Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization.

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4-activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP-modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP-modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)-expressing Escherichia coli was cultured onto PAR4-AP-modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP-modified titanium.

Structural alterations of the superior temporal gyrus in schizophrenia: Detailed subregional differences.

BACKGROUND: Reduced gray matter volumes in the superior temporal gyrus (STG) have been reported in patients with schizophrenia. Such volumetric abnormalities might denote alterations in cortical thickness, surface area, local gyrification or all of these factors. The STG can be anatomically divided into five subregions using automatic parcellation in FreeSurfer: lateral aspect of the STG, anterior transverse temporal gyrus of Heschl gyrus (HG), planum polare (PP) of the STG, planum temporale (PT) of the STG and transverse temporal sulcus. METHODS: We acquired magnetic resonance imaging (MRI) 3T scans from 40 age- and sex-matched patients with schizophrenia and 40 healthy subjects, and the scans were automatically processed using FreeSurfer. General linear models were used to assess group differences in regional volumes and detailed thickness, surface area and local gyrification. RESULTS: As expected, patients with schizophrenia had significantly smaller bilateral STG volumes than healthy subjects. Of the five subregions in the STG, patients with schizophrenia showed significantly and marginally reduced volumes in the lateral aspect of the STG and PT of the STG bilaterally compared with healthy subjects. The volumetric alteration in bilateral lateral STG was derived from both the cortical thickness and surface area but not local gyrification. There was no significant laterality of the alteration in the lateral STG between patients and controls and no correlation among the structures and clinical characteristics. CONCLUSIONS: These findings suggest that of five anatomical subregions in the STG, the lateral STG is one of the most meaningful regions for brain pathophysiology in schizophrenia.

Ultrasonographic findings in cubital tunnel syndrome caused by a cubitus varus deformity.

We have retrospectively reviewed the clinical, preoperative ultrasonographic, and operative findings of eight patients who had tardy ulnar nerve palsy caused by a cubitus varus deformity. The mean varus angle on the affected side was 23°. With preoperative ultrasonography, the anterior dislocation of the ulnar nerve from the medial epicondyle was detected in dynamic scanning of short-axis images, and long-axis images revealed nerve compression and kinking in the proximal border of the flexor carpi ulnaris. Operative findings revealed compression of the ulnar nerve by a fibrous band, which was released in all cases. The cause of the tardy ulnar nerve palsy in this series of patients was constriction by a fibrous band and kinking in the proximal border of the flexor carpi ulnaris due to ulnar nerve dislocation from compression resulting from the forward movement of the medial head of the triceps brachii muscle.

Neurodevelopmental abnormalities associated with severe congenital neutropenia due to the R86X mutation in the HAX1 gene.

OBJECTIVE: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. METHODS: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. RESULTS: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376-434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. CONCLUSIONS: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.

Effects of nutritional supplementation with antioxidant vitamins and minerals and fish oil on antioxidant status and psychosocial stress in smokers: an open trial.

Nutritional supplement foods containing antioxidant vitamins and minerals and fish oil (mainly docosahexaenoic acid, DHA, C22:6n-3), referred to as capsules, were administered to seven smokers every day for 34 days. Concentrations of antioxidant vitamins and minerals in serum, activity of superoxide dismutase in plasma and the concentration of 8-isoprostane (8-epi-prostaglandin F(2) alpha) in the urine showed an increase or a tendency to increase after the end of administration. The frequency of subjects showing poor state of psychological health evidenced by a total score of 8 points or more on the General Health Questionnaire (30-item edition) scale was 42.9%, although there was a significant decrease to 14.3% upon completion of administration of the capsules. These biochemical and psychological changes were mostly returned to the basal level one month after the end of administration of the capsules. The results suggest that administration of antioxidant vitamins and minerals and fish oil to smokers resulted in an increase in antioxidant capacity. Effectiveness in alleviating psychosocial stress likely to be attributable to DHA was also observed.

[Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering].

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.

Hepatic portal venous gas associated with nonocclusive mesenteric ischemia in a hemodialysis patient.

Hepatic portal venous gas (HPVG) has been rarely described in chronic hemodialysis patients. We report a case of HPVG in a 59-year-old female patient with hemodialysis-dependent chronic renal failure due to diabetes who presented with acute onset of abdominal pain. Abdominal CT demonstrated the presence of gas in the portal veins. However, on laparotomy, no evidence of bowel necrosis or perforation could be found. HPVG seemed to be caused by nonocclusive mesenteric ischemia (NOMI), an increasingly recognized complication in hemodialysis patients. The patient responded favorably to intravenous hyperalimentation and antibiotics.

Multiple probes reveal a native-like intermediate during low-temperature refolding of ubiquitin.

We investigate the refolding of ubiquitin Phe45Trp/Ile61Ala (Ub(*)I61A) in a low-temperature, high-viscosity buffer, where folding is slowed so that apparent two-state and three-state mechanisms are readily distinguishable. Ub(*)I61A forms a compact ensemble rapidly (as judged from stopped-flow, small-angle X-ray scattering) with a secondary structure signature similar to that of the native state (as judged from stopped-flow circular dichroism from 215 nm to 250 nm), but the fluorescence signature still resembles the guanidinium-denatured state. The compact ensemble forms over a range of solvent and temperature conditions. The native fluorescence signature, which requires the tryptophan residue to be packed tightly, is acquired at least 500 times more slowly. Molecular dynamics simulations at 495 K show no contraction of the backbone in ethylene glycol buffer compared to pure aqueous buffer, and no significant effect on the local backbone structure of the unfolded protein. Only at higher simulation temperature does a backbone contraction appear. Thus, it appears unlikely that the aqueous ethylene glycol buffer fundamentally changes the folding mechanism of ubiquitin. We suggest that ubiquitin forms a compact ensemble with native-like secondary structure, but without tight packing, long before the native state.

Exercise-induced acute renal failure with renal hypouricemia: a case report and a review of the literature.

A previously healthy 16-year-old boy developed acute renal failure following a track race at a local athletic meeting. Several hours after the run, he expressed pain in the loins with nausea and vomiting. After 3 sessions of hemodialysis, he was referred to our hospital. On admission, serum creatinine was elevated to 2.3 mg/dl without an increase in serum uric acid level. After recovery from acute renal failure (ARF), hypouricemia (0.7 mg/dl) became evident in the patient. One year later, he suffered from ARF after a track race with the highest creatinine levels of 1.1 mg/dl. In order to clarify the cause and prognosis of ARF with renal hypouricemia, we summarized the clinical features in 18 patients previously described and our patient. Serum uric acid levels after recovery from ARF were below 1.0 mg/dl in all patients. Renal biopsy in 9 patients showed acute tubular necrosis in 8 patients and uric acid nephropathy in 1. The short-term prognosis of these patients seemed good, although 5 patients needed to undergo hemodialysis in their ARF courses. However, the recurrence of ARF episodes occurred in 6 patients (31.6%) including our patient, indicating that prevention of ARF might be necessary in these patients. More information is required to establish guidance for prevention of ARF.

Refolding of beta-lactoglobulin studied by stopped-flow circular dichroism at subzero temperatures.

Refolding of bovine beta-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%-buffer 55% at -15 degrees C, the isomerization rate from the kinetic intermediate rich in alpha-helix to the native state is approximately 300-fold slower than that at 4 degrees C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At -28 degrees C, we observed at least three phases; the fastest process, accompanied by an increase of alpha-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of alpha-helix content with the rate of 2 s(-1), and the third phase, accompanied by a decrease of alpha-helix content. This last phase, corresponding to the isomerization process at -15 degrees C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid alpha-helix formation and their concurrent collapse are common even in proteins rich in beta-structure in their native forms.

Clinical implications of duplicated mtDNA in Pearson syndrome.

We report on a seven-year-old Japanese boy with Pearson syndrome, which is characterized by refractory sideroblastic anemia with vacuolization of marrow precursors and dysfunction of the exocrine pancreas, and caused by mitochondrial (mt) DNA deletions and duplications. Although analysis with Southern hybridization on his bone marrow cells at age one year or on the muscle at age five years did not detect any duplications of mtDNA, an analysis after death at age seven years detected them in the kidney, heart, and even in the bone marrow. Using long PCR to specifically amplify duplicated mtDNA, we found duplications in all biopsy and postmortem samples, indicating that duplications had been present in the patient since his early life, and that the number of duplications increased with age. The results indicate some dynamism in the mtDNA duplication and that the dynamism may imply clinical importance.

Antithrombotic activity of AT-1015, a potent 5-HT(2A) receptor antagonist, in rat arterial thrombosis model and its effect on bleeding time.

The antithrombotic activity of N-[2-(4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidino)ethyl]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate (AT-1015; a 5-HT(2A) receptor antagonist) was studied in a photochemically induced arterial thrombosis (PIT) model in the rat femoral artery, and in the tail transection bleeding time test. Ticlopidine (an antiplatelet agent) and sarpogrelate (a selective 5-HT(2A) receptor antagonist) were studied as reference compounds. Pretreatment with AT-1015 (1 mg/kg, p.o.) significantly prolonged the time required to occlusion of the artery with thrombus, and the effect (3 mg/kg, p.o.) persisted for 24 h with significant inhibition of 5-HT-induced vascular contraction. Ticlopidine and sarpogrelate also significantly prolonged the time to occlusion at 100 mg/kg, p.o. Sarpogrelate (300 mg/kg, p.o.) showed the similar antithrombotic efficacy to AT-1015 (3 mg/kg, p.o.), while the effect disappeared within 6 h. No significant bleeding time prolongation was observed at 10 mg/kg of AT-1015, which is 10 times higher than the antithrombotic effective dose; whereas ticlopidine significantly prolonged bleeding time at the same dose as the antithrombotic effective dose. These results suggested that AT-1015 is a potent and long-acting oral antithrombotic agent in this model, which may be elucidated by its potent and long-acting inhibition of vasoconstriction through 5-HT(2A) receptor.

Inhibitory effects of a newly synthesized 5-HT2 receptor antagonist, AT-1015 (N-[2-[4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidino]-ethyl]-1-for myl-4-piperidinecarboxamide monohydrochloride monohydrate), on contraction and relaxation of pig coronary arteries induced by 5-HT and alpha-methylserotonin: comparison with ketanserin.

Inhibitory effects of a newly synthesized 5-HT2 receptor antagonist, AT-1015 (N-[2-[4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidinolethyl ]-1-formyl-4-piperidinecarboxamide monohydrochloride monohydrate) on contraction and relaxation of coronary arteries of pig hearts mediated by 5-HT2 subtypes were evaluated and these results were compared with those of ketanserin. Contraction and relaxation were determined by adding 5-HT or alpha-methylserotonin (alpha-Me-5-HT) as agonists. Although ketanserin induced rightward shifts of contraction, AT-1015 inhibited the maximal response. In addition, ketanserin inhibited relaxation induced by high concentration of agonists, but there were no inhibitory effects of AT-1015 on relaxation. Thus, these results suggest that AT-1015 is a strong non-competitive 5-HT2 antagonist in porcine coronary arteries and that this drug clearly exhibited different effects on the contraction and relaxation of coronary arteries of pig hearts from those of ketanserin.

AT-1015, a novel serotonin (5-HT)2 receptor antagonist, blocks vascular and platelet 5-HT2A receptors and prevents the laurate-induced peripheral vascular lesion in rats.

The serotonin (5-HT2A) antagonistic activities and the protective effect on laurate-induced peripheral vascular lesions of AT-1015, a novel 5-HT2 receptor antagonist, were investigated. In platelet aggregation, AT-1015 selectively inhibited in vitro 5-HT2A receptor-mediated aggregation, and the activity was almost equivalent to that of ketanserin (5-HT2A/2C receptor antagonist) and 100 times more potent than sarpogrelate (5-HT2A receptor antagonist). AT-1015 also inhibited 5-HT2A receptor-mediated aggregation by oral administration in rat, and the dose required for inhibition was equivalent to ketanserin. In a 5-HT-induced vasoconstriction study in rat, AT-1015 slightly reduced maximal contraction and caused a rightward shift of the concentration-response curve (pKB value, 9.5), which was unlike competitive inhibitors such as ketanserin and sarpogrelate (pA2 value, 9.3 and 8.7, respectively). Moreover, the ex vivo inhibitory activity significantly remained after oral administration (1 mg/kg). In the rat peripheral vascular lesion model, AT-1015 (1 mg/kg, p.o.) effectively prevented progression of peripheral lesions, and it was more potent compared with ketanserin, sarpogrelate, and cilostazol. These results suggest that AT-1015 is a potent 5-HT2A receptor antagonist, and its insurmountable antagonism may be relevant to its therapeutic potential in peripheral vascular disease.

GroES co-chaperonin small-angle X-ray scattering study shows ring orifice increase in solution.

GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin. Here we studied the GroES structure in solution using small-angle X-ray scattering (SAXS). The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution. The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring-like structure from 8 A in the crystal to 21 A in solution.

pH-dependent unfolding of aspergillopepsin II studied by small-angle X-ray scattering.

Aspergillopepsin II (EC 3.4.23.6) secreted from the fungus Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase. It consists of two polypeptide chains (i.e., a heavy chain and a light chain), which are bound noncovalently to each other. The pH titration analysis using small-angle X-ray scattering (SAXS) as well as circular dichroism (CD) and gel filtration indicated that the enzyme was unfolded around a neutral pH with concomitant dissociation of the two chains. Detailed analyses showed that the midpoint pH values for the unfolding are not coincident with one another (pH 6.1 in circular dichroism and gel filtration, pH 6.4 in zero-angle intensity of SAXS, pH 6.8 in radius of gyration). The difference between these values suggested the existence of an intermediate state during the unfolding. Further analyses of the SAXS data showed that the heavy chain just after the dissociation still kept molecular compactness and that it gradually increased its dimensions as the pH was further raised. Noncoincidence of the two phenomena (i.e., chain dissociation and swelling) led to elucidation of a novel intermediate state during unfolding, which was confirmed by the subsequent singular value decomposition (SVD) analysis.

Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein.

The urea-induced equilibrium unfolding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left harpoon I1 right harpoon over left harpoon I2 right harpoon over left harpoon U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P. J., Bilsel, O., and Matthews, C. R. (1999) Protein Sci. 8, 1623-1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. Size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of approximately 24 A for the native state of alphaTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 A, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at approximately 3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from approximately 38 to approximately 52 A. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self-association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to approximately 54 A at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical for the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N right harpoon over left harpoon I1 and I1 right harpoon over left harpoon I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 right harpoon over left harpoon U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincón, G., Gualfetti, P. J., and Matthews, C. R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respective conformational ensembles.

Interhospital network system using the worldwide web and the common gateway interface.

We constructed an interhospital network system using the worldwide web (WWW) and the Common Gateway Interface (CGI). Original clinical images are digitized and stored as a database for educational and research purposes. Personal computers (PCs) are available for data treatment and browsing. Our system is simple, as digitized images are stored into a Unix server machine. Images of important and interesting clinical cases are selected and registered into the image database using CGI. The main image format is 8- or 12-bit Joint Photographic Experts Group (JPEG) image. Original clinical images are finally stored in CD-ROM using a CD recorder. The image viewer can browse all of the images for one case at once as thumbnail pictures; image quality can be selected depending on the user's purpose. Using the network system, clinical images of interesting cases can be rapidly transmitted and discussed with other related hospitals. Data transmission from relational hospitals takes 1 to 2 minutes per 500 Kbyte of data. More distant hospitals (e.g., Rakusai Hospital, Kyoto) takes 1 minute more. The mean number of accesses our image database in a recent 3-month period was 470. There is a total about 200 cases in our image database, acquired over the past 2 years. Our system is useful for communication and image treatment between hospitals and we will describe the elements of our system and image database.

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.