Metabolism of prostaglandins in spontaneously hypertensive rats: NAD"-dependent 15-hydroxyprostaglandin dehydrogenase activity is decreased in kidney and increased in lung.

Renal, pulmonary and gastric NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase activities were determined in both spontaneously hypertensive and normotensive rats at 6 and 12 weeks of age. Renal enzyme activity in hypertensive rats was only 30–40% of that present in normotensive controls at both ages. In contract, pulmonary enzyme activity in hypertensive animals was twice as active as that in normal controls. There was no significant difference in gastric enzyme activity. NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase activity, the enzyme responsible for the conversion of vasoinactive PGF metabolites to PGE metabolites, also failed to show any difference in two types of rat kidneys. The results indicate that, in hypertension, prostaglandin inactivation is impaired in kidney but is facilitated in lung.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

Synthetic peptides representing sequence 39 to 59 of rat V beta 8 TCR fail to elicit regulatory T cells reactive with V beta 8 TCR on rat encephalitogenic T cells.

Subpathogenic doses of syngeneic autoreactive T cells protect experimental animals against associated autoimmune disease. Preferential use of the TCR of encephalitogenic T cells suggests that this molecule serves as the target for immunoregulation in experimental autoimmune encephalomyelitis (EAE). Whether peptides derived from the V beta 8 of the rat TCR elicit regulatory T cells and produce the same vaccinating effect against EAE as do whole T cells remains unknown. Here we show that immunization of Lewis rats with V beta 8(39-59), a peptide representing residues 39 to 59 of the rat V beta 8 TCR, does not induce the production of regulatory T cells reactive to the intact TCR V beta 8 containing this sequence. Moreover, animals that had recovered from both actively induced EAE and transferred EAE did not generate regulatory T cells that recognized the V beta 8(39-59) peptide. Further, transfusion of large doses of peptide-specific T cells did not protect the animals from EAE. Our results suggest that the V beta 8(39-59) peptide may comprise so-called cryptic epitopes, which function as immunogens only when dissociated from large protein complexes.     

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.     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.     

Optimization of batch processes involving simultaneous enzymatic and microbial reactions

Two general models for batch simultaneous enzymatic and microbial reaction (SEMR) processes are presented, the second derived from and simpler than the first and accounting for enzyme denaturation. Using the second model and parameter values from the literature, simulation was used to examine a range of enzyme addition rate strategies (in which the rate was a linear function of time) for a relatively fast ethanol fermentation and for a longer duration citric acid fermentation, both using cellulose as the substrate. For the ethanol process it is optimal (for a specific objective function which accounts for product value and enzyme cost) to add all the enzyme at the beginning of the process. But for the citric acid process a linearly decreasing enzyme addition rate, coupled with the addition of a small fraction of the enzyme at time zero, is better than pure batch operation or operation with the best constant enzyme feed rate.     

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.     

Lipid conjugates of antiretroviral agents. II. Disodium palmityl phosphonoformate: anti-HIV activity, physical properties, and interaction with plasma proteins

Disodium palmityl phosphonoformate, a novel lipid phosphoester of the anti HIV agent phosphonoformate (foscarnet), inhibits HIV replication in H9 cells and syncytia formation in MOLT-3 cells as effectively as foscarnet itself, as shown by dose-response data from assays for expression of p17 and p24 viral antigens and syncytia formation. Protein binding studies indicate that in serum, the derivative exists bound to albumin and the lipoproteins, and would therefore be likely to exhibit improved serum lifetime in vivo.     

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.