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We report successful surgery for a thrombosed St. Jude Medical (SJM) valve 16 years after the initial mitral valve replacement even under conditions of satisfactory anticoagulation therapy. A 61-year-old-female had intermittent claudication and was admitted to our hospital for examination. The prosthetic valve sounds were normal to auscultation and the left ankle-pressure index was decreased to 0.6. Transthoracic echocardiography revealed no mitral regurgitation and a mean mitral valve gradient of 6-7 mmHg. Furthermore, transesophageal echocardiography revealed that one of the leaflets of the prosthetic valve was entirely immobilized at the closing position and a mobile soft tissue mass, 5 mm in diameter, was detected at the atrial side of the obstructed leaflet. Although 96,0000 IU of urokinase was administered intravenously for a week, we could not confirm any change in leaflet mobility. At the time of surgery, the posterior leaflet of the SJM valve, which was implanted at an anatomical orientation, was obstructed at the closing position with old and fresh thrombi. We decided upon replacement with a CarboMedics 29 M prosthetic valve. Postoperative medication consisted of warfarin plus low-dose aspirin. Generally, valve thrombosis occurs within 5 years after valve replacement. However, valve thrombosis is possible even in a reliable SJM valve and as long as 16 years after replacement. Therefore, the implantation of an SJM valve at an anti-anatomical orientation might lower the incidence of valve thrombosis in addition to life-long anticoagulation therapy.

We prepared anti-plasma hyaluronan binding protein (PHBP) mouse monoclonal antibodies and studied the fragmentation profile of PHBP with them. PHBP is present in human plasma as a single polypeptide chain (70 kDa). During the purification, PHBP partially fragmentated into the 50-kDa N-terminal fragment and the 27-kDa C-terminal fragment. After the incubation of the purified PHBP, the 70-kDa precursor form was completely cleaved to the 50- and 27-kDa fragments, followed by the 50-kDa to the 26-kDa, and the 27-kDa to the 17-kDa plus the 8-kDa fragments, respectively. Because the purified PHBP contained no other detectable proteins and PHBP has a typical serine protease domain, we concluded that the fragmentation of PHBP was caused by own serine protease activity. PHBP cleaved the C-terminal side of Arg in the peptide effectively and that of Lys weakly. The results of the pre-incubation experiments of PHBP suggested that the single-chain form of PHBP is a precursor, the two-subunit structure is an active form and the three- or four-chain structure is an inactive form of a serine protease.

Background: During the cardiovascular surgeries in elderly people, only a few cases can avoid the homologous blood transfusion, because of their preoperative anemic tendency and low hemopoietic abilities. We examined the capability to avoid the homologous blood transfusion in over 75 year old patients by the preoperative autologous blood collection. Sixty-six patients underwent scheduled cardiovascular surgery between January 1996 and December 1999. The groups were divided into three categories of preoperatively collected autologous blood amounts: high-amount (800-1,200 ml), medium-amount (200-800 ml), and low-amount (0 ml). Each group was divided into two subgroups in according to the use of cardiopulmonary bypass (CPB). There were no differences among the each group in age, body weight, or preoperative and postoperative day-7 hematocrit values., Results: Only 21.2% of patients could donate the expected blood amounts preoperatively. Mean volume was 641 ml. In groups used CPB, no patient was transfused homologous blood in high-amount group. On the contrary, 100% patients were donated in medium and low amount groups. In groups operated without CPB, homologous blood transfusion was required 14.3% in high-amount group, 25.0% in medium-amount group, and 83.3% in low-amount group., Conclusion: It seems that predonation of more than 800 ml may be sufficient to avoid the homologous blood transfusion in using CPB operation and more than 400 ml in non using CPB operation.

The inhibitor for the serine protease activity of plasma hyaluronan binding protein (PHBP) was purified from human plasma by polyethylene glycol (PEG) fractionation, diethylaminoethyl (DEAE)-Sephacel ion-exchange chromatography, Phenyl Toyopearl 650M hydrophobic chromatography, Bio Gel A-0.5 m gel-filtration and hydroxyapatite chromatography. The serine protease activity of PHBP was measured with Boc-Phe-Ser-Arg-methylcoumarine amide (MCA) as the synthetic substrate of PHBP. The results of the amino acid sequence analyses of the purified PHBP inhibitor indicated that it was C1 inhibitor of the serpin family. C1 inhibitor formed a complex with PHBP, suggesting that it is the actual inhibitor of PHBP in human plasma. On the other hand, dextran sulfate and phosphatidylethanolamine enhanced the auto-fragmentation and the serine protease activity of pro-PHBP, but kaolin did not. These results suggested that the serine protease activity of PHBP was regulated in a similar manner to that of factor XII of the coagulation system.

Plasma hyaluronan biding protein (PHBP) is a novel serine protease, which has an amino acid sequence homology to that of hepatocyte growth factor activator (HGFA), and has a similar domain structure to that of urinary plasminogen activator (u-PA), found in human plasma. We searched the PHBP substrate in human plasma by measuring the digested protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that fibrinogen and fibronectin were the major substrates of PHBP. PHBP cleaved the alpha-chain at multiple sites and the beta-chain between lysine53 and lysine54 but not the gamma-chain of fibrinogen. Therefore, PHBP did not initiate the formation of the fibrin clot and did not cause the fibrinolysis directly. PHBP did not cleave (activate) prothrombin and plasminogen, but it converted the inactive single chain urinary plasminogen activator to the active two chain form.

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 microM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.

Two patients had percutaneous cardiopulmonary support (PCPS) used as a bridge to emergency surgery. A 66-year-old man admitted with profound cardiogenic shock underwent direct stenting under PCPS with the diagnosis of acute myocardial infarction of the left main trunk, with the intention of performing revascularization as soon as possible. Subsequently, double coronary artery bypass grafting was successfully accomplished. A 69-year-old woman, admitted with acute heart failure due to critical aortic stenosis, manifested cardiogenic shock while undergoing catheterization. PCPS was immediately instituted until the acute deterioration of her hemodynamic state could be reversed, and was continued uneventfully till aortic valve replacement was performed. These results suggest that the current PCPS system is an effective response to acute circulatory collapse and will contributed to the improved survival of patients.

Objective and Design: In the present study, the involvement of the binding of IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) and actin in phagocyte activity was investigated., Materials and Methods: The actin polymerization and the phagocytic activity of the polymorphonuclear (PMN) cells were studied in the presence of IHRP., Results: IHRP inhibited the polymerization of actin and the phagocytic activity of the PMN cells., Conclusion: 1) IHRP may bind to actin released from the damaged cells and suppress its toxic action by preventing the formation of actin fibril. 2) IHRP may bind to cell surface actin on PMN cells and inhibit their phagocytic activities. 3) From these results, IHRP may act as an anti-inflammatory protein.

We have previously reported the isolation of human gelatin-binding protein 28 (GBP28) gene which is specifically expressed in adipose tissue. The transcriptional activity of the flanking region of the GBP28 gene was examined by the transient transfection of promoter-luciferase reporter constructs into 3T3 adipocytes and electrophoretic mobility shift assay. This revealed the existence of a protein which binds to the 5'-flanking region of the GBP28 gene in nuclear extracts from human adipose tissue, but not in nuclear extracts from mouse liver. The C/EBP sites contained in this region are thought to take part in the regulation of GBP28 gene expression.

The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 by encoding a protein of 475 as with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted as sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.

A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.

In a previous study, we showed that geranylgeraniol (GGO) is a potent inducer of apoptosis in human leukemia cells, including HL60 promyelocytic leukemia cells. The present study describes the effects of activators of protein kinase C (PKC) on GGO-induced apoptosis in various lines of leukemia cells. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and diacylglycerol (DG) inhibited the GGO-induced morphological changes that are characteristic of apoptosis and the DNA fragmentation. Similar effects were observed with other lines of human and murine leukemia cells such as ML1, U937, M1 and P388. Flow cytometric analysis also revealed that both TPA and DG prevented GGO-induced DNA degradation in a dose-dependent manner. These inhibitory effects of TPA and DG were antagonized by inhibitors of PKC such as H-7 and staurosporin, and by amiloride, an inhibitor of Na+/H+ antiporter. In contrast to the inhibitory effects of TPA and DG on GGO-induced apoptosis, 4alpha-TPA, which is unable to activate PKC, failed to prevent GGO-induced DNA fragmentation. However, the selective activator of PKC-beta, 12-deoxyphorbol 13-phenylacetate 20-acetate, significantly inhibited GGO-induced DNA fragmentation. Our results suggest that PKC, and in particular the PKC-beta isoenzyme, might be involved in the process of GGO-induced apoptosis.

We have found that GGO is a potent inducer of apoptosis in various human tumor cell lines, including a myeloid multipotential leukemia K562 cell line which is resistant to the induction of apoptosis by various apoptosis-inducing agents and conditions. Polyprenylalcohols with isoprenyl units shorter than the geranylgeranyl group or longer than farnesyl group were less effective in inducing apoptosis in myeloid leukemia HL-60 cells and polyprenylketones had no apoptosis-inducing activity. The fragmentation of DNA in HL-60 cells by GGO was so rapid, no significant effect on the cell cycle was observed. [3H]GGO was taken up by HL-60 cells in 3 h and was incorporated into several proteins. The expression of c-myc and bcl-2 decreased significantly within 3 h of the start of the treatment of HL-60 cells with 50 microM GGO.

We have investigated the stimulatory effect of recombinant human macrophage colony-stimulating factor (rhM-CSF) on interleukin-8 (IL-8) production by human peripheral blood monocytes. When monocytes were prepared from peripheral blood and cultured for 24 hours, the average amount of IL-8 in the culture medium was about 8.9 +/- 3.2 ng/2 x 10(5) cells. In contrast, the production of IL-8 by monocytes increased to a level of 19.2 +/- 4.7 ng/2 x 10(5) cells in response to rhM-CSF. This induction by rhM-CSF was dose-dependent. Northern blot analysis showed that expression of IL-8 at the pretranslational level was enhanced after M-CSF treatment. Kinetic studies showed that secretion of IL-8 from monocytes was enhanced within 2 hours after exposure to rhM-CSF, and a saturation level, which was reached around 48 hours, was two-fold higher than that of cells without M-CSF treatment. In addition, conditioned medium of M-CSF-stimulated monocytes activated the chemotaxis of human neutrophils, and this activity was significantly inhibited by anti-IL-8 antibody. These results, taken together, suggest that M-CSF can affect many cellular functions through regulation of IL-8 expression in monocytes.

A previously healthy 8-month-old girl developed exanthem subitum and acute encephalopathy with status epilepticus, quadriplegia and bilateral abducens nerve palsies. Human herpesvirus-6 DNA was found in the cerebrospinal fluid by the polymerase chain reaction at the acute stage. Cranial computed tomography showed low density areas in the thalami and in the cerebellar and abducens nuclei. The distribution of the lesions was consistent with acute necrotizing encephalopathy. As for the thalamic lesions, a T2 weighted magnetic resonance image on the 24th day of the illness demonstrated low signal intensity surrounded by high intensity; 99mTc-ECD SPECT showed hypoperfusion, which suggested irreversible tissue damage. The patient is now 1 year 6 months old and has spastic quadriparesis with mental retardation and abducens nerve palsies.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).

In order to supplement our previous report on the reliability and validity of slow vertex response (SVR) audiometry, the detectability of the response, the agreement among scorers and test-retest reliability were studied in 6 children aged 2-4 years who have sensorineural hearing loss of 60-75 dB for the test frequency (1 000 Hz). Mean percentage of positive response increased from 20.6 to 62.2% as stimulus intensity was increased from 60 to 80 dB. Such a sharp increase in the detectability of the response at a level of stimulus intensity close to the auditory threshold of the subjects was not observed in normal children. The results indicate that SVR audiometry is more reliable and valid in children with sensorineural hearing loss than in normal children.

The purpose of this study is to investigate how differences in post shape, storage time and condition after removal affect impression accuracy with respect to dimension and shape. The following results were obtained for various elastic impression materials which were tested using a metal model with five different post shapes. 1. Alginate, polysulfide (regular type) and agar-agar showed substantial deformation due to undercut in post shape. Polysulfide (injection type) and polyether showed slight deformation due to undercut. Silicone (both condensation and addition types) showed very little deformation regardless of post shape. 2. As for elapse of time after removal, dimensions of all impressions decreased in varying degrees depending on type of materials. 3. When impressions were placed horizontally, increased deformation was observed as time elapsed with both the polysulfide and the alginate. Increased deformation was not observed with elapse of time for other impression materials. While the above results show that accurate impressions with little deformation at time of and after removal are obtainable depending on the types of materials, nothing currently available fully satisfies the demand for the type of impression materials that would slightly decrease in dimensions with no deformation for best results. This finding indicates the need to develop new impression materials to better satisfy the demand.

As the maximal K+-conductance (or K+-channel density) of the Hodgkin-Huxley equations is reduced, the stable resting membrane potential bifurcates at a subcritical Hopf bifurcation into small amplitude unstable oscillations. These small amplitude solutions jump to large amplitude periodic solutions that correspond to a repetitive discharge of action potentials. Thus the specific channel density can act as a bifurcation parameter, and can control the excitability and autorhythmicity of excitable membranes.

The effects of horizontal as well as vertical optokinetic stimuli on the body's center of gravity in normal adults were investigated by using Jungtype optokinetic stimulator, a straingage platform and a minicomputer. The following results were obtained. With increasing speed of the optokinetic stimulation, the total length of the center of gravity gradually increased up to 60 deg/sec and then decreased. Meanwhile there was a standard two-phasic course in the position of the center of gravity. The ratio of the anteroposterior component to the lateral component in the movement of the center of gravity was below 1.00 in horizontal optokinetic stimulation, while in vertical stimulation it was over 1.00 and showed the maximum deviation of the position at about 60 deg/sec of optokinetic stimulation. The frequency spectra of the movement of the center of gravity showed some peaks at around 0.3 Hz and 1.0 Hz during optokinetic stimulation. The averaged divisional frequencies were calculated to show the change in the frequency spectra in digital values.