed markedly from those described in the literature for epidermal cells in vivo and for normal human keratinocytes in vitro. Furthermore, cyanogen bromide cleavage of purified envelopes from transformed and normal keratinocytes revealed a completely different peptide pattern. This indicates that the exact molecular composition of the cornified envelope may not be strictly determined and may vary according to the availability of potential substrate proteins at the very moment when the cross-linking enzyme, the plasma membrane associated transglutaminase, becomes functional.

Cell Line↗

PubMed1986

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis.

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.

Adult↗

PubMed1985

Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes.

When confluent cultures of the transformed human keratinocyte line SV-K14 are shifted to serum-free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross-linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane-bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.

Acyltransferases↗

PubMed1985

High-yield purification of plasma membranes from transformed human keratinocytes in culture.

The density pertubation technique with cationic silica microbeads was applied to prepare highly purified plasma membranes from cultured human keratinocytes. Trypsinized cells were coated successively with the beads (diameter approximately 50 nm, gravity greater than 2 g/cm3) and polyacrylic acid before they were lysed by osmotic shock and mechanical shear. The plasma membranes remained in the form of large open sheets which could easily be separated from other cell organelles and the cytosol by low-speed centrifugation. The membrane preparation was characterized by scanning and transmission electron microscopy, marker enzyme activities, one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis, and the specific beta-adrenergic receptor count. A yield of 79 +/- 9% was calculated by comparing the amount of beta-adrenoceptors in the purified membrane preparation with that of a crude cellular particulate fraction. The specific beta-adrenoceptor count of these two preparations was 1.2 +/- 0.02 and 0.2 +/- 0.05 pmol/mg protein, respectively, indicating a 6-fold improved purification with this microbead technique. The purified membranes were essentially free from contamination of other cell organelles.

Cell Line↗

PubMed1985

Localization of bullous pemphigoid antigen (BPA) in isolated human keratinocytes.

In early studies, the bullous pemphigoid antigen (BPA) has been localized extracellularly in the lamina lucida in the basement membrane zone. However, trypsin-dissociated basal cells can be tagged with bullous pemphigoid sera (BPS). By immunofluorescence, BPA appears located at the dermal pole of basal cells (BC). This may indicate that when BC are separated from the underlying matrix molecules, chunks of BPA remain attached to them. In the present study, fresh crude initial suspensions (CIS) of epidermal cells were prepared by trypsin-EDTA dissociation. The cells were smeared and air-dried. Polar fluorescent cells (i.e., BC) amounted to 42% +/- 7%. CIS were then passed through a fluorescence-activated cell sorter (FACS). In the fluorescent-positive fractions selected by FACS, 34% +/- 7% only of the BC were present. FACS-negative cell fractions were smeared on glass slides, air-dried, and restained with BPS + fluorescein isothiocyanate; 66% +/- 10% of BC were present in these fractions. This is evidence that trypsin-isolated BC comprise two subpopulations: one with BPA directly accessible, the other not. Viability tests and tissue culture studies indicated that the FACS-positive cell fractions were not viable. BPA was extracted from CIS, FACS-positive, and FACS-negative fractions and immunoblotted against BPS. Identical blots were found. FACS-negative cell fractions were treated with heparitinase, nitrous acid, methanol-chloroform, or EDTA without modifying the number of reacting cells. When BC were treated with Triton X-100 or permeabilized by successive freezings and thawings, the number of positive cells became comparable to those obtained by air-drying smears. Finally, BPA was localized on the intracellular part of hemidesmosomes of BC by immunoelectron microscopy. To see whether BPA was also present extracellularly, suction blisters were raised in minipigs and BPS injected into the blister cavity. BPA was found attached to all cells of the cellular roof but not to the dermal base of the blisters. When pieces of skin kept overnight in cold trypsin were reacted with BPS, BPA was found on both sides (epidermal and dermal) of the split. It is concluded that BPA has two localizations: one extracellular, essentially labile which accumulates at the dermal-epidermal junction; the other essentially stable which remains on the intracellular part of basal cell hemidesmosomes and which can be detected after permeabilization of the cells.

Antigens↗

PubMed1983

The increased phosphorylation of ribosomal protein S6 in Arbacia punctulata is not a universal event in the activation of sea urchin eggs.

Eggs of the sea urchins Arbacia punctulata (Ap), Lytechinus pictus (Lp), and Strongylocentrotus purpuratus (Sp) were labeled to equilibrium with 32PO3-4. Approximately 65-70% of the label in extractable adenine nucleotides comigrates chromatographically with ATP. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels show that each species possesses a distinct complement of phosphate-exchangeable phosphoproteins. No changes in the phosphoprotein composition are detected in Lp and Sp eggs as a result of fertilization or development for 2.5 hr (with the possible exception of a 43,000 Mr protein in Lp). In Ap, increases in the phosphorylation of bands at Mr's 30,000, 55,000, and 105,000 are seen during the first 10 min postinsemination. The 30,000 Mr band in Ap eggs has previously been identified as ribosomal protein S6 and the hypothesis presented that its increased phosphorylation may be an important step in the activation of protein synthesis at fertilization (D. G. Ballinger and T. Hunt, 1981, Dev. Biol. 87, 277-285). In Lp and Sp eggs S6 (identified by two-dimensional PAGE) is heavily phosphorylated in the unfertilized state and the extent of labeling does not increase after fertilization. If the increased phosphorylation of S6 seen in Ap is indeed related to translational activation, then these results suggest that different sea urchin species may rely on different mechanisms for the activation of protein synthesis.

Animals↗