Binding analysis of peptides that recognize preferentially cis-azobenzene groups of synthetic polymers.

Peptides identified by the PD method against a film surface composed of azobenzene-containing synthetic polymers were characterized by QCM measurements. Among the four peptides analyzed, the c16 peptide with the sequence Trp-His-Thr-Leu-Pro-Asn-Ala showed the highest binding affinity to the films rich in cis-azobenzene groups. SPR served to determine the association/dissociation constants of the c16 peptide against the trans- and cis-azobenzene groups. The binding constants were estimated to be 1.3 × 10(5) and 1.4 × 10(6) /M, respectively, indicating a high specificity of the c16 peptide for cis-azobenzene conformer. Then mutants of the c16 peptide were synthesized and characterized to gain information on the structural requirements for the cis-form specificity. An Ala-scan clearly revealed that all the amino acid residues of the c16 peptide were essential for the specificity. More importantly, the results with peptides of inverted sequence and containing Gly insertions confirmed that the specificity is strictly derived from the primary sequence of the c16 peptide.

Gamma knife surgery for brain metastases: indications for and limitations of a local treatment protocol.

OBJECTIVE: The purpose of this retrospective study was to evaluate results of a local treatment protocol using gamma knife surgery (GKS) for brain metastases without upfront whole brain radiation therapy (WBRT). METHODS: Results for 521 consecutive patients satisfying the following 3 criteria were analysed: 1) a maximum of 3 tumours with a diameter of 25 mm or more; 2) no prior WBRT; 3) no surgically in accessible large (>30 mm) tumours. Large tumours were surgically removed and all smaller lesions were treated by GKS without up front WBRT. New lesions, detected with follow-up MRI, were appropriately treated with repeat GKS. Overall survival (OS), neurological survival (NS), qualitative survival (QS) and new lesion-free survival (NLFS) curves were calculated and the prognostic values of covariates were obtained. OS and NS were compared according to tumour number. RESULTS: In total, 1023 separate sessions were required to treat 4562 lesions. The primary organs were lung in 369 patients, gastro-intestinal tract in 70, breast in 33, urinary tract in 24, and others/unknown in 25. The median OS period was 9.0 months. On multivariate analysis, the significant prognostic factors for OS were found to be extracranial disease (risk factor: active), Karnofsky performance status (KPS) score (<70) and gender (male). NS and QS at one year were 85.6% and 73.0%, respectively. The only significantly poor prognostic factor for NS was carcinomatous meningitis. NLFS at 6 months was 68.9%. For both OS and NS, the differences between a few (10) tumours had a significantly poorer prognosis than those with

Embolization of the Middle Meningeal Artery for Refractory Chronic Subdural Haematoma. Usefulness for Patients under Anticoagulant Therapy.

SUMMARY: Endovascular embolization of the middle meningeal artery was performed in two cases of refractory chronic subdural haematoma (CSDH) after repeated burr hole and irrigation surgeries. The embolization prevented expansion of the CSDH in both cases, and the haematoma disappeared completely in one case. The expansion of CSDH is considered to result from repeated bleeding from the macrocapillaries on the haematoma capsule. Embolization of the middle meningeal artery appears to be useful to eliminate the blood supply to this structure.

Synthesis and lectin recognition of polystyrene core-glycopolymer corona nanospheres.

Polymeric nanospheres with a polystyrene core and a glucosyloxyethyl methacrylate (GEMA) oligomer corona were synthesized by the free radical coplymerization of styrene (M(1)) plus a GEMA macromonomer (M(2)) at various molar ratios (M(1)/M(2) = 50-150) in the presence of AIBN (1 mol % to the total monomer) in an ethanol/water (3/2, v/v) solvent. The size of the nanospheres was controlled from 300 to 620 nm by altering the monomer ratio. The size distributions were significantly narrow. The amount of glucose conjugated per unit surface area of the nanosphere, which was analyzed by the anthron-sulfuric acid method, was 1.01-2.28 microg cm(-1), which increased with an increase in size. The transmittance of a solution of dispersed nanospheres (the corresponding glucose concentration was 73 microM) increased by the addition of the glucose-binding protein concanavalin A (Con A) (1-50 microM), indicating that the nanospheres were being precipitated by the cross-linking of ConA. An enzyme-linked lectin assay (ELLA) revealed that Con A bound to the glucose on the nanospheres 250-700-fold more than to monomeric glucose. The binding activity to the nanospheres was less than that to a GEMA oligomer, and decreased with an increase in the amount of GEMA oligomer grafted onto the nanosphere, possibly because of steric hindrance of the lectin binding to the glucose on the nanospheres. The polystyrene core-glycopolymer corona nanosphere is a useful material for studying sugar-biomolecule recognition.

Left ventricular relaxation abnormality is detectable by analysis of the relaxation time constant in patients with atrial fibrillation.

Left ventricular (LV) contractility is constantly changing during atrial fibrillation (AF), which is dependent on the force-interval relationships. However, no information has been available on LV relaxation in patients with both AF and impaired LV systolic function. LV pressure was measured with a catheter-tipped micromanometer and the time constant of isovolumic LV pressure decline (tau(bf)) was calculated with best exponential fitting from more than 10 consecutive beats. Patients with AF (5 with mitral valvular disease, 6 with idiopathic dilated cardiomyopathy, and 1 with no underlying disease) were subdivided into 2 groups: group A, with ejection fraction (EF) <0.5 (n=7); and group B, with EF > or =0.5 (n=5). Linear correlation coefficients (r) between tau and RR2, RR2/RR1, LV peak systolic pressure (peak LVP) were calculated. Although tau did not show a discrepancy between the 2 groups, tau(bf) correlated better with RR2/RR1 only in the group A patients. The relation between tau and peak LVP showed a good correlation with a steep slope (R, Deltatau/Deltapeak LVP) only in the group A patients (accentuated afterload-dependence). R was significantly different between the 2 groups. Thus, a beat-to-beat analysis of tau may be a practical and feasible way for detecting LV relaxation abnormality in patients with AF.

HSP 47 and collagen mRNA expression in L929 cells adhered to lipid films.

L929 cell adhesion on various lipid films prepared by Langmuir-Blodgett methods (LB method) were studied. L929 cells adhered to every lipid film similar to tissue culture poly(styrene) (TCPS). The mRNA expression of both collagen and HSP47 in adherent cells were evaluated by the RT-PCR method. mRNA expression of collagen was not altered during adhesion and proliferation duration, while HSP47 mRNA expression changed depending on culture time. L929 cells adhered to L-alpha-dipalmitoylphosphatidylcholine (DPPC)-films showed little HSP47 mRNA expression. It was suggested that DPPC films regulate L929 cell function via unique serum protein adsorption.

Hydroxyapatite deposition by alternating soaking technique on poly(vinyl alcohol)-coated polyethylene films.

A poly(vinyl alcohol) (PVA)-coating on polyethylene films, prepared by repetitive adsorption/drying in an aqueous PVA solution, accelerated hydroxyapatite (HAp) deposition by an altemate soaking in aqueous solutions containing Ca2+ and PO4(3-) ions. X-ray photoelectron spectra of the surface of the HAp-deposited film showed the presence of calcium and phosphorus of a suitable peak ratio for HAp formation. X-ray diffraction analyses also revealed peaks corresponding to HAp. Scanning electron microscopic observation showed the surface of the HAp layer to be smooth, with nano-ordered dotted threads in networks. A simple PVA coating on a surface will serve as a novel system for accelerated HAp formation via alternating soaking.

Treatment of asymptomatic unruptured intracranial aneurysms. A clinical decision analysis.

SUMMARY: The indication of preventive surgery for patients who harbor asymptomatic un ruptured intracranial aneurysms remains controversial. To evaluate the benefit of this treatment, we investigated the management outcome in 128 patients with 157 unruptured aneurysms. Surgery was planned in patients 70 years old or younger without serious systemic complications. A total of 77 patients underwent surgery including four endovascular interventions, and conservative management was chosen in 51 patients. There was no mortality and 6.5% morbidity as postoperative results, and no complication was found after endovascular treatment. Among the patients in conservative management, four patients suffered from subsequent rupture during the total follow-up period of 148 person-years. The annual rupture rate was estimated at 2.7%. According to the clinical decision analysis based on our data, preventive surgery is beneficial for a Japanese 70 years old or younger. However, the expected utility decreases if the rupture rate is set at 0.5% or 0.05%, posing a doubt about the benefit of the surgery. Decision analysis provides an aid for logical and objective choice in the management of unruptured aneurysms. The actual risk of rupture has a major impact on decision making in therapeutic strategy.

Polymyxin B binds to anandamide and inhibits its cytotoxic effect.

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Study on complex formation between recombinant human thrombomodulin fragment and thrombin using surface plasmon resonance.

Human thrombomodulin (hTM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. The affinity constant of recombinant human soluble TM (rhs-TM) and a peptide containing active site of hTM fragment (f-hTM) with thrombin were determined using the surface plasmon resonance. The interaction of f-hTM with thrombin could be analyzed by a simple model, whereas the association and the dissociation steps of rhs-TM with thrombin consisted of at least two kinds of interaction phases. The dissociation constant for complex (K(D)) of f-hTM and thrombin was determined to be 205 nM, which was more than twice as high as that of rhs-TM (6.7 and 75 nM). The lower affinity of f-hTM was not due to the slow association rate but to the rapid dissociation rate. It comes clear that f-hTM interacts with thrombin rapidly.

Gamma knife treatment for multiple metastatic brain tumors compared with whole-brain radiation therapy.

OBJECT: The purpose of this retrospective study was to compare the effectiveness of gamma knife radiosurgery (GKS) for multiple cerebral metastases with that of whole-brain radiation therapy (WBRT). METHODS: Ninety-six consecutive patients with cerebral metastases from nonsmall cell lung cancer were treated between 1990 and 1999. The entry criteria were the presence of between one and 10 multiple brain lesions at initial diagnosis, no surgically inaccessible tumors with more than a 30-mm diameter, no carcinomatous meningitis, and more than 2 months of life expectancy. The patients were divided into two groups: the GKS group (62 patients) and the WBRT group (34 patients). In the GKS group, large lesions (> 30 mm) were removed surgically and all other small lesions (< or = 30 mm) were treated by GKS. New distant lesions were treated by repeated GKS without prophylactic WBRT. In the WBRT group, the patients were treated by the traditional combined therapy of WBRT and surgery. In both groups, chemotherapy was administered according to the primary physician's protocol. The two groups did not differ in terms of age, sex, initial Karnofsky Performance Scale (KPS) score, type, lesion number, and size of lesion, systemic control, and chemotherapy. Neurological survival and qualitative survival of the GKS group were longer than those of the WBRT group. In multivariate analysis, significant poor prognostic factors were systemically uncontrolled patients, WBRT group, and poor initial KPS score. CONCLUSIONS: Gamma knife radiosurgery without prophylactic WBRT could be a primary choice of treatment for patients with as many as 10 cerebral metastases from nonsmall cell cancer.

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.

OBJECTIVES: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. METHODS: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique. RESULTS: PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. CONCLUSIONS: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

Cellular basis for the acute inhibitory effects of IL-6 and TNF- alpha on excitation-contraction coupling.

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF- alpha). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic actions of interleukin-6 (IL-6) and TNF- alpha in the same model. We studied the effects of cytokines on [Ca(2+)](i)transients (using indo-1), cell shortening (CS) (using a video motion detector) and the L-type Ca(2+)channel current (I(Ca)) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF- alpha (500 U/ml) decreased both peak systolic [Ca(2+)](i)(IL-6: 0.43+/-0.01 to 0.40+/-0.01, n=5, P<0.05; TNF- alpha : 0.42+/-0.02 to 0.39+/-0.02, n=5, P<0.05) and the amplitude of CS (IL-6: 7.5+/-0.9 to 6.2+/-0.5 micrometers, n=5, P<0.05; TNF- alpha : 6.7+/-0.7 to 5.8+/-0.7 micrometers, n=5, P<0.05) without detectable reductions in I(Ca)(IL-6: 0.9+/-0.1 to 0.9+/-0.1 nA, n=4, N.S.; TNF- alpha : 1.1+/-0.3 to 1.1+/-0.2 nA, n=4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl- L arginine (300 micromol/l), blocked the effects of IL-6 but not of TNF- alpha. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both I(Ca)(2.8+/-0.5 to 2. 0+/-0.3 nA) and the amplitude of CS (10.4+/-2.4 to 7.5+/-1.9 micrometer) within 5 min. TNF- alpha also clearly depressed I(Ca)(2.9+/-0.9 to 2.3+/-0.7 nA) and the amplitude of CS (7.0+/-1.4 to 5.5+/-1.3 micrometer) in beta -adrenergic stimulated cells. TNF- alpha significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 micromol/l) mimicked those of TNF- alpha on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF- alpha did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.

Direct observation of fibrinogen-heparinoid complexes formation using surface plasmon resonance.

We analyzed the binding of heparinoid or heparin with fibrinogen by real-time measurement using surface plasmon resonance technology. Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate] and dextran sulfate were used as heparinoids. The binding ability of each sulfated polymer was estimated by having each polymer-containing buffer interact with the sensor chip surfaces that had immobilized fibrinogen. Dextran sulfate and poly(GEMA) sulfate showed high affinity to the fibrinogen in this experiment, while the heparin did not. All of the dextran sulfates were desorbed from its surface, while about 30% of the poly(GEMA) sulfate remained on the immobilized fibrinogen upon the addition of NaCl to the buffer which was done in order to analyze the desorption of poly(GEMA) sulfate or dextran sulfate from the surface of the fibrinogen. These data show that the type of binding between fibrinogen-poly(GEMA) sulfate was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was caused not only by an electrostatic but also by a hydrophobic force. These results suggest that the interaction mechanism of heparinoids with fibrinogen was different from that of heparin.

Synthesis of polystyrene nanospheres having lactose-conjugated hydrophilic polymers on their surfaces and carbohydrate recognition by proteins.

We prepared a poly(vinylamine)-grafted polystyrene nanosphere by the free radical polymerization of poly(N-vinylacetamide) macromonomer and styrene with AIBN in ethanol and subsequent acid hydrolysis. We conjugated lactose on the nanosphere by an amide linkage and found that the lactose was recognized by a galactose-specific lectin RCA120, which was analyzed by enzyme-linked lectin assay (ELLA). The binding ability of the lectin to lactose on the nanosphere was at least 10(2) times larger than that to a monomeric lactose. Binding ability was clearly affected by the lactose density on the nanosphere and showed a maximum value at the suitable surface density of lactose. We found that the carbohydrate-conjugated nanosphere would be a useful biomaterial for a site specific drug carrier.

Life-style related factors and idiopathic dilated cardiomyopathy--a case-control study using pooled controls.

A case-control study was conducted to investigate how basic habits of life including dietary habit, physical activity, cigarette smoking, and drinking, are involved in the development of idiopathic dilated cardiomyopathy (DCM). Collection of cases was entrusted to the clinical research group of DCM, and national pooled controls established by sex and age category by the epidemiological research group of intractable diseases were used to ensure representativeness of the controls. Fifty-eight cases of DCM which developed in and after January 1991 were collected, and 5,912 controls matched with the cases by residential area, sex, and age were selected. Analysis of the results of the study showed that items in the questionnaire suggestive of viral infection, such as "susceptibility to common cold" and "susceptibility to diarrhea", items concerning dietary habit, including "taking no breakfast", "ingestion of salty food", and "ingestion of fatty food", and such items as "cigarette smoking" and "lack of sleep" tended to be observed in the case group at significantly higher frequencies. Since viral infection has been suspected as a causative factor of DCM, further research of this area is thought to be of particular importance for determining the etiology of DCM.

Concanavalin A-immobilized polystyrene nanospheres capture HIV-1 virions and gp120: potential approach towards prevention of viral transmission.

To establish an effective tool for the prevention of HIV-1 transmission, lectin-immobilized polystyrene nanospheres were synthesized and examined for their HIV-1 capture activity. When concanavalin A (Con A) was immobilized on the surface of polystyrene nanospheres (400 nm in diameter) with poly(methacrylic acid) branches and incubated with HIV-1 suspension at room temperature for 60 min, the nanospheres (Con A-NSs) achieved a >3.3 log and a 2.2 log reduction of viral infectivity in HIV-1 (IIIB strain) suspension at a concentration of 2 and 0.5 mg/ml, respectively. Meanwhile, Con A-free nanospheres, which were not immobilized with Con A, achieved only a 0.29 log reduction at 0.5 mg/ ml. Con A-NSs (2 mg/ml) could also reduce the gp120 level of III(B) and HE strains to <7.1% and 5.5% of each control, respectively. The combination of Con A-NS treatment followed by filtration with a microporous membrane efficiently removed virion-free gp120 as well as infectious viral particles from HIV-1 suspension. Electron microscopic examination demonstrated that HIV-1 virions were trapped on the surface of Con A-NSs. Thus, Con A-NSs can capture HIV-1 virions and gp120 with high affinity, and may have potential as an effective tool for the prevention of HIV-1 transmission.

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. METHODS: An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 micrograms/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. RESULTS: Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6 +/- 5.1-fold increases (n = 6, p < 0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P < 0.05) as well as the intracellular cGMP contents (+75%, P < 0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 microM). CONCLUSION: As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.