Tongue-palatal contact changes in patients with skeletal mandibular prognathism after sagittal split ramus osteotomy: an electropalatography study.

The purpose of this study was to investigate the changes in tongue-palatal contact patterns using electropalatography (EPG) before and after sagittal split ramus osteotomy (SSRO) in patients with mandibular prognathism. Nine clients who underwent SSRO for mandibular setback and seven control subjects were participated in this study. Tongue-palatal contact patterns for /t/, /s/ and /k/ production were investigated using EPG before surgery and 3 months after surgery. The mean value of whole total of palate contact (WT) in the maximum contact frame was examined before and after SSRO. The correlation quantity between the change of center of gravity (COG) value and the amount of mandibular setback was also evaluated. The mean value of WT for /t/ and /s/ significantly increased after SSRO, and the EPG pattern became normal. However, a remarkable change in WT for /k/ was not observed, and the mean value was significantly larger in the SSRO group before and after surgery than in the control group. A negative correlation between COG variation and the amount of mandibular setback for /t/ and positive correlation for /s/ was observed. This study demonstrated that tongue-palatal contact patterns for /t/ and /s/ articulation improved clearly after SSRO. There was a significant correlation between COG variation and the amount of mandibular setback. However, no significant change was detected through perceptual assessment before and after SSRO. Further investigation is needed to determine whether these results will change over time.

The effect of magnetic field during freezing and thawing of rat bone marrow-derived mesenchymal stem cells.

Previous studies showed that a programmed freezer with magnetic field can maintain a high survival rate of mesenchymal stem cells (MSCs). The purpose of this study was to evaluate the influences of magnetic field during freezing and thawing on the survival of MSCs isolated from rat bone marrow. The cells were frozen by a normal programmed freezer or a programmed freezer with magnetic field (CAS-LAB1) and cryopreserved for 7 days at -150 °C. Then, the cells were thawed in the presence or absence of magnetic field. Immediately after thawing, the number of surviving or viable cells was counted. The cell proliferation was examined after 1-week culture. Cryopreserved MSCs which were frozen by a normal freezer or a CAS freezer were transplanted into bone defects artificially made in calvaria of 4-week-old rats. Non-cryopreserved MSCs were used as a control. The rats were sacrificed at 8, 16, or 24 weeks after transplantation and the bone regeneration area was measured. Proliferation rates of MSCs after 1 week were significantly higher in the CAS-freezing-thawing group than in the CAS-freezing group. The extent of new bone formation in the CAS-freezing-thawing group tended to be larger than in CAS-freezing group 24 weeks after transplantation. These results suggest that a magnetic field enhances cell survival during thawing as well as freezing.

Cranial suture-like gap and bone regeneration after transplantation of cryopreserved MSCs by use of a programmed freezer with magnetic field in rats.

Mesenchymal stem cells (MSCs) can be used for regeneration of various organs and tissues. A previous study revealed that cryopreserved MSCs, which were frozen by a programmed freezer with a magnetic field (Cells Alive System: CAS) and cryopreserved for 7 days in a -150°C deep freezer, can maintain high survival and proliferation rates while retaining both adipogenic and osteogenic differentiation abilities. The purpose of this study was to examine MSC viability and tissue regenerative ability after long-term cryopreservation using a CAS freezer. MSCs were isolated from rat femora bone marrow and cryopreserved in a -150°C deep freezer (CAS group) or directly cryopreserved in a deep freezer (Direct group). After 3 years, the cells were thawed and the number of viable cells was counted. Cell proliferation was also examined after 14 days in culture. For histological examination, forty 4-week-old Fischer 344 male rats received bone and sagittal suture defects with a diameter of 6.0mm, and MSCs (CAS or Direct group) cryopreserved for 1 year were grafted with membranes. Non-cryopreserved MSCs (Control group) were transplanted to an additional twenty rats. The rats were sacrificed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope and the extent of bone regeneration was measured. Our results indicate that MSCs survival and proliferation rates were significantly higher in the CAS group than in the Direct group. In the Control and CAS groups, a large amount of new bone formation and a suture-like gap was identified 24 weeks after transplantation, whereas only a small amount of new bone formation was observed in the Direct group. These results suggest that the CAS freezer is amenable to long-term cryopreservation of MSCs, which can be applied to the regeneration of various tissues, including bone tissue with suture-like gap formation.

Cryopreservation of osteoblasts by use of a programmed freezer with a magnetic field.

In order to determine a suitable condition for osteoblasts cryopreservation, murine osteoblasts were freezed by programmed freezer with a magnetic field (CAS freezer). After 7 days cryopreservation at -150°, the number of survival cells immediately after thawing and the growth rate of cultured cells for 48 hours were examined. Gene and protein expression of alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) were compared between cryopreserved and non-cryopreserved groups. As a result, a plunging temperature of -30°, a hold-time at -5° for 15 minutes and a 0.1 mT of magnetic field led to the largest survival and growth rate. Moreover, there was no significant difference in ALP, OPN and BSP mRNA and protein expression between cryopreserved and control groups. From these results, it was suggested that the CAS freezer is available for osteoblast cryopreservation and bone tissue banking can be established in the future.

Effects of cryopreservation with a newly-developed magnetic field programmed freezer on periodontal ligament cells and pulp tissues.

The purpose of this study was to evaluate the effects of long-term cryopreservation on the isolated human periodontal ligament cells (PDL) and pulp tissues. In the first part of study, 10 freshly extracted teeth were selected and divided into two groups. In the cryopreserved group, the teeth were frozen for 5 years using a programmed freezer combined with a magnetic field, known as Cells Alive System "CAS". As for the control group, freshly extracted teeth were used. In each group, extracted PDL tissues were cultured and gene expression and protein concentration of collagen type I, alkaline-phosphatase (ALP) and vascular endothelial growth factor (VEGF) was compared between the two groups. In the second part, pulp tissues were obtained from 10 mature and immature third molars which were freshly extracted or cryopreserved for three months. Expression of VEGF and nerve growth factor (NGF) mRNAs and the protein concentration in the supernatant were investigated. Results indicated that long-term cryopreservation with the use of CAS freezer cannot affect the growth rate and characteristics of PDL cells. There was no significant difference in VEGF expression and VEGF and NGF protein concentration of pulp cells derived from cryopreserved teeth with immature apex and control group with mature root formation. Finally, proper PDL regeneration and appropriate apexogenesis after transplanting magnetically cryopreserved immature tooth was clinically confirmed. These findings demonstrate that teeth banking with the use of magnetic field programmed freezer can be available for future autotransplantation as a treatment modality for replacing missing teeth.

Pacemaker-associated infection caused by ST81/SCCmec IV methicillin-resistant, vancomycin-intermediate Staphylococcus aureus in Japan.

A 76-year-old Japanese man was admitted to hospital for treatment of fever and skin lesion at the implantation site of his pacemaker. During his hospitalization, vancomycin-intermediate Staphylococcus aureus (MIC 4 µg/mL) with reduced susceptibility to daptomycin was isolated from venous blood. This isolate was identified as methicillin-resistant S. aureus with SCCmec IV and was genotyped as sequence type 81, coa VIIa and spa type t7044, harbouring blaZ, aac(6')-aph(2″) and enterotoxin(-like) genes sea, seb, sek, sel, selx and selw. The patient was successfully treated with daptomycin, minocycline and sulfamethoxazole/trimethoprim. We describe the identification of sequence type 81/SCCmec IV vancomycin-intermediate S. aureus from pacemaker-associated septicaemia.

Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinase-type proteins.

Oral administration of clinical-type high molecular weight urokinase (HMW-UK) in a single dose of 30,000-60,000 International Units (IU) in enteric-coated capsules, in a group of normal human subjects, induced a plasma fibrinolytic state suggesting transport of HMW-UK across the intestinal tract. Other groups of human subjects were given a single dose of 120,000 IU daily of pure HMW-UK for 1 d and 7 d together with a placebo dose, all of the ingredients except urokinase (UK), daily for 7 d. UK-type proteins were isolated from the plasma of blood samples drawn 6 h after administration of the final dose, by a sequential two-step affinity chromatography method first with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose and second with a specific rabbit anti-HMW-UK-IgG-Sepharose. The yield of UK-type proteins from the 7-d group, 0.79 mg/dl, was approximately twofold greater than that obtained from either the placebo or 1-d groups. The specific plasminogen activator activity of the 1-d and 7-d groups were similar, 508 and 537 IU/mg protein, respectively; negligible plasminogen activator activity could be detected in the placebo group. The kinetics of activation parameters of human Glu-plasminogen of the UK-type protein, isolated from the 1-d group, were similar to those obtained with urinary HMW-UK. The UK-type proteins isolated only from the 7-d group showed, in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a major protein band of molecular weight 53,000 in the same position as HMW-UK. In addition, two other major protein bands of approximately 140,000 and approximately 120,000 mol wt were found in the 7-d placebo-, and 1-d groups, and also in the 7-d group. The 53,000 mol wt protein, about 50% of the total protein in the 7-d group, was further purified by preparative SDS-polyacrylamide gel electrophoresis, and found to be a two-chain protein with a specific activity of 1,241 IU/mg protein. The protein showed common antigenic determinants with urinary HMW-UK. The oral administration of 120,000 IU HMW-UK to human subjects for 7 d stimulated the synthesis of a UK-type protein of 53,000 mol wt, probably the zymogen, from either the liver or vascular endothelium, which was released into the circulation, and converted into active UK by circulating plasmin. The induction of the fibrinolytic state produced the conversion of native circulating Glu-plasminogen into the degraded Lys-plasminogen form, which was found in large amounts in the plasma of the 7-d group. The new plasma components, e.g., the 53,000-mol wt UK-zymogen and Lys-plasminogen, could play an important role in clot resolution of the fibrin-thrombus in thromboembolic diseases. Oral administration of HMW-UK has been shown to be clinically effective in patients with cerebral thrombosis in a multicenter double blind study.

A thiol protease inhibitor released from cultured human malignant melanoma cells.

A thiol protease inhibitor (TPI) was found in culture media of human malignant melanoma cells (Bowes) at 1.5 to 2.3 units/day/flask (full sheet, 75 sq cm). This amount well exceeded that for cultured nonmalignant cells (human fetal lung fibroblasts). In the intracellular region of the melanoma cells, TPI activity was localized mainly in the cytosol fraction. The difference in specific activities between the intracellular and extracellular TPI and the TPI accumulation in the culture media indicated that cultured melanoma cells release TPI. Partial purification and characterization of the TPI by column chromatography using Sephadex G-150, papain-Sepharose, and Sephadex G-50, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed two distinct TPIs with molecular weights of 56,000 and 9,800 to 10,800. The latter (main) TPI had a high specificity for thiol proteases and was heat stable (60 degrees for 60 min), like previously reported normal human TPIs. The inhibitor, however, differed from normal human TPIs in that it had a lower molecular weight than any normal TPI, was unable to inhibit bromelain, and exhibited a mosaic pattern; namely, the low-molecular-weight TPI resembled liver-type TPI but the pH stability curve resembled serum-type TPI. The thiol protease, cathepsin B, was not detected in culture media of this human melanoma cell line.

Dynamics of protein conformational fluctuation in enzyme catalysis with special attention to proton transfers in serine proteinases.

We have provided a quantum mechanical model for proteinase-catalyzed peptide, amide and ester hydrolysis. The model rests on electron and atom transfer theory, but incorporates the dynamics of conformational nuclear modes as a new element. The model is applied to acylation, but can straightaway be extended to deacylation, and is substantiated by recent structural and kinetic data for proteinase enzyme catalysis. The role of the conformational modes is found to be two-fold. First, the crystallographic distances for the proton transfers involved are far too large for direct transfer. His-57 mobility, handled stochastically, to bring the donor and acceptor groups within suitable reach, is therefore a crucial element of the theory. Secondly, the charge alignment in the Asp-102/His-57/tetrahedral intermediate system implies that the curvature of the potential surface along the conformational coordinates in this state is much lower than in the initial enzyme-substrate and final acyl states. A consequence of this is that the activation energy liberated after the first proton transfer is not dissipated, but stored in the conformational system and used in the second proton transfer step.

Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein.

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.

Purification and partial characterization of two forms of urinary trypsin inhibitor.

Crude urinary trypsin inhibitor was obtained by DEAE-cellulose column chromatography from normal fresh urine. By the purification of the crude urinary inhibitor on successive chromatography methods using Sephacryl S-200, DEAE-cellulose, CM-Sepharose CL-6B and Sephadex G-100, we detected two forms of urinary trypsin inhibitor: form I and form II. The specific activity of form I increased approx. 4-fold with a recovery of 60%, as compared to that of crude urinary trypsin inhibitor. N-terminal amino acids of form I and form II were determined to be alanine and valine, respectively. Molecular weights of forms I and II were estimated to be 67000 and 28000 by gel filtration on Sephadex G-100 and to be 43000 and 19000 by SDS-polyacrylamide gel electrophoresis. S-carboxymethylated form I migrated as a single band corresponding to a molecular weight of 59000 in SDS-polyacrylamide gel electrophoresis. From the results of the determination of a single N-terminal amino acid of form I and a single band of S-carboxymethylated form I, it is indicated that it is composed of single polypeptide chain. And the present study suggests that form I is a native form of trypsin inhibitor in normal human urine and form II is a fragmented product from form I in the purification steps.

A method using fibrin-fixed Blue Dextran for determining the plasmin and plasmin inhibitor activities in human plasma.

The fibrinolytic activity of plasmin was determined by incubating with fibrin-fixed Blue Dextran as a substrate, the Blue Dextran released being proportional to the plasmin activity. The applicability of this method for rapid and accurate evaluation of fibrinolytic activity was demonstrated by dose-response curves with purified plasmin, plasmin generated by urokinase in human plasma and euglobulin. The method can also be used to determined plasmin inhibitors in plasma.

Correlation of tissue and plasma RANTES levels with disease course in patients with breast or cervical cancer.

The beta-chemokine RANTES was measured in plasma in 43 patients with breast cancer and in 23 patients with cervical cancer, and the RANTES content in primary tumors, tumor metastatic to lymph nodes, and clinically normal skin or pelvic mucosa was measured. In addition, plasma levels were determined in all of the patients for the platelet-derived chemokine beta-thromboglobulin (beta-TG) and for IFN-gamma, interleukin (IL)-2, IL-4, IL-5, and IL-10, along with serum IgE levels and blood eosinophils. Plasma RANTES levels were found to be higher in order of stages IV, III, II, and I of each cancer except for stage I. A marked increase in plasma RANTES level (> 10,000 pg/ml) was found in 27% of patients with progressive malignancy but in none of those in clinical remission. The platelet RANTES content was correspondingly decreased in those patients with increased plasma RANTES levels. Beta-TG showed a pattern similar to RANTES both in plasma and platelets, but with much less dramatic differences between patients with different stages of disease. Other allergic parameters, IgE, eosinophils and plasma IFN-gamma, IL-2, -5, and -10, were not elevated in the cancer patients. The RANTES content was markedly elevated in the primary tumor and metastatic lesions (lymph node or skin) from all of the patients with breast or cervical cancer, irrespective of the plasma RANTES level. In addition, in patients with progressive breast or cervical cancer, but not in patients thought to be cured of these tumors, the RANTES content was markedly increased in clinically normal tissue taken from near the operative site several months postoperatively, as well as in intact skin or mucosa taken perioperatively near the excised tumor. This study suggests an as-yet-undefined but important role played by RANTES in carcinogenesis, as well as the possibility that a RANTES assay in tissue surrounding a tumor or postoperative tumor site may help predict prognosis in these patients.

Immunochemical determination of the serum protein reacting with antibody against human urinary trypsin inhibitor by single radial immunodiffusion: use of polyethylene glycol.

A single radial immunodiffusion method is described for determining the serum protein which reacts with antibody against the urinary trypsin inhibitor, but does not react with anti-inter-alpha-trypsin inhibitor antibody. Since the amount of this protein could not be determined with an agar plate containing antibody alone, we first prepared an agar plate containing 4% polyethylene glycol 6 000 and 1 mg of anti-urinary trypsin inhibitor gamma-globulin and confirmed that the amount of this protein could be measured accurately from the sizes of precipitin rings after 72 h incubation at room temperature. Levels of this serum protein, alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were measured in normal human serum by the single radial immunodiffusion method, and it was confirmed that the level of this serum protein did not correlate with the levels of these 3 other proteins.

The effects of metal ions on esterase activities of urokinase.

The esterase activity of highly purified human urokinase on Nalpha-acetylglycl-L-lysine methyl ester is strongly inhibited by 1 X 10(-5) to 1 X 10(-2)M Cu++, Hg++, Ni++, Co++, Fe+++, and Mn++ solutions, whereas Na+, K+, Ca++, and Mg++ are weakly effective. This inhibition is parallel with the inhibition of activation of plasminogenby urokinase. There is no simple linear relation between inhibiton and concentration. Addition of ethylenediaminetetraacetate or electrodialysis fully reactivates the inhibited enzyme. These results are discussed in relation to similar effects of ions on trypsin.

Studies on the effect of plasminogen activator on the interaction between alpha 2-macroglobulin and plasmin.

Six different plasmins were prepared by incubating human plasminogen with various amounts of streptokinase or urokinase. It was confirmed that the six different plasmins possessed similar caseinolytic activities, and the inhibitory effects of a alpha 1-antitrypsin on caseinolytic activities of the six different plasmins were all the same. On the other hand, interactions between the six different plasmins and alpha 2-macroglobulin were complicated. Plasmins activated by cleavage of plasminogen were almost immediately or effectively inhibited by alpha 2-macroglobulin. However, plasmin activated by complex formation of plasminogen with streptokinase was not so immediately or effectively inhibited by alpha 2-macroglobulin. It was supposed that the difference between these two results on the interaction between plasmin and alpha 2-macroglobulin might be due to the difference in molecular form of plasmin. In the present study, it was also confirmed that streptokinase or urokinase, in free form in the reaction mixture, interfered with the interaction between plasmin and alpha 2-macroglobulin. The cause for such interference was discussed.

Studies on the activation mechanism of fibrinolytic enzyme system in plasma by human pancreatic elastase.

In the present study, the activation mechanism of fibrinolytic enzyme system in plasma by human pancreatic elastase was investigated. It was confirmed that human pancreatic elastase not only converted the co-existing plasminogen to low molecular weight-plasminogen which could be easily activated by the activator, but also inhibited alpha 2-macroglobulin and alpha 2-plasmin inhibitor which are antiactivators or fast reacting antiplasmins, and consequently, induced the activation of the fibrinolytic enzyme system in plasma.

Low molecular weight trypsin-plasmin inhibitors isolated from papain treated urinary trypsin inhibitor.

Papain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chrymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotryspin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10(-7) - 2.12 X 10(-6) M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors of plasmin was thought to be different from that to trypsin or chymotrypsin.

A simple rapid separation of C1-esterase using an immunoadsorbent column.

An immunoadsorbent-antibody (egg albumin-Sepharose-antibody) column was found to be suitable for the rapid separation of C1-esterase from normal human serum. About 1.54 mg of C1-esterase, with a specific activity of 447 units/mg was obtained in voer 80% yield from the 20 ml of human serum.

Activation of a new plasma enzyme by antigen-antibody reaction.

The change of enzyme activity in immunized rabbit plasma after addition of the homologous antigen was examined. The activities of N alpha-tosyl-L-arginine methyl ester (TAMe) and N alpha-tosyl-L-lysine methyl ester (TLMe) hydrolysis increased about 15 to 18 days after immunization. This increase was especially marked before the maximal rise of antibody content, and is thought to be related to the IgM antibody not to the IgG antibody. Enzyme activation was strongly inhibited by chelation of Ca2+ with 5 mM disodium ethylenediamine tetraacetate (EDTA), but not by other protease inhibitors, such as epsilon amino-caproic acid (epsilon-ACA), bovine lung kallikrein inhibitor (Trasylol) or soybean trypsin inhibitor (SBTI).