Muc5ac Mucin Expression During Rat Skin Development

Some mucin genes have been detected during human embryonic and fetal organ development; however, little is known about mucin expression in epidermal development, neither in humans nor in other species. The present research was developed to explore Muc5ac skin expression during pre- and post-natal rat development. Immunohistochemistry (IHC), Western blotting (WB) and RT-PCR were employed. By IHC, Muc5ac protein was found early in embryonic epidermis from day 13 of gestation until seven days after birth when the surface epidermis became negative and the reaction was restricted to secreting sebum cells. In coincidence with IHC findings, WB analysis showed a band at approximately 200KDa at the same periods of development. Results were also confirmed by RT-PCR.Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report that confirmed Muc5ac expression during skin development.Key words: Muc5ac, skin, rat, development     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

Calcium-dependent protein modulator of cyclic nucleotide phosphodiesterases from mouse epidermis.

1. A heat-stable modulator protein was partially purified from mouse epidermis. The protein stimulated modulator-depleted cyclic AMP phosphodiesterase from bovine brain in the presence of Ca2+. 2. DEAE-cellulose chromatography of epidermal extracts demonstrated the presence of two main phosphodiesterase activities that hydrolysed both cyclic AMP and cyclic GMP. A minor peak was eluted between 0.1 and 0.3 M-sodium acetate and a major peak was eluted between 0.3 and 0.45 M-sodium acetate. 3. Cyclic AMP phosphodiesterase activity eluted at low salt concentrations was markedly activated by the epidermal modulator protein in the presence of Ca2+. Storage of the enzyme led to a decrease in its sensitivity to the protein modulator. 4. Treatment of mouse skin with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate, which leads to an increase in epidermal cyclic nucleotide phosphodiesterase activity, did not alter the amount of modulator present in soluble epidermal extracts. The tumour promoter decreased the amount of modulator extractable from particulate epidermal preparations with Triton X-100.     

Insulin-induced tyrosine-phosphorylation in intact rat adipocytes

Insulin-induced tyrosine-phosphorylation in intact isolated rat adipocytes was studied using immunoblotting method with antiphosphotyrosine antibodies. Insulin-stimulated adipocytes were solubilized with Triton X-100. The lysate was incubated with wheat germ agglutinin, then with hydroxylapatite. Insulin stimulated tyrosine-phosphorylation of a 95 KDa protein which adsorbs to wheat germ agglutinin and appears to be the beta-subunit of the insulin receptor. Among the proteins adsorbed to hydroxylapatite, tyrosine-phosphorylation of 170 KDa and 60 KDa proteins was stimulated. 170 KDa was also stimulated by polyclonal anti-insulin receptor antibodies B-10 Ig G, IGF-I and H2O2. The detection of these proteins in rat adipocytes may lead to the elucidation of a common signal transduction pathway in insulin-responsive cells.     

A protein in rat prostatic interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostatechromatin(residual proteins)contain proteins able tointeract specifically with androgen-receptor complex andis,therefore,a part of the acceptor complex.Amongresidual proteins,a 97 KDa protein has been found whichbinds signifieantly to a genomic fragment containingan androgen-regulated gene coding for a 22 KDa protein.The biological significance of this binding in androgenaction need to be further studied. A mini-plasmid clone containing 22 KDa proteincoding sequence was cloned into Charon 4A genomiclibrary from which a 5.7 Kb genomic fragment wasisolated,identified by hybridization with a 5' and a 3'cDNA probes,and shown to contain the 5' flankingsequence.Restriction enzyme treatment of this fragmentyielded a 4.7 Kb restriction fragment representingthe 5' upstream region and a 1.0 Kb containing part ofthe coding sequence.Deletion studies indicated that the97 KDa protein bound only to a subclone of about 300 bpsegment.Furthermore,gel shifting experiment supportedits DNA-prptein binding.     

Reversible inhibition by DMSO of hydrocortisone-induced keratinization of chick embryonic skin

Hydrocortisone, at a physiological concentration of 10^?8 M, induces keratinization of chick embryonic tarsometatarsal skin in a chemically defined medium in 4 days [1]. The presence of 1–4% DMSO with hydrocortisone reversibly prevented this keratinization. DMSO suppressed the appearance of epidermal structural protein, which was preferentially induced by hydrocortisone. It also suppressed hydrocortisone-induced epidermal transglutaminase activity; which was presumably responsible for polymerization and decrease in solubility of epidermal protein in keratinization, and it suppressed increase of epidermal protein. When DMSO was added to differentiated skin or added concomitantly with a higher concentration of hydrocortisone, epidermal transglutaminase activity was suppressed. Electron microscopic studies showed that hydrocortisone induced tonofilament bundles and keratinized cells with cellular envelopes, which are all characterestic of α-type keratinization of chick embryonic skin [2], and that DMSO inhibited hydrocortisone induced keratinization and kept the epidermis in an undifferentiated state. Moreover, DMSO inhibited epidermal DNA synthesis and increase in thickness of the epidermis during culture of hydrocortisone-treated skin, indicating that it suppressed cell proliferation as well as cell differentiation. DMSO by itself at 1 or 2 % did not affect epidermal cell differentiation, but suppressed cell proliferation when compared with untreated control.     

Evidence for a regulatory action of vanadate on protein phosphorylation in brain microvessels

We investigated the action of vanadate on protein phosphorylation in microvessels isolated from rat brain. We found that a stimulation of protein phosphorylation from 32P-ATP occurs, in the presence of different concentrations of vanadate, 10(-3) M being the most effective dose. This action was time-dependent, and it was more evident after 60 s of treatment. The contribution of ATPase inhibition caused by vanadate appears to be negligible. In addition a stimulation of cAMP-dependent protein kinase activity was observed. The pattern of protein phosphorylation showed that exposure to 10(-3) M vanadate resulted in a nonspecific stimulation of protein phosphorylation concomitantly with a selective inhibition of the 55 KDa protein phosphorylation. The nature of this protein is also discussed.     

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.     

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

Release of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.     

Phosphorylation of aortic plasma membranes by protein kinase C

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca²⁺ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca^2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca²⁺.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - K_m Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate     

Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland.

A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A.     

Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.