the collagen-binding protein of streptococcus mutans is involved in haemorrhagic stroke - drying equipment

Although several risk factors for stroke have been identified
The third reason is unknown.
Here, we found a strain of the deformed chain that expresses collagen.
Binding protein (CBP)
It is a potential risk factor for bleeding stroke.
Infection of Type K S
Deformed bacteria, but not the standard strain, will aggravate the cerebral hemorrhage in mice. Serotype k S.
Deformation bacteria gather at the damaged site, but not in the opposite hemisphere, suggesting that bacteria interact with the damaged blood vessels.
For high-the most important factor
Toxicity is the expression of CBP and is a common feature of most k-type strains.
Detection frequency of CBP-expressing S.
Deformation bacteria in patients with bleeding stroke were significantly higher than those in the control group.
Strains isolated from patients with bleeding stroke exacerbated the bleeding in the mouse model, suggesting that they were bleeding strokes-associated.
The use of recombinant CBP can lead to increased bleeding.
According to our data, CBP in the United States
Deformation bacteria are directly related to bleeding stroke.
All animal experiments in this study are in line with the guidelines for experimental animal care and use published by the National Institutes of Health, it was also approved by the Graduate School of Dentistry of Osaka University and the Animal Care and Use Committee of the Hamamatsu University School of Medicine institution.
The study programme using human samples was approved by the Graduate School of Dentistry of Osaka University, binsong General Hospital and the ethics committee of sutian city hospital.
All subjects were informed of the agreement and given written consent prior to their participation in the study.
Neurosurgery experts strictly select patients with cerebral hemorrhage using MRI and other diagnostic criteria.
We also chose a stroke (
Including arterial porridge
Patients diagnosed with an ischemic stroke without bleeding.
Collect oral samples from patients from several institutes and hospitals. Age-matched non-
Stroke subjects who have no history of stroke, heart failure, diabetes, arthritis, liver disease, kidney disease, stomach disease, etc. , define hypertension or anemia as a control group.
The main strains used in this study were shown.
In addition, 58 clinical strains (blood isolates; =13; oral isolates; =45)
In this study, we selected it from our laboratory inventory.
All strains were grown in brain and heart infusion broth (Difco)
Erythromycin was added to the screening of mutant strains.
Strain with collagen
Binding activity (
Phillips, Cowen 1 and Newman)
It is also cultivated in brain heart infusion broth. The collagen-
The performance was evaluated in combination with the methods described by Waterhouse and Russell, and some modifications were made.
Activity was evaluated under fixed conditions of 2 mg type I collagen and 1 × 10 bacterial cells.
The results of each strain were expressed in percentage compared to TW871 binding (defined as 100%.
By impedance method, platelet aggregation test was performed using mouse whole blood with aggregator (Whole-
C540 (Baxter) blood aggregation instrument).
To put it simply, whole blood was taken from mice (ICR, male, 8-weeks-old, CLEA)
And a mixture of different numbers of bacterial cells (
Colony formation unit (CFU) 10-10)
The whole blood was incubated at 37 °c for 5 minutes and then collagen was added (
Natural collagen fiber (type I), Chrono-log).
Calculate the aggregation rate of each strain by impedance (Ω)
The value of adding or not adding bacterial cells, expressed as a percentage of the vehicle.
In addition to whole blood aggregation, we performed a platelet aggregation test using human PRP.
Human PRP is made by healthy volunteers and standard strains MT8148 or TW871 (
Final concentration; 10 cells ml)was added. Collagen (4 μg)
Added after 5 minutes of incubation with MT8148 or TW871, 20-min.
Use the aggregator to measure the viewing period.
Maximum platelet aggregation rate and prolonged delay time
Use PRP to evaluate the time at which the aggregation begins.
The surface charge of the bacteria was evaluated as Zita-
Potential value using the zeta-
Potential Analyzer (ELSZ-
Electronic Mass Grave)
Automatically calculate the tower-
The potential obtained from the electrodelicity using the Smoluchowski equation. Overnight-
The cultured bacterial cells were washed and adjusted to 10 CFU with PBS and applied to the analyzer, which measured the Zetas-
The potential of the cell is automatically displayed at five standard points.
The result is represented by an average.
The mouse middle cerebral artery endothelial injury was induced by light irradiation and some modifications were made ().
Bacterial suspension (
10 cells per body)
In PBS, administered via jugular vein and then in the photo Bangladeshi Red
Illuminated with xenon lamps equipped with heating devices
Absorption filter (0.
540 W cm, wavelength nm, binsong photon)
Use 1 for 10 minutes. 5-
Millimeter diameter fiber mounted on a micro manipulator. Twenty-
After 4 hours of bacterial infection, mice were killed and the brain was removed.
Computer analysis calculates the total bleeding area of all brain slices and expresses it with mm according to this method (
Olympus DP70 DP controller).
Gelatin zymography Gursoy is based on the method-Ozdemir .
Made some changes.
Simple, tissue samples 24 h bacteria or vehicles are homogenized at 6-10mm Tris-HCl, 150 m NaCl, 1% Nonidet P-40, 0. 1% SDS and 0.
1% deoxic acid, pH 7.
4 buffer solution containing protease inhibitor.
Then, the samples were separated using gelatin enzymes
Kit (Cosmo Bio).
Brain tissue was removed from mice 3 hours after cerebral hemorrhage was induced and used for electron microscope observation in the bleeding area.
Simply fix the bleeding brain with 2% propolis.
Then cut and remove the brain, including part of the obstacle, and then fix again with 1% of the four sulfur chromium
Oxidation and dehydration with ethanol series.
By means of a freezing fracture device with liquid nitrogen, the sample is frozen and broken into two to four pieces.
The fracture surface is perpendicular to the brain surface and includes bleeding.
The broken samples are dried using a freeze-drying device with N-alcohol and adhere to the sample stage using a conductive slurry with a cross section at the top, and then treated with an osmium coating as conductive.
The completed samples were observed by SEM.
Comparison of bacterial membranes using three methodsdimensional re-
Computed tomography (CT)of TEM (JEM 1220: JEOL).
From the tilt range of 60 ° to 60 °, TEM images of bacteria were taken at a magnification of 150,000.
CT image of three-
The 3D reconstruction is carried out with the Radon transform software.
These CT images can be displayed in any direction.
The infection bacteria of several organs were detected by PCR, as shown below.
Complete DNA, such as the injured or uninjured cerebral hemisphere, lungs, liver and intestines, is extracted from the removed tissue.
PCR using-
Specific Primer groups described earlier ().
The detection limit of bacteria in each specimen is 5-50 cells.
To confirm the presence of a viable strain in the tissue, each removed tissue was squeezed into sterile PBS, and both the original and diluted liquids were striped on Mitis
Saliva Agar (Difco)
Peptide containing bacteria (
100 units per milliliters; Sigma-Aldrich)
This is the selected medium plate.
Male SHRSP mouse (12-weeks-old, SLC)
Placed in a climate-controlled (
Temperature 22-24 °C, humidity 40-60%)light-
Adjustment room with 12-h light: 12-h dark cycle.
All rats consumed SP diets (Funahashi).
Get free rat week 0.
5% saline solution was maintained throughout the experiment.
The rats were divided into two groups, TW295-
Management and vehicles-
Control group.
TW295 was administered via tail vein on 1, 3, 8 and 10 days (). Non-
The first dose of TW295 or one day before and after the vehicle, the invasive blood pressure was measured with the tail sleeve.
The weight and nerve score of each rat were also measured at each intravenous dose.
Nerve injury scores were evaluated for each animal based on the previously described method.
To put it simply, the forelimb and hind legs of the animal were observed, the motor function of the forelimb, the posture response, and the posture of each animal.
Each item scored from 0-3 and the most serious animals scored 15.
At 15 days, the animal died under the anesthesia of E. obarbital sodium.
The brain is removed after pre-formed saline perfusion and 6 coronary slices are selected in advance (3 from the front.
5mm to the first 13. 5 mm)
The thickness is 2mm, which is made using the brain matrix (RBS-
Neuroscience).
For each section, the bleeding area is determined using computer image analysis via Photoshop and image J software.
Restructuring Cnm is produced as previously described.
In short, the coding region of the TW871 strain Cnm was amplified by PCR and subcloned into the expression vector pET-42a(+)(Novagen).
Then, the obtained plasmid is transformed into E. Coli (DE3).
These creatures grew up in Loria.
Bertani Broth containing card and antibiotics from 37 °c to mid-term
Index stage. Isopropylthio-β-d-galactoside (Wako Chemicals)
Then add and give a final concentration of 1.
0mm, culture was incubated for another 3 h to induce GST-Cnm protein synthesis.
After that, the cells were collected and suspended in 10mm phosphate buffer and ultrasound was performed on ice.
Culture medium was obtained and purified using reduced gluten 4B column (GE Healthcare). The GST-
Fusion protein treated with precipitated protein (GE Healthcare)
SDS-PAGE at 4 °c and use GeBA flex-tube (
Biological Application of genes.
By inhibiting the adhesion of TW295 to the collagen layer, the collagen binding activity of the recombinant Cnm protein was confirmed ()
, And the dose was determined to be 10 and 100 μg according to the inhibition curve.
The recombinant Cnm protein was then applied to a mouse cerebral hemorrhage model as described above.
Statistical analysis with Prism 4 (
Software).
Fisher's protection difference is minimal, student's-
Test, regression analysis and Bonferroni method after ANOVA (ANOVA)were performed.
When