Association of a basic 25K protein with membrane coating granules of human epidermis

Keratinocytes of the upper granular layers contain unique round-to-oval granules, 100-500 nm in diameter, in their peripheral cytoplasm. These granules (known as membrane coating granules [MCG], or lamellar granules) fuse with the apical cell surface of uppermost granular cells and discharge their contents into the intercellular space, where they are believed to play a role in establishing the permeability barrier of the epidermis and possibly in regulating the orderly desquamation of terminally differentiated keratinocytes. Using two monoclonal antibodies originally prepared against hair follicle antigens, we have identified a 25K epidermal protein in association with both MCG-like granules in the peripheral cytoplasm of granular cells as well as MCG-derived intercellular material. This protein is relatively basic (pI greater than 8), largely aqueous soluble, methionine deficient, and is relatively abundant in epidermis (comprising up to approximately 0.1% of soluble proteins). Its distribution is restricted to the granular layer of keratinized (cornified) stratified squamous epithelia. The identification of this protein component opens new avenues for studying the molecular mechanisms underlying the establishment of permeability barrier and/or regulation of desquamation.     

Effect of a new synthetic free radical scavenger, 2-octadecyl ascorbic acid, on the mortality in mouse endotoxemia

Oxygen-derived free radicals have been implicated as mediators of cellular injury in several model systems. In order to clarify the role of oxygen radicals in endotoxemia, we measured the serial lipid peroxide changes resulting from systemic radical reactions using a newly developed colormetric method. To determine the effect of a free radical scavenger on mortality in endotoxemia, a new synthetic scavenger, 2-Octadecylascorbic acid (CV-3611), which overcome the detrimental properties (circulation half-life and cell penetration) of native SOD, was used in the model of mouse endotoxemia induced by the i.p. administration of E-coli endotoxin (10 mg/kg). Serial LPO (Lipid Peroxide) changes revealed significant elevations from the basal level of 4.52 +/- 0.79 nmol/ml to 10.5 +/- 2.04 nmol/ml at 2h (P less than 0.05), 12.0 +/- 2.44 nmol/ml at 8h (P less than 0.05), 32.8 +/- 12.5 nmol/ml at 12h (P less than 0.05) and 13.6 +/- 2.40 nmol/ml at 24h (P less than 0.05) following i.p. administration of E-coli. The circulation half life of CV-3611 was checked by a reversed-phase HPLC after 10 mg/kg s.c. administration. The level of CV-3611 reached peak levels of 0.54 +/- 0.10 micrograms/ml at 1h and 0.52 +/- 0.20 micrograms/ml at 2h then gradually decreased to the level of 0.04 +/- 0.004 micrograms/ml at 6h and to a non-detectable level at 24h after s.c. administration. Increased survival was seen at 2 days (P less than 0.001) after E-coli endotoxin administration in the CV-3611 treated group compared to the control group. These results suggest that oxygen derived free radicals contribute to mortality in mouse endotoxemia and that antioxidants such as CV-3611 may provide a new therapeutic avenue by improving survival of patients with gram-negative bacterial sepsis.     

Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the P_FR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. P_R and P_FR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.05–1.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofP_FR was at 728–730 nm and that of P_R was at 666–667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the P_Ror P_FR form did not result in degradation of the molecule. Therate of dark reversion of P_FR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate. ²Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990)     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Identification of Surface-Exposed Parts of Red-Light- and Far-Red-Light- Absorbing Forms of Native Pea Phytochrome by Limited Proteolysis

Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either theP_R or the P_FR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the P_R or the P_FR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of P_FR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for P_R (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the P_R and P_FR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992)     

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.     

Detection of the changes in protein distribution of rat plasma induced by carbon tetrachloride administration by means of two-dimensional electrophoresis

The changes in rat plasma protein distribution after carbon tetrachloride administration were examined using two-dimensional electrophoresis, utilizing isoelectric focusing in polyacrylamide gel in the first dimension and pore gradient polyacrylamide gel electrophoresis in the second dimension. Drastic changes in amount of protein were observed at more than 20 spot positions including those of transferrin, Gc-globulin and low-density lipoprotein. The time course of the changes was examined, and the most drastic changes were observed at 2 days after carbon tetrachloride administration.     

Characterization of a keratinocyte-specific extracellular epitope of desmoglein. Implications for desmoglein heterogeneity and function.

Despite the presumed importance of desmoglein, a 160-kDa glycoprotein, in desmosome formation and its possible involvement in certain blistering skin diseases, the precise location and function of this protein have not yet been firmly established. We describe here the characterization of a new monoclonal antibody, AE23, against an extracellular epitope of desmoglein. Both the AE23 epitope and another epitope, defined by the previously characterized DG3.4 antibody, reside on a 160-kDa human epidermal desmoglein as evidenced by their identical solubility profile, their coexistence in a 130-kDa desmoglein degradative product, their coadsorption by an AE23 immunoaffinity column, and the identical changes in the two antigens' electrophoretic mobility after air oxidation and deglycosylation. The AE23 epitope is resistant to various endoglycosidases, suggesting that sugar moieties are not involved. Characterization of several proteolytic fragments of this epidermal desmoglein enabled us to map the DG3.4 epitope to a 96-kDa intracellular domain and the AE23 epitope to an extracellular domain flanked by the plasma membrane and the distal N-glycosylation site(s). However, these two epitopes do not always coexist on the same desmoglein molecule. For example, tissue surveys showed that although the DG3.4 epitope is present in the desmogleins of all epithelial cell types, the AE23 epitope is limited to normal keratinocytes. Moreover, electron microscopic localization data indicate that whereas the DG3.4 epitope is detected in the submembranous plaques of desmosomes, the AE23 epitope is present in the intercellular space of both desmosomal and nondesmosomal areas. These results raise the possibility that there exist several biochemically closely related isoforms of desmoglein, one (AE23+/DG3.4+) restricted to epidermal desmosomes, one (AE23+/DG3.4-) uniformly distributed along the keratinocyte cell surface, and another (AE23-/DG3.4+) present in desmosomes of simple epithelia and basal cells of cultured keratinocytes. The uniform distribution of at least one desmoglein-related antigen in the intercellular space of keratinocytes coupled with the realization that different isoforms of desmogleins form a subfamily of cadherins suggest that desmoglein(s) may play a more general role in keratinocyte adhesion than previously appreciated.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.