Multidrug resistance gene 1 expression in salivary gland adenocarcinomas and oral squamous-cell carcinomas.

In combined chemotherapy for head-and-neck cancer (HNC), salivary gland-cell adenocarcinoma (SGA) shows insufficient clinical outcome, and it has been suggested that the sensitivity and/or the mechanism of resistance to anti-cancer drugs are different between SGA and oral squamous-cell carcinoma (SCC). The aim of our study was to clarify whether P-glycoprotein (P-gp) expression is associated with multidrug resistance (MDR) in HNC and the difference in the process of its development between SGA and SCC. In immunohistochemical analysis, P-gp expression was found in the ductal cells of salivary glands but not in oral mucosal epithelium. In cancer tissues, a few SCC cells in 12 of 37 and most cells in all SGAs expressed P-gp. The intensive P-gp expression was significantly found in SGA compared with SCC. In an in vivo chemotherapeutic model using tumor-bearing nude mice, P-gp expression in counterparts was observed in only a few cells of the HSY line, while no P-gp expression was observed in Hepd cells. However, P-gp expression was developed in both HSY and Hepd cell lines after vincristine (VCR) treatment. RT-PCR showed that the mean ratios of mdr1 mRNA expression levels in HSY clones were 3.7-fold higher than those in Hepd clones after VCR treatment, while each cell line exhibited both induction and activated production of P-gp. These results suggest that P-gp-related MDR in SGA is an inherent phenotype caused by both high levels of P-gp induction and activated P-gp production during VCR treatment, while that in SCC is an acquired phenotype chiefly caused by induction of P-gp.

Crystal structure of cambialistic superoxide dismutase from porphyromonas gingivalis.

The crystal structure of cambialistic superoxide dismutase (SOD) from Porphyromonas gingivalis, which exhibits full activity with either Fe or Mn at the active site, has been determined at 1.8-A resolution by molecular replacement and refined to a crystallographic R factor of 17.9% (Rfree 22.3%). The crystals belong to the space group P212121 (a = 75.5 A, b = 102.7 A, c = 99.6 A) with four identical subunits in the asymmetric unit. Each pair of subunits forms a compact dimer, but not a tetramer, with 222 point symmetry. Each subunit has 191 amino-acid residues most of which are visible in electron density maps, and consists of seven alpha helices and one three-stranded antiparallel beta sheet. The metal ion, a 3 : 1 mixture of Fe and Mn, is coordinated with five ligands (His27, His74, His161, Asp157, and water) arranged at the vertices of a trigonal bipyramid. Although the overall structural features, including the metal coordination geometry, are similar to those found in other single-metal containing SODs, P. gingivalis SOD more closely resembles the dimeric Fe-SODs from Escherichia coli rather than another cambialistic SOD from Propionibacterium shermanii, which itself is rather similar to other tetrameric SODs.

A change of the metal-specific activity of a cambialistic superoxide dismutase from Porphyromonas gingivalis by a double mutation of Gln-70 to Gly and Ala-142 to Gln.

Gln-70, which is located near the active-site metal, is conserved in aligned amino acid sequences of iron-containing superoxide dimutases (Fe-SODs) and cambialistic SOD from Porphyromonas gingivalis, but is complementarily substituted with Gln-142 in manganese-containing SODs (Mn-SODs). In order to clarify the contribution of this exchange of Gln to the metal-specific activity of P. gingivalis SOD, we have prepared a mutant of the enzyme with conversions of Gln-70 to Gly and Ala-142 to Gln. The ratio of the specific activities of Mn- to Fe-reconstituted P. gingivalis SOD increased from 1.4 in the wild-type to 3.5 in the mutant SODs. Furthermore, the visible absorption spectra of the Mn- and Fe-reconstituted mutant SODs more closely resembled that of Mn-specific SOD than that of the wild-type SOD. We conclude that a difference in configuration of the Gln residues of P. gingivalis SOD partially accounts for the metal-specific activity of the enzyme.

Inactivation and destruction of conserved Trp159 of Fe-superoxide dismutase from Porphyromonas gingivalis by hydrogen peroxide.

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5% of its original activity after a 15 min incubation at 25 degrees C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20% of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properties of the iron center in the Fe-SODs.

High-performance liquid chromatographic determination of prolylcarboxypeptidase activity in monkey kidney.

A simple HPLC procedure for the determination of prolylcarboxypeptidase activity in monkey kidney was established with Cbz-Pro-Ala used as substrate. Decrease of the substrate and increase of the product were stoichiometrically related to each other. Heat treatment at 60 degrees C freed the enzyme preparation of contaminating activities. Data on substrate specificity and influence of inhibitors suggested this method was sensitive for the determination of prolylcarboxypeptidase without the use of a radioactive substrate.

Purification and characterization of tripeptide aminopeptidase from bovine dental follicles.

Tripeptide aminopeptidase (EC 3.4.11.4) was purified from bovine dental follicles by a series of chromatographies. Purified enzyme had a specific activity of 59.5 units per mg protein with L-prolyl-glycylglycine as substrate. The pH optimum was 8.0. The purified native enzyme had a Mr of 230,000 and was shown to be a tetramer of subunit Mr of 58,000. The isoelectric point was pH 7.0. The enzyme was inhibited by 5-5'-dithio-bis (2-nitrobenzoic acid), o-phenanthroline, and bestatin. Substrate specificity studies indicated that the enzyme specifically hydrolyzes the N-terminal amino acid residue from tripeptides only.

The cDNA and protein sequences of mouse lactate dehydrogenase B. Molecular evolution of vertebrate lactate dehydrogenase genes A (muscle), B (heart) and C (testis).

Mouse lactate dehydrogenase-B cDNAs were isolated from cDNA libraries of macrophage (ICR strain) and thymus (F1 hybrid of C57BL/6 and CBA strains), and their nucleotide sequences determined. The lactate dehydrogenase-B cDNA insert of thymus clone mB188 consists of the protein-coding sequence (1002 nucleotides), the 5' (46 nucleotides) and 3' (190 nucleotides) non-coding regions, and poly(A) tail (19 nucleotides), while macrophage clone mB168 contains a partial lactate dehydrogenase cDNA insert from codon no. 55 to the poly(A) tail. Seven silent nucleotide substitutions at codon no. 142, 143, 186, 187, 241, 285 and 292, as well as a single nucleotide change in the 3' non-coding region, were found between these different strains of mice. The predicted sequence of 333 amino acids, excluding initiation methionine, was confirmed by sequencing and/or compositional analyses of a total of 103 (31%) amino acids from tryptic peptides of mouse lactate dehydrogenase-B protein. The nucleotide sequence of the mouse coding region for lactate dehydrogenase B shows 86% identity with that of the human isoenzyme, and only eight of the 139 nucleotide differences resulted in amino acid substitutions at residues 10, 13, 14, 17, 52, 132, 236 and 317. The rates of nucleotide substitutions at synonymous and nonsynonymous sites in the mammalian lactate dehydrogenase genes are calculated. The rates of synonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart) are considerably higher than the average rate computed from human and rodent genes. The rates of nonsynonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart), particularly the latter, are highly conservative. The rates of synonymous and nonsynonymous substitutions for the lactate dehydrogenase-C gene are about the same as the average rates for mammalian genes. A phylogenetic tree of vertebrate lactate dehydrogenase protein sequences is constructed. In agreement with the previous results, this analysis further indicates that lactate dehydrogenase-C gene branched off earlier than did lactate dehydrogenase-A and lactate dehydrogenase-B genes.

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes.

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.

Metal ion inactivation and chelator stimulation of Streptococcus mitis arginine aminopeptidase.

Activation of Streptococcus mitis (ATTC 9811) arginine aminopeptidase resulted in removal of the metal(s) from the enzyme molecule, and the action of the heavy metal ion in the inactivation process was shown to involve formation of a chelate complex between the enzyme molecule and metal or oxidation of functional group(s) on the enzyme surface. The enzyme also underwent activation by bovine serum albumin, amino acids, phosphate, and citric acid, which are probable physiological chelators.

Coordinate repression of arginine aminopeptidase and three enzymes of the arginine deiminase pathway in Streptococcus mitis.

Streptococcus mitis contains two arginine aminopeptidases (I and II) as an arginine-supplying system and the arginine deiminase pathway as an arginine-utilizing system. The levels of arginine aminopeptidase I and three enzymes of the arginine deiminase pathway were suppressed by glucose in an apparently coordinate manner. Enzyme II appeared to be constitutive.

Inorganic pyrophosphatase activity of purified bovine pulp alkaline phosphatase at physiological pH.

At physiological pH, the hydrolytic activity of purified bovine pulp alkaline phosphatase toward phosphorus compounds was observed to be in the order of inorganic pyrophosphate greater than beta-glycerophosphate greater than phosphorylcholine greater than p-nitrophenylphosphate greater than glucose-6-phosphate. Optimum pH of the enzyme toward inorganic pyrophosphate was shown to be 8.5, with around 60% of the activity at pH 7.5. The activity was increased by the addition of Mg2+, but a different pattern of activation was observed between pH 7.5 and 8.5.

Analysis and identification of human parotid salivary proteins by micro two-dimensional electrophoresis and Western-blot techniques.

Using this method, without denaturing agents, over 60 protein components in 2 microliter (about 4 micrograms of protein) of unconcentrated parotid saliva could be detected. After two-dimensional separation, the spots of albumin, secretory IgA, IgG, acid phosphatase, amylase and plasma-originating protein were identified by antibodies with the Western-blot technique.

Two-dimensional electrophoresis in the analysis of a mixture of human sublingual and submandibular salivary proteins.

Complex protein mixtures of unstimulated human sublingual and submandibular saliva were fractionated by two-dimensional (2-D) gel electrophoresis, visualized by silver staining and then analysed by immunostaining. Specific proteins were identified by incubation with specific antibody and peroxidase-conjugated second antibody (Western blot). Electrophoresis and silver staining revealed over 50 protein components in 2 microliter of unconcentrated mixture. The Western-blot technique allowed detection of protein spots of plasma origin when an antibody against whole serum was used, but only the albumin spot could be found. Albumin, secretory IgA, acid phosphatase and alpha-amylase were identified with specific antibodies.

Sandwich enzyme-immunoassay for dipeptidyl aminopeptidase IV in the serum of people with oral cancer.

A highly-sensitive sandwich enzyme immunoassay for serum dipeptidyl aminopeptidase IV (DAP IV) was developed; its level in sera of patients with oral cancer [1.50 +/- 0.35(SD)mg/l serum] was significantly lower than in normal subjects [2.27 +/- 0.77(SD)mg/l serum].

Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV.

Kinetic studies of pig kidney dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5) were carried out using substrates possessing a side-chain of different length at the P2 position (or amino-terminal position in this case) such as Lys-, Arg-, Phe-, Met-, Ser-, His-, Glu- and Gly-Pro-pNA. The hydrolytic coefficient (Kcat/Km) has determined in the order Met- greater than Glu- greater than Ser- greater than His- greater than Phe- greater than Lys- greater than Gly- greater than Arg-, indicating a gradual increase with elongation of the side-chain from 0.03 to 0.60 nm followed by a decline when side-chain length approached 0.70 nm. Thus, the most probable depth of the side-chain pocket at the S2 subsite of the enzyme is proposed to be 0.50-0.60nm.

Immunochemical properties and intracellular localization of two molecular forms of arginine aminopeptidase in Streptococcus mitis ATCC 9811.

Streptococcus mitis contains two multiple forms of arginine aminopeptidase (I and II) which differ from each other with respect to their content, immunochemical properties and cellular localization. Immunological analyses by Ouchterlony double immunodiffusion and immunoprecipitation showed an antigenic difference between each form by the use of antisera specific for each enzyme. The amounts of enzymes I and II within the cell were estimated to be 230 +/- 4.3 and 646 +/- 20 ng/mg protein (+/- S.D.), respectively, using a standard curve of purified enzyme in a single radial immunodiffusion assay. When intact cells were treated with the cell wall lytic enzyme, N-acetylmuramidase, though both enzymes were solubilized, a time lag was observed for the solubilization of enzyme II. Enzyme I was detected only in the cell wall fraction and showed no detectable associated with the membrane. Although most of the enzyme II activity was recovered in the cell wall fraction, a slight amount (7.5%) of the total activity was also found in the membrane fraction.