Glycated high-density lipoprotein induces apoptosis of endothelial cells via a mitochondrial dysfunction.

Glycation of plasma proteins may contribute to an excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is nonenzymatically glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelium dysfunction in diabetes. This study set out to clarify whether glucose-modified HDL affects the function of endothelial cells by examining the apoptosis of cultured human aortic endothelial cells (HAECs) exposed to a glycated-oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation of HAECs with 100 microg/ml of gly-ox-HDL for 48 h showed apoptotic features, such as cell shrinkage, membrane blebbing, and concentration and fragmentation of the nucleus, and the degree of apoptosis was dose-dependent on the glucose used in the preparation of gly-ox-HDL. Stimulation of HAECs with gly-ox-HDL elicited a marked increase in caspase 3 activity and the expressions of active caspase 3 and caspase 9, whereas concomitant treatment with a caspase 3 inhibitor significantly blocked gly-ox-HDL-induced apoptosis of HAECs. The release of cytochrome c into cytosols markedly increased in HAECs during the treatment with gly-ox-HDL. The increased expressions of Bax and Bad were detected in HAECs incubated for 24 h with gly-ox-HDL, but gly-ox-HDL failed to interfere with the expression of Bcl-2 and Bcl-x. Moreover, in vitro experiments with HDL (gly-HDL) glycated in the presence of 2 mM EDTA and Cu(2+)-oxidized HDL suggested that the apoptotic effect of gly-ox-HDL on endothelial cells might be due to an additional oxidative modification of gly-HDL. Taken altogether, additional oxidation of HDL under hyperglycemic conditions may induce endothelial apoptosis through a mitochondrial dysfunction, following the deterioration of vascular function.

alpha-Amylase expressed in human liver is encoded by the AMY-2B gene identified in tumorous tissues.

BACKGROUND: An alpha-Amylase in human liver is detected with an anti-human salivary amylase antibody, but the enzyme activity is very low. We previously found that the rat liver contained an amylase which differed from the enzyme of mice. In this study, we characterized the human liver amylases biochemically and immunohistochermically. METHODS AND RESULTS: Although the amylase activity of human liver was much lower than that of rat, protein moiety and sugar chains of the human amylase were identified as similar to the rat liver enzyme with an anti-human salivary amylase antibody and by concanavalin A (Con A) affinity chromatography. Liver amylases from human and rat were the same size, 50 kDa, on Western blot analysis and had the same isoelectric points. The cytoplasm of hepatocytes was moderately stained immunohistochemically with the anti-human salivary amylase antibody. Intrahepatic bile ducts were also stained weak-to-moderately. RT-PCR, with a specific primer for the consensus sequence of human amylases, amplified a single 474-bp product from the human liver total RNA. The PCR product was sequenced and referred to the homology. Thirteen bases in the 434-bp fragment of the human liver amylase differed from the corresponding region of the AMY-1 gene transcript and the deduced amino acid sequence differed at five residues. The human liver amylase cDNA sequence was identical to the corresponding cDNA of the AMY-2B, which was known to expressed in tumorous tissues. In situ hybridization revealed the expression of AMY-2B mRNA in non-tumorous human liver. CONCLUSIONS: The present results suggest the possibility that a novel amylase detected in tumorous tissues and encoded by the AMY-2B gene is a liver-specific amylase expressed in the human liver.

Effects of prosthetic valve placement on mitral annular dynamics and the left ventricular base.

Insertion of a rigid mitral prosthesis impairs the function of the mitral annulus and induces systolic narrowing of the left ventricular outflow tract (LVOT). To study this mechanism, we investigated dynamic changes in the left ventricular (LV) base, which consists of the mitral annulus and LVOT orifice. In seven patients with mechanical mitral valve prostheses and eight normal subjects, the image of the LV base was reconstructed three-dimensionally and its dynamic change during systole was studied. In the patients, the rigid prosthetic valve (=mitral annulus) tilted toward the left ventricle with a hinge point at the posterior mitral annulus during systole. The left ventricular base exhibited contraction, but the size of the prosthetic valve was constant. As a consequence, the prosthetic valve occupied more of the left ventricular base, which resulted in narrowing of the LVOT. In the normal subjects, the mitral annulus did not interfere with the region of the LVOT orifice during systole as the mitral annulus underwent both dorsiflexion and contraction. Thus, fixation of the mitral annulus induces an anti-physiologic motion of the annulus. Conscious preservation of annular flexibility in mitral valve surgery is important in avoiding potential dynamic LVOT obstruction.

Characterizations of recombinant human tartrate-resistant acid phosphatase from osteosarcoma: comparison study between recombinant and placental proteins.

We cloned the human tartrate-resistant acid phosphatase (TRAP) gene from human osteosarcoma cells (Saos-2), and produced recombinant human TRAP (rhTRAP) using a baculovirus vector expression system. RhTRAP from Sf9 culture medium was purified by cation exchange chromatography, gel filtration and affinity chromatography. The molecular mass and amino acid composition of the rhTRAP were consistent with the deduced amino acid composition from the TRAP gene. The N-terminal amino acid sequence of rhTRAP was identical to that of TRAP purified from osteoclastoma and hairy cell leukemia spleen. The monoclonal antibodies generated against rhTRAP also reacted to human placental TRAP (pTRAP). The optimum pH of rhTRAP and pTRAP were pH 5.0-5.5 and pH 6.0-6.5, respectively. The enzymatic activities of rhTRAP and pTRAP were activated by reducing agents such as 2-mercaptoethanol, dithiothreitol and ascorbic acid. The activities of rhTRAP and pTRAP were enhanced by Fe2+ ions, but were inhibited by Fe3+ ions. The present results indicate that rhTRAP has similar properties to the native human TRAP, and suggest that the enhancement of TRAP activity by reducing agents might be expressed via the reduction of Fe ions at the metal center.

Annular stabilization in mitral repair without a prosthetic ring.

BACKGROUND AND AIM OF THE STUDY: Annular stability is not guaranteed after mitral repair without a prosthetic ring. We introduce a newly developed plication technique and detail its stabilizing effect on the mitral annulus after Gerbode plasty. METHODS: Patients suffered degenerative mitral valve prolapse with chordal rupture restricted to the middle scallop of the posterior leaflet. Between 1986 and 1997, 102 patients underwent Gerbode plasty with or without annular reinforcement with a pericardial strip or modified Paneth plasty (group C). The mean (+/- SD) follow up was 70.4 +/- 41.1 months. Since 1996, 32 patients have undergone a newly developed annuloplasty technique (group N), where a pericardial strip was tightly anchored to the bilateral trigones and posterior annulus, which was folded by Gerbode plasty. With the final anchoring suture the intention was to prevent plication breakdown of this portion. Mean follow up for this group was 17.6 +/- 7.1 months. Progression of mitral regurgitation after surgery in both groups was studied. RESULTS: In group C, postoperative progression of mitral valve regurgitation occurred in 41.1% of patients (5.9% to grade 1, 17.6% to grade 2, 17.6% to grade 3). Among these patients, reoperation was due to plication breakdown of the Gerbode plasty in six cases (5.9%), and to either chordal rupture or annular dilatation in 10 cases each (9.8%). In contrast, no reoperation due to recurrent severe mitral regurgitation was needed in group N. Progression of mitral regurgitation after surgery was seen in six patients (two to grade 1; four to grade 2). CONCLUSION: The newly developed annuloplasty technique may be useful in stabilizing the mitral annulus after Gerbode plasty.

Induction of rat alkaline phosphatase isozymes bearing a glycan-phosphatidylinositol anchor modified by in vivo treatment with a benzimidazole derivative linked to ethylbenzene.

Serum alkaline phosphatase (ALP) is detected in soluble-form as a result of translocation from the membrane site by cleavage at the glycosyl-phosphatidylinositol moiety (GPI anchor). It is known that membrane-bound ALP (mALP) can be detected in serum in certain pathological and physiological conditions, and that it can be solubilized in vitro to soluble-ALP (sALP) by phosphatidylinositol-specific phospholipase C (PIPLC), phospholipase D, bile salt, detergent, etc. We observed a marked increase in ALP activity in the serum of rats given a benzimidazole derivative by gavage, and detected it as slow-migrating ALPs (SM-ALPs), which were mALP-like but resistant to PIPLC and n-butanol treatment on disc PAGE. On the other hand, ficin treatment made SM-ALPs shift to the sALP position. The molecular size of the SM-ALPs was smaller than that of sALP on sodium dodecyl sulphide-polyacrylamide slab-gel electrophoresis (SDS-PAGE), and immunoreactivity revealed the intestinal type. SM-ALPs were also detected in the duodenum and jejunum. The main sugar chain structure of SM-ALPs was the biantennary complex-type, which was coincided with intestinal sALP sugar chain. These results suggest that intestinal ALPs induced by the benzimidazole derivative were modified in their C-terminus or GPI anchor region and modification of this region may also participate in translocation into the bloodstream.

Lipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effect: reduction of MRNA levels for interleukin-1beta, interleukin-6, cyclooxygenase-2, and p22phox by regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) in primary endothelial cells.

We examined the effects of four 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (pravastatin, simvastatin, fluvastatin, and cerivastatin) on the production and expression of inflammatory cytokines and on enzyme expression involving prostaglandin and superoxide production in cultured human umbilical vein endothelial cells (HUVEC). All HMG-CoA reductase inhibitors significantly reduced interleukin-1beta and -6 mRNA expression and their protein levels in the culture medium, and also inhibited cyclooxygenase-2 mRNA expression and their protein levels. And these drugs induced peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma mRNA expression and their protein levels in HUVEC and hepatocyte. Moreover, the mRNA levels of p22phox, a 22-kD subunit and the protein levels of p47phox, a 47-kD subunit of nicotine adenine dinucleotide phosphate (NADPH) oxidase, was decreased by treatment with either simvastatin, fluvastatin or cerivastatin, and this effect was reversed by mevalonate, geranylgeraniol, farnesol, and cholesterol. The changes induced by HMG-CoA reductase inhibitors might be due to regulation of cellular cholesterol content level, cellular cholesterol metabolic pathway, and cellular PPARalpha activity, which was related with inflammation. This unique anti-inflammatory effect in addition to its hypolipidemic action, may be beneficial in preventing the vascular complications that are induced by hyperlipidemia.

[Detection of anti-Chlamydia trachomatis antibody by means of enzyme immunoassay using synthetic peptide antigen].

Newly developed diagnostic kits for the detection of Anti-Chlamydia trachomatis, Peptide-Chlamvdia (LOY: Meiji Milk Products Co., Ltd., Tokyo; for IgG and IgA), were evaluated using the microimmunofluorescence assay (MIF) as the gold standard. These results were also compared to results of testing by Sero-IPALISA and immunoblot (I-B). Detection by LOY in based on enzyme immunoassay with synthetic peptides as the antigen. Thirty serum samples from pediatric patients and 130 serum samples from gynecology patients were used. All 26 pediatric samples that were positive for Chlamydia pneumoniae IgG antibody tested negative with LOY, indicating that the presence of the antibody against C. pneumoniae did not affect the assay by LOY. For 90 gynecological samples, the total, the positive and the negative agreement rates for IgG were quite high; i.e. 87.8%, 90.0% and 70.0% (LOY vs MIF), 85.6%, 85.0% and 90.0% (Sero-IPALISA vs MIF), and 92.0%, 94.9% and 70.0% (I-B vs MIF), respectively. On the other hand, many cases of MIF (-) and LOY (+) discrepancy were seen in IgA detection. In order to better understand the basis for such disagreement. 34 serum samples were collected from patients whose cervical samples were negative for the Chlamydia group antigen based on the assay with IDEIA-Chlamydia. They were then assayed by MIF and LOY. The total, the positive and the negative agreement rates for IgG were 91.2%, 100% and 90.9%, while the total and the negative agreement rates for IgA were 88.2% and 88.2% (there were no IgA positive cases). Furthermore, 6 serum samples (1 case of MIF (+) LOY (+) and 5 cases of MIF (-) LOY (+)) were provided to determine whether LOY detects C. trachomatis specific IgA antibody. Increasing amounts of C. trachomatis serovar L2 were added to the serum samples resulting in a progressive decrease in their reactivity in the LOY assay. These results lead us to speculate that LOY can reveal even low levels of C. trachomatis specific IgA antibody. In conclusion, LOY can be used as an useful kit for detecting C. trachomatis antibody.

[Small increases of the serum alkaline phosphatase activity and malignant changes].

In order to see the clinico-pathological significance of the small increase of serum alkaline phosphatase (ALP) activity alone in routine laboratory examinations, we picked up 76 new out-patients of adult within the two-fold high ALP level in contrast to the reference range in the Akita University Hospital for one year, and then studied the relation between the histories and serum ALPs of 33 patients whose ALP had increased pathologically or decreased after intervention such as surgical operations. The ratio of the patients with malignant tumors to the tested all patients was 13 to 76 (17%). Immunochemical analysis of sera for the cancers of the lung, larynx and prostate showed that the small amount of the placental isozyme or intestinal-like isozyme was expressed selectively. It may be useful for earlier detection of malignancy to analyze the cancer-associated ALPs, when the small and sequential increase of the serum ALP activity is found in routine laboratory examinations.

Changes in intestinal alkaline phosphatase isoforms in healthy subjects bearing the blood group secretor and non-secretor.

We found the high molecular mass intestinal alkaline phosphatase (HIAP) and normal molecular mass intestinal alkaline phosphatase (NIAP) in serum at fasting and after fatty meal by use of 6.0% polyacrylamide gel electrophoresis (PAGE) in the presence of 1% Triton X-100. HIAP only appeared in serum of Lewis blood group secretors ¿Le(a-b+)¿, and HIAP levels were dependent on ABO blood groups. Among the secretors, the highest activities of HIAP in fasting serum were observed in subjects with blood groups O and B (8.6+/-1.4 U/1; mean+/-SD) and the lowest activities were associated with blood group A (0.7+/-0.2 U/1; mean+/-SD), and the HIAP activities did not change after fatty meal. In contrast, NIAP was present in the serum of both secretors and non-secretors regardless of ABO blood group. Trace amounts of NIAP remained in fasting serum; however serum NIAP activities rose sharply after fatty meal. The remaining ratios of NIAP activity at fasting and 9 h after fatty meal of secretors were approximately the same as those of non-secretors. The electrophoretic mobility on PAGE or the apparent molecular mass estimated by gel filtration of serum NIAP in secretors was slightly different from that in non-secretors. In addition, HIAP can be normalized to NIAP on PAGE in the absence of Triton X-100, and the electrophoretic mobility of normalized-NIAP was identical to that of original NIAP in secretors. Accordingly, it can be concluded that the structure of serum NIAP in the secretor was different from that in the non-secretor, because HIAP is only formed by serum NIAP in the secretor. These results suggest that differences in serum NIAP in the secretor and the non-secretor may be closely related to the appearance of IAP in the circulation.

Bezafibrate has an antioxidant effect: peroxisome proliferator-activated receptor alpha is associated with Cu2+, Zn2+-superoxide dismutase in the liver.

Administration of bezafibrate in rats significantly reduced the levels of plasma thiobarbituric acid-reactive substances (TBARS) in comparison with those obtained in rats fed a soy or lard chow. Moreover, an elevation of in vitro conjugated diene production and linoleic acid levels in the high-density lipoproteins and low-density lipoproteins induced by a soy or lard chow, was reduced by bezafibrate administration. In addition, the liver Cu2+, Zn2+-superoxide dismutase (SOD) gene expression showed a significant positive correlation with the liver peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA level (R=0.769, p<0.0001). This unique characteristic of bezafibrate, which possesses both a hypolipidemic effect and antioxidant activity, may be beneficial in preventing vascular complications in hyperlipidemia.

The relationship between the mitral annulus and left ventricular outflow tract.

Mitral annular inflexibility due to rigid prostheses (ring or valve) has long been considered to contribute to the mechanism of dynamic left ventricular outflow tract (LVOT) obstruction after mitral repair or replacement. In clarifying the geometric relationship between LVOT orifice and mitral valve annulus (MVA) in eight normal subjects, the authors have endeavored to show how that a rigid mitral prosthesis might obstruct the LVOT based on the assumption that any rigid prosthesis necessarily follows the motion of the posterior half of the MVA (MVApost) in the course of every heart beat. During systole, the relationship between the MVApost and the approximated plane of the LVOT orifice was constant. However, with the respect to the relationship between the LVOT orifice and the approximated plane of the MVApost (PI-MVApost), the intersection between the two shifted toward the apex during systole. Assuming the prosthesis is aligned on the MVApost with the same orientation as the PI-MvApost, this shift implies a reduction in the effective size of the LVOT orifice due to the prosthesis. The calculated obstruction rate was 24.9% (0 ms), 30.9% (100 ms), 35.5% (200 ms), and 45.4% (300 ms). These results indicate the importance of maintaining the flexibility of the MVA after mitral valve surgery.

Mitral annular flexibility.

An analysis of three-dimensional movement of the mitral valve annulus (MVA) may address the question of geometrical change after mitral valve repair to preserve mitral annular function. Conventionally, annular contraction has been studied for this purpose. We investigated this geometrical change occurring in the anterior half of the MVA and discuss its clinical significance. Three-dimensional images of the MVA during systole were reconstructed from magnetic resonance images of eight normal subjects. The posterior half of the MVA exhibited translational motion. We assume that this portion, exhibiting translational motion as well as contraction, purely follows the motion of the left ventricular contraction. Compensating for the discrepancy between the motion of the aortic root and that of the posterior half of the MVA, the anterior half exhibited a flexible change in shape during systole, thus maintaining a sufficient left ventricular outflow tract (LVOT). The increase in the extent of displacement of the anterior MVA from the posterior half of the MVA during systole, which was 3.6 +/- 1.0 mm (mean +/- SD), indicates the annular flexibility. The preservation of annular flexibility may prevent LVOT obstruction. Further geometrical analysis of patients after mitral repair will clarify annular function as presented in this article.

The inflexible mitral annulus after valve prosthesis. Inherent risk of dynamic left ventricular outflow tract obstruction.

Although chordal preserving mitral valve replacement is beneficial to cardiac function, the loss of flexibility of the annulus and consequent translational motion of the valve prosthesis during systole may cause potential left ventricular outflow tract (LVOT) obstruction after surgery. The extent of the flexibility of the mitral valve annulus (MVA) necessary for the prosthetic valve to prevent potential LVOT obstruction was determined. The three dimensional images of the MVA at 0, 100, 200, and 300 msec delay from the electrocardiogram R wave were reconstructed from cine-mode magnetic resonance images in eight normal subjects. In the lateral view of the MVA, the dorsal flexion angle (DFA) was defined. This angle implies the extent of the flexion of the anterior half of the MVA in relation to the posterior half. The data (mean +/- SD) for the DFA were 31.7 +/- 5.4 degrees (0 msec), 36.4 +/- 4.5 degrees (100 msec), 39.0 +/- 3.8 degrees (200 msec), and 43.6 +/- 2.6 degrees (300 msec), whereas the systolic increase in DFA was 11.9 +/- 3.2 degrees. The flexibility observed in normal mitral annuli is relevant to prosthetic mitral valves.

A variant alkaline phosphatase detected in a patient with lung cancer.

A variant alkaline phosphatase (ALP), with heat-sensitivity characteristics similar to that of the bone type, was found in the serum of a patient suffering from lung cancer. In disc polyacrylamide gel electrophoretic studies most of this enzyme had migrated to the region corresponding to liver ALP, with the remainder affecting bone ALP. Like kidney ALP, this ALP was markedly inhibited by 0.5 mmol/l L-cysteine. The K(m) of this ALP for p-nitrophenylphosphate was 0.39 mmol/l, similar to that of kidney ALP. The sugar moiety of this enzyme bore greater resemblance to that of kidney ALP than liver or bone ALP. However, immunoprecipitation of this particular ALP was strong with a monoclonal antibody against liver ALP and moderate with an antibody against bone ALP.

A precursor form of human kidney gamma-glutamyl transferase in normal and cancerous tissues, and its possible post-translational modification.

We have found three molecular forms of human gamma-glutamyl transferase (GGT) in normal and renal cell carcinomatous tissues, and also have reported the marked differences in the sugar chains of GGTs between normal and cancerous tissues by serial lectin affinity chromatographies. In this study, the peptide maps of three purified GGTs (79 kDa, 50 kDa and 25 kDa) obtained by lysylendopeptidase digestion, the subcellular localization of GGTs, and the sugar chains of GGTs were compared between normal and cancerous tissues. According to the results, the total peptide bands of the digested 79 kDa component represented the sum of those of the digested 50 and 25 kDa components on 12.5% SDS-PAGE. In addition, C-terminal and N-terminal amino-acid sequences of the 79 kDa protein were the same as the sequences of light and heavy subunits, respectively, suggesting that the 79 kDa component is of the precursor form of the 50 kDa mature heavy and 25 kDa light subunits, respectively. On the other hand, the GGT activity in renal cell carcinomatous tissues was significantly increased in the microsomal fraction and decreased in the soluble fraction compared with that of normal tissues. Meanwhile, the sugar moiety of GGTs in the respective subcellular fractions was obviously different between normal and cancerous tissues. In particular, a reduced multiantennary complex type sugar chain and an elevated high-mannose or hybrid-type sugar chain in the microsomal fraction were observed in the GGT in cancerous tissues.

A variant alkaline phosphatase found in a case of gastric carcinoma with super bone scan.

A rare case of gastric carcinoma associated with increased serum variant alkaline phosphatase activities is presented. A 54 year old man had extremely high serum alkaline phosphatase activity (18,607 U/l) with normal calcium and phosphate concentrations. His bone scintigram showed abnormal findings, 'super bone scan'. He was diagnosed as having Borrmann type 4 gastric carcinoma with diffuse bone metastases by examinations of the upper gastrointestinal tract and iliac bone biopsy. The alkaline phosphatase isozyme of this patient was of the bone type as measured by cellulose acetate membrane electrophoresis and the placenta/bone type by agarose gel electrophoresis, respectively. Immunoelectrophoresis and the immunoprecipitation method using monoclonal antibodies against various alkaline phosphatase isozymes, however, showed that his serum alkaline phosphatase had the liver type antigenicity. Furthermore, it had a larger molecular size and different sugar chains compared with the common liver type alkaline phosphatase. These findings suggest that a unique variant alkaline phosphatase was produced by gastric cancer cells, which is possibly an explanation for the high serum alkaline phosphatase activities in this patient.

Carbohydrate-mediated recognition of a circulating placental alkaline phosphatase-immunoglobulin M complex.

We detected an abnormal alkaline phosphatase (AP) electrophoretically in the serum of a patient with rheumatoid arthritis, who had a macromolecular AP linked with immunoglobulin M (IgM) bearing a kappa light chain. The IgM isolated from the AP-IgM complex in the patient's serum reacted apparently with all of the AP isozymes tested, i.e. those originating in the liver, bone, intestine and placenta, but the alpha-mannosidase-treated IgM from the patient's serum bound to placental AP (PAP) alone. This suggests that untreated IgM recognizes multivalent epitopes of the AP and that the complex of AP with alpha-mannosidase-treated IgM is a specific antibody-antigen complex. In order to investigate further the multivalent binding capacity for the PAP-untreated IgM complex, we prepared a monoclonal antibody (MoAb) against PAP and identified it as an IgM with a kappa light chain. The binding affinities and their circulating half-lives of the synthetic complexes of PAP and respective MoAbs were examined with and without treatment with several glycosidases. The untreated MoAb bearing IgM had binding affinity for all of the AP isozymes tested, while alpha-mannosidase-treated IgM attached only to PAP, the same as the IgM isolated from the PAP-IgM complex in the patient's serum. The circulating clearance of the PAP-IgM complex in rabbits was faster than either component alone. In addition, the PAP-IgM complex treated with alpha-mannosidase was found to have the shortest half-life of all the complexes of PAP and Igs treated with the several glycosidases tested. These results suggest that the formation of the PAP-IgM complex as an enzyme-linked antibody and the clearance of the complex in vivo are dependent on the sugar moieties of the Igs.