Clinical and immunoserological features of 7 cases of anti-p200 pemphigoid / 中华皮肤科杂志

Objective:To analyze clinical and immunoserological features of patients with anti-p200 pemphigoid.Methods:Clinical data were collected from patients with confirmed anti-p200 pemphigoid in Hospital of Dermatology, Chinese Academy of Medical Sciences from January 2015 to October 2021, and their clinical and immunoserological characteristics were retrospectively analyzed.Results:Seven patients with anti-p200 pemphigoid were included. Indirect immunofluorescence on salt-split skin (IIF-SSS) showed that serum IgG antibodies of the 7 patients were located in the dermis of the salt-split skin, and Western blot analysis with dermal extracts as substrates revealed a protein band with a relative molecular mass of 200 000. Four patients presented with classic bullous pemphigoid-like skin lesions, 2 initially presented with eczematous lesions, and 1 presented with linear IgA bullous dermatosis-like skin lesions. Circulating IgG antibodies could recognize the recombinant laminin γ1 C-terminal region in 6 cases. Four patients received different doses of systemic glucocorticoids, 1 of whom was resistant to high-dose systemic glucocorticoids (equivalent to 1.4 mg·kg -1·d -1 prednisone) ; 2 responded well to minocycline and dapsone; 1 was lost to follow-up. Four patients achieved complete remission and discontinued the treatment at a mean follow-up of 22.5 months; 2 received complete remissiona on minimal therapy at a mean follow-up of 8 months. Conclusion:Patients with anti-p200 pemphigoid presented with heterogeneous clinical manifestations, and the recombinant C-terminal fragment of laminin γ1 can serve as a reliable antigen substrate for the detection of autoantibodies in patients with anti-p200 pemphigoid; some patients can eventually achieve complete remission off treatment.

Differential Expression of Integrin ß1 in Two Brain Injury Models of Rats.

ABSTRACT: Objective To study the characteristics of positive expression of integrin ß1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin ß1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin ß1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin ß1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin ß1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin ß1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin ß1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.

Increased calcium-mediated cerebral processes after peripheral injury: possible role of the brain in complex regional pain syndrome.

BACKGROUND: Among various diseases that accompany pain, complex regional pain syndrome (CRPS) is one of the most frustrating for patients and physicians. Recently, many studies have shown functional and anatomical abnormalities in the brains of patients with CRPS. The calcium-related signaling pathway is important in various physiologic processes via calmodulin (CaM) and calcium-calmodulin kinase 2 (CaMK2). To investigate the cerebral mechanism of CRPS, we measured changes in CaM and CaMK2 expression in the cerebrum in CRPS animal models. METHODS: The chronic post-ischemia pain model was employed for CRPS model generation. After generation of the animal models, the animals were categorized into three groups based on changes in the withdrawal threshold for the affected limb: CRPS-positive (P), CRPS-negative (N), and control (C) groups. Western blot analysis was performed to measure CaM and CaMK2 expression in the rat cerebrum. RESULTS: Animals with a decreased withdrawal threshold (group P) showed a significant increment in cerebral CaM and CaMK2 expression (P = 0.013 and P = 0.021, respectively). However, groups N and C showed no difference in CaM and CaMK2 expression. CONCLUSIONS: The calcium-mediated cerebral process occurs after peripheral injury in CRPS, and there can be a relationship between the cerebrum and the pathogenesis of CRPS.

A Role for MMP-10 (Matrix Metalloproteinase-10) in Calcific Aortic Valve Stenosis.

OBJECTIVE: Aortic valve (AV) calcification plays an important role in the progression of aortic stenosis (AS). MMP-10 (matrix metalloproteinase-10 or stromelysin-2) is involved in vascular calcification in atherosclerosis. We hypothesize that MMP-10 may play a pathophysiological role in calcific AS. Approach and Results: Blood samples (n=112 AS and n=349 controls) and AVs (n=88) from patients undergoing valve replacement were analyzed. Circulating MMP-10 was higher in patients with AS compared with controls (P<0.001) and correlated with TNFα (tumor necrosis factor α; rS=0.451; P<0.0001). MMP-10 was detected by immunochemistry in AVs from patients with AS colocalized with aortic valve interstitial cells markers α-SMA (α-smooth muscle actin) and vimentin and with calcification markers Runx2 (Runt-related transcription factor 2) and SRY (sex-determining region Y)-box 9. MMP-10 expression in AVs was further confirmed by RT-qPCR and western blot. Ex vivo, MMP-10 was elevated in the conditioned media of AVs from patients with AS and associated with interleukin-1ß (rS=0.5045, P<0.001) and BMP (bone morphogenetic protein)-2 (rS=0.5003, P<0.01). In vitro, recombinant human MMP-10 induced the overexpression of inflammatory, fibrotic, and osteogenic markers (interleukin-1ß, α-SMA, vimentin, collagen, BMP-4, Sox9, OPN [osteopontin], BMP-9, and Smad 1/5/8; P<0.05) and cell mineralization in aortic valve interstitial cells isolated from human AVs, in a mechanism involving Akt (protein kinase B) phosphorylation. These effects were prevented by TIMP-1 (tissue inhibitor of metalloproteinases type 1), a physiological MMP inhibitor, or specifically by an anti-MMP-10 antibody. CONCLUSIONS: MMP-10, which is overexpressed in aortic valve from patients with AS, seems to play a central role in calcification in AS through Akt phosphorylation. MMP-10 could be a new therapeutic target for delaying the progression of aortic valve calcification in AS.

Differential Expression of Integrin β1 in Two Brain Injury Models of Rats / 法医学杂志

Objective To study the characteristics of positive expression of integrin β1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin β1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin β1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin β1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin β1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin β1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin β1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.

Relationship between Expression Changes of CB2R and Wound Age of Brain Contusion in Mice.

ABSTRACT: Objective To investigate the expression of cannabinoid type 2 receptor （CB2R） at different time points after brain contusion and its relationship with wound age of mice. Methods A mouse brain contusion model was established with PCI3000 Precision Cortical Impactor. Expression changes of CB2R around the injured area were detected with immunohistochemical staining, immunofluorescent staining and Western blotting at different time points. Results Immunohistochemical staining results showed that only a few cells in the cerebral cortex of the sham operated group had CB2R positive expression. The ratio of CB2R positive cells gradually increased after injury and reached the peak twice at 12 h and 7 d post-injury, followed by a decrease to the normal level 28 d post-injury. The results of Western blotting were consistent with the immunohistochemical staining results. Immunofluorescent staining demonstrated that the changes of the ratio of CB2R positive cells in neurons, CB2R positive cells in monocytes and CB2R positive cells in astrocytes to the total cell number showed a single peak pattern, which peaked at 12 h, 1 d and 7 d post-injury, respectively. Conclusion The expression of CB2R after brain contusion in neurons, monocytes and astrocytes in mice suggests that it is likely to be involved in the regulation of the biological functions of those cells. The changes in CB2R are time-dependent, which suggests its potential applicability as a biological indicator for wound age estimation of brain contusion in forensic practice.

Time-dependent Expression and Distribution of AChE during the Skin Incised Wound Healing in Mice.

ABSTRACT: Objective To study the time-dependent expression and distribution of acetylcholinesterase （AChE） during skin incised wound healing in mice, and discuss its effect in wound healing as well as the feasibility of using it as a reference index for wound age estimation. Methods A total of 45 C57BL/KsJ mice were randomly divided into one control group and eight incised groups. The skin incised wound model was established in the incised groups with samples of skin wounds taken at 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 10 d and 14 d post-injury respectively, while the uninjured skin tissue was extracted in the control group. Expression and distribution of AChE in skin samples were detected by immunohistochemistry, double immunofluorescence and Western blotting. Results Immunohistochemistry results indicated that AChE was mainly detected in infiltrating polymorphonuclear cells （PMNs） 6 to 12 h post-injury. A large number of AChE-positive mononuclear cells （MNCs） were observed 1 to 3 d post-injury. The AChE-positive cells were mainly fibroblastic cells （FBCs） 5 to 14 d post-injury. The ratio of the AChE-positive cells increased initially 6 h post-injury, and reached the peak at 1 d post-injury. Double immunofluorescent staining showed that the majority of AChE-positive MNCs and FBCs expressed macrophage marker and myofibroblast marker, respectively. Western blotting results showed that the relative expression level of AChE in the incised group was higher than that in the control group averagely, reached the peak at 1 d post-injury, then reached a second peak at 7 d post-injury. Conclusion The expression of AChE is found in PMNs, macrophages and myofibroblast during skin wound healing, which indicates it might be involved in the adjustment of inflammatory response and fibrotic repair after injury. Moreover, combined use of various methods for the detection of the expression of AChE would provide reference for skin wound age estimation.

Id-1 expression in colorectal adenocarcinoma tissues and its clinical significance

SUMMARY BACKGROUND: This study aims to investigate the expression of Id-1 in human colorectal adenocarcinoma tissues and explore its correlation with the clinical pathological parameters of colorectal cancer. METHODS: The Id-1 mRNA and protein expression levels of 50 specimens of normal colorectal tissues and 50 specimens of colorectal adenocarcinoma tissues were detected using reverse-transcription polymerase chain reaction and western blot. Furthermore, Id-1 protein was detected using immunohistochemistry. The correlation between the expression of Id-1 and clinicopathologic features was analyzed. RESULTS: The mRNA expression level of Id-1 in colorectal adenocarcinoma tissues and normal colorectal tissues was 0.96 ± 0.03 vs. 0.20 ± 0.04, respectively; and the difference was statistically significant (P=0.011). Furthermore, Id-1 protein expression was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (0.82 ± 0.04 vs. 0.31 ± 0.02, P=0.020). In addition, the positive protein expression rate of Id-1 was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (72.00% vs. 24.00%, X2=23.431, P=0.000). The expression of Id-1 was correlated with the depth of tumor invasion, TNM stage, lymph node metastasis, vessel invasion, and liver metastasis (P<0.01). However, this expression was not correlated with tumor size and differentiation degrees (P>0.05). CONCLUSIONS: The high Id-1 expression in colorectal adenocarcinoma tissues play an important role in the process of cancer, and is expected to become a new tumor monitoring indicator for clinical diagnosis, treatment, and prognosis judgment.

RESUMO OBJETIVO: O objetivo deste estudo é investigar a expressão de Id-1 em tecidos de adenocarcinoma colorretal em humanos e investigar sua correlação com os parâmetros patológicos clínicos de câncer colorretal. MÉTODOS: Os níveis de expressão de proteína e mRNA Id-1 em 50 amostras de tecido colorretal normal e 50 amostras de tecido de adenocarcinoma colorretal foram detectados através de reação em cadeia de polimerase precedida de transcrição reversa e western blot. Além disso, a proteína Id-1 foi detectada através de imuno-histoquímica. A correlação entre a expressão de Id-1 e características clínico-patológicas foi analisada. RESULTADOS: O nível de expressão de mRNA Id-1 em tecidos de adenocarcinoma colorretal e tecidos colorretais normais foi de 0,96 ± 0,03 versus 0,20 ± 0,04, respectivamente; a diferença foi estatisticamente significativa (P= 0,011). Além disso, a expressão da proteína Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (0,82 ± 0,04 versus 0,31 ± 0,02, P= 0,020). Além disso, a taxa de expressão positiva de proteínas Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (72,00% vs. 24,00%, X2=23,431, p=0,000). A expressão de Id-1 foi correlacionada com a profundidade da invasão tumoral, estágio TNM, metástases linfonodais, invasão vascular e metástase hepática (P<0,01). Todavia, essa expressão não se correlacionou com o tamanho do tumor e graus de diferenciação (P>0,05). CONCLUSÃO: A alta expressão de Id-1 em tecidos de adenocarcinoma colorretal desempenham um importante papel no processo do câncer, e é esperado que se torne um novo indicador de monitoramento de tumores para o diagnóstico clínico, tratamento e estimativa de prognóstico.

HMGB1 concentration measurements in trauma patients: assessment of pre-analytical conditions and sample material.

BACKGROUND: HMGB1 is a mediator of systemic inflammation in sepsis and trauma, and a promising biomarker in many diseases. There is currently no standard operating procedure for pre-analytical handling of HMGB1 samples, despite that pre-analytical conditions account for a substantial part of the overall error rate in laboratory testing. We hypothesized that the considerable variations in reported HMGB1 concentrations and kinetics in trauma patients could be partly explained by differences in pre-analytical conditions and choice of sample material. METHODS: Trauma patients (n = 21) admitted to a Norwegian Level I trauma center were prospectively included. Blood was drawn in K2EDTA coated tubes and serum tubes. The effects of delayed centrifugation were evaluated in samples stored at room temperature for 15 min, 3, 6, 12, and 24 h respectively. Plasma samples subjected to long-term storage in - 80 °C and to repeated freeze/thaw cycles were compared with previously analyzed samples. HMGB1 concentrations in simultaneously acquired arterial and venous samples were also compared. HMGB1 was assessed by standard ELISA technique, additionally we investigated the suitability of western blot in both serum and plasma samples. RESULTS: Arterial HMGB1 concentrations were consistently lower than venous concentrations in simultaneously obtained samples (arterial = 0.60 x venous; 95% CI 0.30-0.90). Concentrations in plasma and serum showed a strong linear correlation, however wide limits of agreement. Storage of blood samples at room temperature prior to centrifugation resulted in an exponential increase in plasma concentrations after ≈6 h. HMGB1 concentrations were fairly stable in centrifuged plasma samples subjected to long-term storage and freeze/thaw cycles. We were not able to detect HMGB1 in either serum or plasma from our trauma patients using western blotting. CONCLUSIONS: Arterial and venous HMGB1 concentrations cannot be directly compared, and concentration values in plasma and serum must be compared with caution due to wide limits of agreement. Although HMGB1 levels in clinical samples from trauma patients are fairly stable, strict adherence to a pre-analytical protocol is advisable in order to protect sample integrity. Surprisingly, we were unable to detect HMGB1 utilizing standard western blot analysis.

Effect of sec-O-glucosylhamaudol on mechanical allodynia in a rat model of postoperative pain

BACKGROUND: This study was performed in order to examine the effect of intrathecal sec-O-glucosylhamaudol (SOG), an extract from the root of the Peucedanum japonicum Thunb., on incisional pain in a rat model. METHODS: The intrathecal catheter was inserted in male Sprague-Dawley rats (n = 55). The postoperative pain model was made and paw withdrawal thresholds (PWTs) were evaluated. Rats were randomly treated with a vehicle (70% dimethyl sulfoxide) and SOG (10 μg, 30 μg, 100 μg, and 300 μg) intrathecally, and PWT was observed for four hours. Dose-responsiveness and ED50 values were calculated. Naloxone was administered 10 min prior to treatment of SOG 300 μg in order to assess the involvement of SOG with an opioid receptor. The protein levels of the δ-opioid receptor, κ-opioid receptor, and μ-opioid receptor (MOR) were analyzed by Western blotting of the spinal cord. RESULTS: Intrathecal SOG significantly increased PWT in a dose-dependent manner. Maximum effects were achieved at a dose of 300 μg at 60 min after SOG administration, and the maximal possible effect was 85.35% at that time. The medial effective dose of intrathecal SOG was 191.3 μg (95% confidence interval, 102.3–357.8). The antinociceptive effects of SOG (300 μg) were significantly reverted until 60 min by naloxone. The protein levels of MOR were decreased by administration of SOG. CONCLUSIONS: Intrathecal SOG showed a significant antinociceptive effect on the postoperative pain model and reverted by naloxone. The expression of MOR were changed by SOG. The effects of SOG seem to involve the MOR.

Time-dependent Expression and Distribution of AChE during the Skin Incised Wound Healing in Mice / 法医学杂志

Objective To study the time-dependent expression and distribution of acetylcholinesterase （AChE） during skin incised wound healing in mice, and discuss its effect in wound healing as well as the feasibility of using it as a reference index for wound age estimation. Methods A total of 45 C57BL/KsJ mice were randomly divided into one control group and eight incised groups. The skin incised wound model was established in the incised groups with samples of skin wounds taken at 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 10 d and 14 d post-injury respectively, while the uninjured skin tissue was extracted in the control group. Expression and distribution of AChE in skin samples were detected by immunohistochemistry, double immunofluorescence and Western blotting. Results Immunohistochemistry results indicated that AChE was mainly detected in infiltrating polymorphonuclear cells （PMNs） 6 to 12 h post-injury. A large number of AChE-positive mononuclear cells （MNCs） were observed 1 to 3 d post-injury. The AChE-positive cells were mainly fibroblastic cells （FBCs） 5 to 14 d post-injury. The ratio of the AChE-positive cells increased initially 6 h post-injury, and reached the peak at 1 d post-injury. Double immunofluorescent staining showed that the majority of AChE-positive MNCs and FBCs expressed macrophage marker and myofibroblast marker, respectively. Western blotting results showed that the relative expression level of AChE in the incised group was higher than that in the control group averagely, reached the peak at 1 d post-injury, then reached a second peak at 7 d post-injury. Conclusion The expression of AChE is found in PMNs, macrophages and myofibroblast during skin wound healing, which indicates it might be involved in the adjustment of inflammatory response and fibrotic repair after injury. Moreover, combined use of various methods for the detection of the expression of AChE would provide reference for skin wound age estimation.

Relationship between Expression Changes of CB2R and Wound Age of Brain Contusion in Mice / 法医学杂志

Objective To investigate the expression of cannabinoid type 2 receptor （CB2R） at different time points after brain contusion and its relationship with wound age of mice. Methods A mouse brain contusion model was established with PCI3000 Precision Cortical Impactor. Expression changes of CB2R around the injured area were detected with immunohistochemical staining, immunofluorescent staining and Western blotting at different time points. Results Immunohistochemical staining results showed that only a few cells in the cerebral cortex of the sham operated group had CB2R positive expression. The ratio of CB2R positive cells gradually increased after injury and reached the peak twice at 12 h and 7 d post-injury, followed by a decrease to the normal level 28 d post-injury. The results of Western blotting were consistent with the immunohistochemical staining results. Immunofluorescent staining demonstrated that the changes of the ratio of CB2R positive cells in neurons, CB2R positive cells in monocytes and CB2R positive cells in astrocytes to the total cell number showed a single peak pattern, which peaked at 12 h, 1 d and 7 d post-injury, respectively. Conclusion The expression of CB2R after brain contusion in neurons, monocytes and astrocytes in mice suggests that it is likely to be involved in the regulation of the biological functions of those cells. The changes in CB2R are time-dependent, which suggests its potential applicability as a biological indicator for wound age estimation of brain contusion in forensic practice.

Evaluation of the value of Western blot analysis based on human epidermal extracts prepared by thermal separation in the diagnosis of bullous pemphigoid / 中华皮肤科杂志

Objective To prepare human epidermal extracts by thermal separation,and to evaluate the value of epidermal extract-based Western blot analysis in the diagnosis of bullous pemphigoid (BP).Methods Human epidermal extracts were prepared by thermal separation from circumcised foreskins of healthy males.Serum samples were obtained from 22 inpatients with BP and 25 inpatients without BP in Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017.These serum samples were subjected to Western blot analysis with epidermal extracts as substrates,as well as to BP180-NC16A enzyme-linked immunosorbent assay (ELISA).Statistical analysis was carried out using chi-square test and Fisher's exact test with the SPSS22.0 software.Results The sensitivities of epidermal extract-based Western blot analysis and BP 180-NC16A ELISA in the diagnosis of BP were 86.36％ (95 ％ CI:64.03％-96.41％) and 95.45％ (95％ CI:75.11％-99.76％) respectively (~ =1.10,P =0.294),and the specificities were 100％ (95％ CI:83.42％-100％) and 92％ (95％ CI:75.11％-99.76％) respectively (x2 =20.8,P =0.149).Epidermal extract-based Western blot analysis in the 22 patients with BP showed a protein band with relative molecular mass (RMM) of 230 000 in 4 patients,a protein band with RMM of 180 000 in 18,a protein band with RMM of 120 000 in 1,and a protein band with RMM of 97 000 in 1.The BP180-NC16A ELISA showed that the antibody titers were more than 50 U/ml in the BP patients with protein bands of RMM of 180 000.Conclusions The epidermal extract-based Western blot analysis mainly showed the protein band with RMM of 180 000 in the patients with BP.The sensitivity of the epidermal extract-based Western blot analysis was lower than that of the BP180-NC16A ELISA,and the epidermal extract-based Western blot analysis tends to be negative when the titer of the autoantibody is low.

Choosing appropriate lysis buffers for protein extraction from acidotic mouse skeletal muscles / 中国组织工程研究

BACKGROUND: RIPA Buffer exhibits different extraction efficiencies of proteins of cells and tissues, which is not appropriate for all samples. OBJECTIVE: To achieve an optimal lysis buffer for skeletal muscle protein extraction in mice of acidosis, and to provide basis for studies on skeletal muscle atrophy. METHEDS: Twenty male healthy C57BL/6 mice, aged 3 months, weighting 25-30 g, were provided by Laboratory Animal Center of Shanxi Medical University. The mice were sacrificed after anesthesia, and the gastrocnemius muscle of lower extremity was isolated. There were two groups: acidosis group was given 10 g of feed mixed with 0.4 mol/L hydrochloric acid (10 mL) , and control group received 10 g of feed mixed with same volume of water, for 7 consecutive days. The effect of RIPA Buffer, Original Buffer and JP Buffer on the skeletal muscle protein extraction in mice of acidosis was compared. The expression levels of AKT, p-AKT (Thr308) , rpS6 and p-rpS6 (Ser235/236) were detected by western blot assay. GLUT4 mRNA expression was examined by RT-qPCR. RESULTS AND CONCLUSION: (1) Different buffers generated different protein-yields. The protein yield was highest in JP Buffer, but the target protein signal was not high. The protein yield was low in RIPA Buffer. Original Buffer could extract sufficient proteins, and had clear band detected by western blot assay. (2) Western blot assay scores in Original Buffer were higher than those of other two buffers. (3) Western blot assay results showed that the extent of phosphorylation in both groups showed no significant changes. (4) GLUT4 mRNA expression level examined by RT-qPCR showed no significant changes in both groups. (5) These results indicate that Original Buffer is optimal lysate of skeletal muscle protein extraction. Inactivated AKT signaling pathway is seen in the short-term hydrochloric acid-induced acidosis group, so whether lengthening acidosis time can activate the signaling pathway. Selecting the optimal lysis buffer for different samples is premise to ensure western blot assay results.

Expressions of transforming growth factor ß1 signaling cytokines in aortic dissection

Abstract Objective: To demonstrate the underlying mechanisms of aortic dissection compared to those of coronary artery disease in terms of the transforming growth factor-beta (TGF-β) signaling pathway. Methods: Twenty consecutive aortic dissection patients and 20 consecutive coronary artery disease patients undergoing a surgical treatment in this hospital were enrolled into this study. The aortic tissues were sampled and the TGF-β1 and its receptor TGF-β receptor I (TβRI) were detected by Western blotting assay. Results: TGF-β1 and TβRI were positively expressed in the aortic tissues in both groups by Western blotting assay. The expressions of the two proteins were significantly higher in the aortic tissue of patients with aortic dissection than in those with coronary artery disease. The quantitative analyses of the relative gray scales of the proteins disclosed close correlations between the expressions of TGF-β1 and TβRI in both the study and control group patients. Conclusions: The aortic remodeling of aortic dissection might differ from that of coronary artery atherosclerosis concerning the nature, mechanism, mode, and activities of TGF-β signaling pathway. The development of aortic dissection could be associated with a significantly enhanced function of TGF-β1/Smad signaling transduction as a result of aortic remodeling incorporating both vascular injury and repair.

Inward Rectifier Potassium Channels (Kir2.x) and Caveolin-3 Domain-Specific Interaction: Implications for Purkinje Cell-Dependent Ventricular Arrhythmias.

BACKGROUND: In human cardiac ventricle, IK1 is mainly comprised Kir2.1, but Kir2.2 and Kir2.3 heterotetramers occur and modulate IK1. Long-QT syndrome-9-associated CAV3 mutations cause decreased Kir2.1 current density, but Kir2.x heterotetramers have not been studied. Here, we determine the effect of long-QT syndrome-9-CAV3 mutation F97C on Kir2.x homo- and heterotetramers and model-associated arrhythmia mechanisms. METHODS AND RESULTS: Super-resolution microscopy, co-immunoprecipitation, cellular electrophysiology, on-cell Western blotting, and simulation of Purkinje and ventricular myocyte mathematical models were used. Kir2.x isoforms have unique subcellular colocalization in human cardiomyocytes and coimmunoprecipitate with Cav3. F97C-Cav3 decreased peak inward Kir2.2 current density by 50% (-120 mV; P=0.019) and peak outward by 75% (-40 mV; P<0.05) but did not affect Kir2.3 current density. FRET (Förster resonance energy transfer) efficiency for Kir2.2 with Cav3 is high, and on-cell Western blotting demonstrates decreased Kir2.2 membrane expression with F97C-Cav3. Cav3-F97C reduced peak inward and outward current density of Kir2.2/Kir2.1 or Kir2.2/Kir2.3 heterotetramers (P<0.05). Only Cav3 scaffolding and membrane domains co-immunoprecipitation with Kir2.1 and Kir2.2 and Kir2.x-N-terminal Cav3 binding motifs are required for interaction. Mathematical Purkinje, but not ventricular, myocyte model incorporating simulated current reductions, predicts spontaneous delayed after-depolarization-mediated triggered activity. CONCLUSIONS: Kir2.x isoforms have a unique intracellular pattern of distribution in association with specific Cav3 domains and that critically depends on interaction with N-terminal Kir2.x Cav3-binding motifs. Long-QT syndrome-9-CAV3 mutation differentially regulates current density and cell surface expression of Kir2.x homomeric and heteromeric channels. Mathematical Purkinje cell model incorporating experimental findings suggests delayed after-depolarization-type triggered activity as a possible arrhythmia mechanism.

Myocardial Upregulation of Cathepsin D by Ischemic Heart Disease Promotes Autophagic Flux and Protects Against Cardiac Remodeling and Heart Failure.

BACKGROUND: Lysosomal dysfunction is implicated in human heart failure for which ischemic heart disease is the leading cause. Altered myocardial expression of CTSD (cathepsin D), a major lysosomal protease, was observed in human heart failure, but its pathophysiological significance has not been determined. METHODS AND RESULTS: Western blot analyses revealed an increase in the precursor but not the mature form of CTSD in myocardial samples from explanted human failing hearts with ischemic heart disease, which is recapitulated in chronic myocardial infarction produced via coronary artery ligation in Ctsd+/+ but not Ctsd+/- mice. Mice deficient of Ctsd displayed impaired myocardial autophagosome removal, reduced autophagic flux, and restrictive cardiomyopathy. After induction of myocardial infarction, weekly serial echocardiography detected earlier occurrence of left ventricle chamber dilatation, greater decreases in ejection fraction and fractional shortening, and lesser wall thickening throughout the first 4 weeks; pressure-volume relationship analyses at 4 weeks revealed greater decreases in systolic and diastolic functions, stroke work, stroke volume, and cardiac output; greater increases in the ventricular weight to body weight and the lung weight to body weight ratios and larger scar size were also detected in Ctsd+/- mice compared with Ctsd+/+ mice. Significant increases of myocardial autophagic flux detected at 1 and 4 weeks after induction of myocardial infarction in the Ctsd+/+ mice were diminished in the Ctsd+/- mice. CONCLUSIONS: Myocardial CTSD upregulation induced by myocardial infarction protects against cardiac remodeling and malfunction, which is at least in part through promoting myocardial autophagic flux.

[Polyclonal antibodies against recombinant dengue virus NS3 protein].

INTRODUCTION: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of themost attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. OBJECTIVE: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. MATERIALS AND METHODS: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. RESULTS: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling. CONCLUSION: The recombinant proteins expressed and the antibodies produced constitute valuable tools for studying DENV infectious processes involving NS3 and for evaluating tests designed to interfere with its functions.

Decreased Adiponectin-Mediated Signaling Through the AdipoR2 Pathway Is Associated With Carotid Plaque Instability.

BACKGROUND AND PURPOSE: Adiponectin, the most abundantly secreted anti-inflammatory adipokine, protects against all stages of atherosclerotic plaque formation by acting on its receptors, AdipoR1 (adiponectin receptor 1) and AdipoR2 (adiponectin receptor 2). Through binding of AdipoR1, adiponectin leads to the activation of the AMPK (adenosine monophosphate-activated protein kinase) pathway, whereas stimulation of PPAR-α (peroxisome proliferator-activated receptor-α) is attributed to the binding of AdipoR2. However, the role of adiponectin and its receptors in plaque instability remains to be characterized. Thus, we aimed to investigate whether the adiponectin-AdipoR pathway is associated with carotid atherosclerotic plaque instability. METHODS: The instability of plaque specimens obtained from patients who underwent a carotid endarterectomy (n=143) was assessed using gold standard histological classifications. RESULTS: Using immunohistochemistry, we showed that adiponectin and AdipoR1/AdipoR2 are expressed in human carotid plaques and that their expression was localized most abundantly in areas of macrophage and foam cell accumulation. Unstable plaques expressed more adiponectin protein (Western blot, P<0.05) and less AdipoR2 mRNA (2.11-fold decrease, P<0.05) than stable plaques, whereas AdipoR1 expression remained similar between stable and unstable plaques. Beyond AdipoR1/AdipoR2 expression, a graded decrease in PPAR-α protein levels was observed in relation to carotid plaque instability (P<0.001), whereas AMPK phosphorylation was increased (P<0.05). Our in vitro model of plaque instability, involving the induction of foam cells from human THP-1 (Tamm-Horsfall protein 1) macrophages treated with acetylated low-density lipoprotein, supported our in vivo conclusions. CONCLUSIONS: An overall abundance of adiponectin with a decrease in AdipoR2 expression and activity was observed in unstable plaques, suggesting that reduced signaling through the AdipoR2 pathway, and not through AdipoR1, may contribute to plaque instability.

Anticuerpos policlonales contra la proteína recombinante NS3 del virus del dengue / Polyclonal antibodies against recombinant dengue virus NS3 protein

Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.