Partial breakdown of glycated alkaline phosphatases mediated by reactive oxygen species.

The lower levels of serum alkaline phosphatase (AP) activity found in patients with diabetes mellitus apparently originate from the selective disappearance or decrease in bone AP activity in the circulation. Hence, we investigated in vitro the effect of glycation on the activities of five AP isozymes. Aseptic incubation with 25 mmol/L of D-glucose and APs rapidly reduced bone and placental AP activities before those of liver, kidney and intestinal enzymes. The resulting bone and placental AP molecules were clearly glycated, according to the result of aminophenylboronic acid affinity chromatography. Furthermore, Western blotting analysis revealed that the placental AP molecule was fragmented, and its partial cleavage took place at Ala154 on the AP molecule. Since glycation of serum proteins causes the generation of reactive oxygen species, the effects of reactive oxygen species on placental AP activity were assayed, and the results indicated that hydroxyl radicals might be a major factor for the specific inactivation of AP activities. The reduction in AP activity by incubation with glucose in vitro was reversed by the further addition of catalase. Furthermore, ferrous ion with hydrogen peroxide, which generates hydroxyl radicals, had an inhibitory effect on AP activities. These findings suggest that the reduced AP activity in diabetic patients might result from partial cleavage of the bone AP molecule by reactive oxygen species induced by glycation.

Use of the highly sensitive PCR method to detect the Herpes simplex virus type 1 genome and its expression in samples from Behçet disease patients.

To study the involvement of the Herpes simplex virus type 1 (HSV-1) in patients with Behçet's disease (BD), PCR was used to detect HSV-1 DNA and RNA (LAT, TK, gB) in peripheral blood leukocytes and oral smears from patients with BD. Out of 32 patients with BD and 30 healthy volunteers, HSV-1 specific DNA was not detected in any case. Using a highly sensitive competitive RT-PCR method, HSV-1 specific mRNA was not detected either in peripheral blood leukocytes from any of the patients or volunteers. In contrast, serum levels of IgG anti HSV-1 antibody were higher in patients with BD; however, there were 70% sero-positives among the control subjects. Even though it has been reported that the highly frequent presence of the HSV-1 genome in peripheral blood is a characteristic feature of BD, our results show that the HSV-1 genome and its expression are not present in peripheral blood in patients with BD. We could not find any evidence that the lesions associated with BD were caused by the direct lytic effects of HSV-1.

Human tissue non-specific alkaline phosphatases: sugar-moiety-induced enzymic and antigenic modulations and genetic aspects.

To investigate the possible role(s) of glycans in human tissue non-specific alkaline phosphatase (TNAP) activity, the iso-enzymes were purified and treated with various exo- and endo-glycosidases. Catalytic activity, oligomerization, conformation and immunoreactivity of the modified TNAPs were evaluated. All TNAPs proved to be N-glycosylated, and only the liver isoform (LAP) is not O-glycosylated. Usually, the kidney (KAP) and bone (BAP) isoenzymes are similar and cannot be clearly discriminated. Differences between the immunoreactivity of KAP/BAP and LAP with a BAP antibody were mainly attributed to the N-glycosylated moieties of the TNAPs. In addition, elimination of O-glycosylations moderately affects the TNAP reactivity. Interestingly, N-glycosylation is absolutely essential for TNAP activity, but not for that of the placental or intestinal enzymes. According to the deduced amino acid sequence of TNAP cDNA, Asn-213 is a possible N-glycosylation site, and our present findings suggest that this sugar chain plays a key role in enzyme regulation. With regard to the oligomeric state of alkaline phosphatase (AP) isoforms, the dimer/tetramer equilibrium is dependent on the deglycosylation of glycosyl-phosphatidylinositol(GPI)-free APs, but not GPI-linked APs. This equilibrium does not affect the AP conformation as observed with CD. With regard to TNAPs, no data were available on the gene expression or nature of the 5'-non-translated leader exon of human KAP, as opposed to BAP and LAP genes. cDNA sequencing revealed that cortex/medulla KAP is genetically related to BAP, and medulla KAP to LAP.

Characterization of two length cDNA for human MSX-2 from dental pulp-derived cells.

Screening of a human dental pulp cells cDNA library with mouse Msx-1 and Msx-2 cDNA probes led to the isolation of human MSX-2. Sequence and Northern Blotting analysis revealed that two different type of transcripts due to the length of 3' untranslated region were expressed in the human dental pulp cells.

Ovariectomy decreases osteogenetic activity in rat bone.

The purpose of this study was to investigate the effect of ovariectomy on osteoinductive activity in the bone in rat. Homograft implantation of decalcified humeral diaphysis from ovariectomized or sham-operated rats was performed and harvested after several time periods. A significant decrease in bone induction was found in terms of soft X-ray photography, alkaline phosphatase activity, mineral content and expression of osteocalcin (BGP; bone gla-protein) in the implants from the ovariectomized group in comparison to those from the sham-operated animals. This result suggested that the level of osteoinductive activity, probably due to bone morphogenetic protein, decreased in ovariectomized animals.

Properties of alkaline phosphatase in the gingival crevicular fluid.

The isoenzymic properties of the alkaline phosphatase (ALP) of the gingival crevicular fluid (GCF) were investigated and compared with those in other cells, such as human polymorphonuclear leukocytes (PMNs), and human periodontal ligament cells (PDLs), and with those of three species of periodontopathic bacteria: Porphyromonas gingivalis 381 (P. gingivalis), Prevotella intermedia ATCC25611 (P. intermedia), and Capnocytophaga sputigena ATCC33123 (C. sputigena). The biochemical properties of the isoenzymes were analyzed by the following methods: enzyme assays, inhibition pattern using three chemical inhibitors, 4 to 20% gradient polyacrylamide gel electrophoresis, thermostability, immunological specificity, and phosphatidylinositol-specific phospholipase C (PI-PLC) treatment. The inhibition experiment showed that ALP of the PMNs and PDLs possessed almost the same enzymatic properties of tissue-nonspecific ALP (bone/liver/kidney; TNSALP), and the ALP of the three species of periodontopathic bacteria possessed specific properties that were different from those of TNSALP, intestinal, or placental ALP. The ALP of the GCF was only slightly susceptible to levamisole (1 mM), L-phenylalanine (20 mM), and SDS (1%). An electrophoresis thermostability test demonstrated that the enzyme activity of the GCF was separated into one or two bands. The main heat-labile slow band contained the phosphatidylinositol (PI)-moiety-anchored ALP and possessed immunological specificity against anti-bone type ALP. The minor fast band was heat stable and showed mobility similar to that in P. gingivalis. These results indicated that the ALP of the GCF consisted of several ALP isoenzyme types whose possible origins are considered to be derived from phosphatidylinositol (PI) anchored ALP and periodontopathic bacterial ALP.(ABSTRACT TRUNCATED AT 250 WORDS)

Voluntary exercise increases osteogenetic activity in rat bones.

The purpose of this study was to investigate the effect of voluntary exercise on osteoinductive activity in rat bone. Sprague-Dawley male and female rats were allowed to exercise freely by running on a treadmill or kept as controls without exercise for 53 days. Decalcified humeral diaphyses from experimental and control rats were implanted intraperitoneally into host rats and harvested after 33 days. A significant increase in bone formation was confirmed in the implanted bone matrices from the running group in comparison with those from control animals by soft X-ray photography and determination of alkaline phosphatase activity and mineral content. Alkaline phosphatase activity in bone and serum was increased by exercise in both male and female animals. The results suggest that osteoinductive activity in the bone was probably due to increased levels of bone morphogenetic protein following voluntary exercise.

Immunological differentiation of human tissue-nonspecific type alkaline phosphatases by a monoclonal antibody to the enzyme of human osteoblast-like cells.

Monoclonal antibodies against alkaline phosphatase [ALP; ortho-phosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1.] of cultured human osteoblast-like cells (HBC) were raised in mice. Immuno-reactions of tissue-nonspecific type ALP from human bone, dental pulp, liver and kidney as well as intestinal and placental types to the monoclonal antibodies were compared by a dot immunoassay and ELISA. One clone was able to recognize antigenic differences among tissue-nonspecific type ALPs in addition to intestinal and placental ALPs; it reacted favorably with ALPs from HBC, human bone, kidney and dental pulp, but not with human liver enzyme. Similarly, the antibody immunoreacted with bone-derived ALP but not with liver-derived enzyme present in human serum. The present monoclonal antibody preparation can be utilized in basic studies as well as in clinical laboratory tests to distinguish minor heterogeneity among human ALPs.

Identification of bone-type alkaline phosphatase mRNA from human periodontal ligament cells.

Tissue-nonspecific-type alkaline phosphatase is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, an alternative noncoding first exon was identified in the liver message which differed from that of the previously known osteoblast-derived cDNA sequence. Although these two mRNAs produce an identical protein, they have different promoter regions. The periodontal ligament tissue expresses a high level of alkaline phosphatase activity. To identify its mRNA type, we isolated a full-length cDNA for alkaline phosphatase from a cultured human periodontal ligament cell expression library, using bone-derived tissue-nonspecific alkaline phosphatase cDNA as a hybridization probe. The size of this clone was 2.5 kb, and its 5' and 3' untranslated sequences were identical to those of the human tissue-nonspecific type isolated from osteoblastic cells but not to those of the liver type. In addition, the same fragments as in bone-derived tissue-nonspecific-type cDNA were detected by the treatment of the cDNA clone with restriction enzymes Hinc II and Pst I. The results suggest that expression of the same alkaline phosphatase isozyme in human periodontal ligament cells may be regulated by the same transcriptional mechanism as in bone.

Modulation of responses to TGF-beta by 1, 25 dihydroxyvitamin D3 in MG-63 osteoblastic cells: possible involvement of regulation of TGF-beta type II receptor.

The effect of TGF-beta 1 on the expression of alkaline phosphatase (ALPase) activity was examined during osteoblastic cell line (MG-63) differentiation induced by 1,25-dihydroxyvitamin D3 (1,25D3). TGF-beta 1 and 1,25D3 were found to enhance ALPase activity. However, preincubation of the cells with 1,25D3 transiently abolished the effects of TGF- beta 1. Kinetics of the complex responses to TGF- beta 1 and 1,25D3 were found to correlate well with that of the expression level of type II receptor for TGF- beta. These results suggest that 1,25D3 may regulate the cellular responses to TGF- beta 1 in part via regulation of functional receptor for TGF- beta.

The nutritional evaluation of globin on maintenance of bone metabolism in ovariectomized osteoporotic rats.

In our previous study, globin was found to be an effective dietary source for increasing bone mineral density (BMD) and mechanical strength. In this study, the bioavailability of the globin preparation was examined to clarify the mechanism of increase in bone density and strength. Six-week-old Sprague-Dawley female rats were ovariectomized and were fed on a low Ca diet for 30 days to produce the experimental osteoporotic rats. Thereafter they were divided into two groups. The BMD and the mechanical strength of bone of the rat group, whose diet was supplemented with globin, were significantly higher than those of the control group. The levels of the serum calcitonin and the bone-type alkaline phosphatase (Alp) activity in serum and bone were also higher, and the tartrate-resistant acid phosphatase (Tr-Acp) activity in serum and bone was lower in the globin group. Moreover, the bone morphogenetic protein activity in bone in the globin group was found to be greater. From these results, it is concluded that alimentary globin is effective for the acceleration of bone formation and the prevention of bone resorption.

Expression of bone morphogenetic protein genes in the human dental pulp cells.

Dental pulp has a potential to induce ectopic bone formation, but little is known about its mechanism. We thought that bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the osteoinductive activity of dental pulp. In order to prove this assumption, we constructed a cDNA library from primary culture cells of human dental pulp (HDP cells), and screened the library with previously cloned cDNAs for mouse BMP-2 and -6 as probes. Three distinct cDNA clones encoding human BMP-2, -4 and -6 were isolated. By Northern blot analysis, specific transcripts of the genes of those BMPs were detected in the HDP cells. It was concluded that the BMPs were expressed in a certain population of dental pulp cells and might play some roles in ectopic bone formation by dental pulp.

Enzymatic and immunological properties of alkaline phosphatase of bullfrog.

Enzymatic and immunological properties of alkaline phosphatase (ALPase) in several tissues of bullfrog (Rana catesbeiana) were investigated. Inhibition and thermal inactivation studies showed that bullfrog ALPases in kidney, liver, and intestine had similar enzymatic properties. In addition, mouse antiserum against bullfrog liver ALPase cross-reacted with kidney and intestine enzymes as well as with liver enzyme. These results suggest that a single phenotype of ALPase exists in all tissues of bullfrog in contrast to two or three isoenzymes in mammals.

Properties of alkaline phosphatase of the human dental pulp.

Enzymatic and immunological properties of alkaline phosphatase [ALP; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP cross-reacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140,000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone.

Detection of minor immunological differences among human "universal-type" alkaline phosphatases.

Two clones of monoclonal antibodies against swine alkaline phosphatase (ALPase; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1), which were useful in distinguishing human kidney and bone ALPases from liver ALPase, were successfully raised in mice. On the other hand, polyclonal antibody cross-reacted not only with human kidney ALPase but also with all other human universal type ALPases. The difference in cross-reactivity of monoclonal and polyclonal antibodies may be caused by the specific antigenicity of human enzymes. The monoclonal antibodies were able to recognize minor heterogeneity that could not be distinguished by their enzymatic properties. The present monoclonal antibody preparations will be utilized for clinical as well as basic investigations to detect minor heterogeneity among universal-type ALPases.

Purification and subunit structure of alkaline phosphatase from bovine enamel organ.

A highly-purified alkaline-phosphatase preparation having a specific activity of 703 U/mg protein was obtained from bovine enamel organ by a series of procedures: butanol extraction, isoelectric and acetone precipitation, ion-exchange, concanavalin A affinity and gel-filtration chromatography. The purified enzyme showed the same properties as kidney-type isozyme and contained carbohydrate moieties which react with concanavalin A. Analysis by polyacrylamide gel electrophoresis showed that the purified enzyme split into half the size of the native molecule (160,000 in mol. wt) after being heated in sodium dodecyl sulphate (SDS) solution and the subunit had no catalytic activity. The results indicate that the enzyme is a dimeric glycoprotein comprised of two identical subunits, each having a molecular weight of 80,000.