[Advances in molecular mechanism and treatment of chronic mucus hypersecretion].

Chronic obstructive pulmonary disease (COPD) is one of the chronic diseases with high morbidity and mortality in China, which imposes heavy economic burden on society. Research has shown that chronic mucus hypersecretion (CMH) is an independent risk factor for persistent clinical symptoms, poor quality of life, rapid decline in lung function, acute exacerbation and increased hospitalization rate in COPD patients. CMH is a clinical phenotype of COPD with specific pathological and physiological changes. At present, the formation mechanism of CMH is not clear. There is a lack of specific and effective targeted treatments. This article aimed to review the latest research findings on CMH at home and abroad from the overview, impact on COPD patients, molecular mechanisms of formation, current treatment status and progress, and discuss potential targets for CMH treatment, to provide new ideas and directions for improving CMH and treating COPD.

[Research progress of chronic obstructive pulmonary disease in young people and pre-COPD].

Conventional wisdom holds that the onset of chronic obstructive pulmonary disease (COPD) is usually in older people. However, with extended follow-up in large cohort studies, the trajectory of lung function development has been gradually delineated, indicating that the decline of lung function may originate early in life. In addition, a large number of studies have shown that people with chronic respiratory symptoms, pulmonary imaging changes and abnormal lung physiology, but not in line with pulmonary function diagnosis of COPD, tend to develop COPD in the future and may have a worse prognosis, suggesting the necessity of early intervention. The GOLD 2022 report proposes a series of terms related to COPD, including COPD in young people and pre-COPD, opening up new opportunities for the prevention, early diagnosis and treatment of COPD. These concepts also guide the study design of the biological mechanism of COPD and the trajectory of disease progression. This article reviews the research progress of COPD in young people and pre-COPD to attract more attention in clinical practice.

[Gadolinium angiography of inner ear in patients with sudden deafness and its clinical analysis].

Objective: To observe the gadolinium imaging findings of inner ear in patients with sudden deafness and to analyze its clinical features. Methods: From November 2017 to July 2020, 21 patients with sudden deafness in the People's Hospital of Dongsheng District, Ordos City were selected as the research objects, including 14 males and 7 females, aged 36-76 years, with a median age of 50 years. The course of disease was 1-19 days, with an average of 5.5 days. The patients received audiology tests, laboratory examination, and intravenous gadolinium angiography, each of whom was scanned twice by 3D-FLAIR sequence: once before intravenous gadolinium injection, and once again 4.5-6.0 h after intravenous gadolinium injection. The following corresponding clinical treatment was given. The imaging manifestations and clinical features were observed. Results: Among 21 cases of sudden deafness in acute stage, the signal intensity of 11 cases was significantly higher than that of the contralateral ear, and 2 cases had vestibular labyrinthine hydrops. In laboratory examination, only 2 cases of total deafness had increased WBC count and faster erythrocyte sedimentation rate, and the rest had no abnormality. The hearing types of 21 patients with sudden deafness were: total deafness in 8 cases, flat decline in 10 cases, low frequency decline in 1 case, high frequency decline in 2 cases. The total effective rate was 57% (12/21). The hearing types of 11 patients with abnormal gadolinium angiography were total deafness in 5 cases, flat decline in 5 cases and high frequency decline in 1 case. The total effective rate was 64% (7/11). Conclusion: Gadolinium angiography is abnormal in some patients with sudden deafness, and the permeability of blood labyrinth barrier may be increased, which is worthy of further study.

[Protective effect of vitamin D in mice with acute liver failure].

Objective: To explore the protective effect of vitamin D in acute liver failure through a mouse model. Methods: Acute liver failure was induced by combining D-galactosamine (D-GalN) lipopolysaccharide (LPS) to observe the effect of long-term vitamin D deficiency on liver injury and inflammatory signals in a mouse model. Acute liver failure was induced by thioacetamide (TAA) to observe the effect of vitamin D deficiency on the survival rate, and further high-dose of vitamin D supplementation protective effect was determined in a mouse model. Liver function was evaluated by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver inflammation by hematoxylin-eosin staining. The expressions of tumor necrosis factor (TNF-α), interleukin (IL) -1ß, NOD-like receptor family, pyrin domain containing 3 (NLRP-3), chemokines (CCL2, CXCL1 and CXCL2), etc. in liver tissues were detected by RT-qPCR. The quantitation of macrophages in liver tissue was detected by immunohistochemistry. The comparison between groups were performed by t-test. The survival curve was analyzed by log-rank (Mantel-Cox) test. Results: Long-term vitamin D deficiency had increased acute liver failure sensitivity in mice, which was manifested by increased blood cell extravasation, massive necrosis of parenchymal cells, up-regulation of TNF-α, IL-1ß, and NLRP-3 mRNA expression (P < 0.05), and increased macrophages quantitation (P < 0.05) in liver tissues. At the same time, vitamin D deficiency had increased the mice mortality rate because of liver injury (P < 0.01). On the contrary, pre-administration of high dose of vitamin D (100 IU/g) had significantly reduced liver injury, inhibited ALT and AST rise (P < 0.01), alleviated liver necrosis, and down-regulated the mRNA expression of inflammatory factors in liver tissues (P < 0.05). Conclusion: Mouse model shows that long-term vitamin D deficiency can aggravate drug-induced acute liver failure and reduce survival rates. Furthermore, high-dose of vitamin D has a certain hepatoprotective effect, which can significantly improve liver necrosis condition and inhibit inflammation. Therefore, adequate vitamin D can retain liver physiological balance to resist liver injury.

[Effect of bone marrow mononuclear cell transplantation on miRNA-21 and miRNA-155 expression in mice with ulcerative colitis].

Objective: To investigate the effect of bone marrow mononuclear cell transplantation on the expression of miRNA-21 and miRNA-155 in mice with ulcerative colitis(UC). Methods: Healthy and clean KM mice aged 6-8 weeks were randomly divided into transplantation group, model group and normal control group with 15 mice in each group. In the transplantation group and model group, dextran sodium sulfate (DSS) was used to establish the model for 24 h. The mice in the transplantation group were injected with 0.4 ml of 4 ', 6-diaminol-2-phenylindole (DAPI) -labeled P3-BM-MNCs cell suspension (3.2×10(6) cells/ml), and the mice in the model group and the normal control group were injected with 0.4 ml phosphate buffer (PBS).UC disease activity index (DAI) was used to test the general condition of mice; HE staining was used to observe the pathological changes of colon tissue; Real-time quantitative PCR was used to detect the expression of miRNA-21 and miRNA-155 mRNA. Results: DAI scores of normal control group, model group and transplantation group were 0 (0,1), 3.1 (2.8,3.3) and 2.7 (2.4,3.1),respectively. Compared with normal control group, the DAI score of model group and transplantation group was higher (P<0.05), and the DAI score of transplantation group was lower than that of model group (P<0.05). The gross scores of tissue injury in normal control group, model group and transplantation group were 0 (0, 1), 3 (3, 4) and 1 (1, 2), respectively,and the pathological scores of tissue injury were 0 (0, 1), 16 (12, 16) and 6 (6, 8), respectively,compared with the normal control group. The tissue injury score of the model group and the transplantation group was higher (P<0.05), and the tissue injury score of the transplantation group was lower than that of the model group (P<0.05). The expression levels of miRNA-21 mRNA in normal control group, model group and transplantation group were 0.87±0.15, 2.38±0.29 and 1.59±0.32, respectively, and the expression levels of miRNA-155 mRNA were 1.87±0.46, 7.38±1.97 and 3.92±0.84, respectively, compared with the normal control group, the expression of miRNA-21 and miRNA-155 mRNA in the model group and transplantation group was higher (P<0.01), the expression of miRNA-21 and miRNA-155 mRNA in the transplantation group was lower than that of the model group (P<0.05). Conclusion: Bone marrow mononuclear cell transplantation can improve the histopathological and DAI scores of mice with UC, which may be related to the down-regulation of miRNA-21 and miRNA-155 mRNA expression.

A study on regulatory mechanism of miR-223 in ulcerative colitis through PI3K/Akt-mTOR signaling pathway.

OBJECTIVE: The aim of this study was to explore the regulatory mechanism of micro ribonucleic acid (miR)-223 in ulcerative colitis (UC) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway. MATERIALS AND METHODS: A total of 36 Sprague-Dawley (SD) rats were randomly divided into three groups, including normal group (n=12), model group (n=12) and inhibitor group (n=12). Rats in the normal group received no treatment. Rats in the model group were used to establish a UC model. Meanwhile, rats in the inhibitor group underwent intraperitoneal injection of inhibitor and establishment of the UC model. Subsequently, specimens were obtained for detection. Immunohistochemistry was applied to measure the expression of mTOR. Western blotting was adopted to determine the relative protein expressions of P85, P110 and phosphorylated Akt (p-Akt). Quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression of miR-223. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized to determine cell apoptosis. Furthermore, an enzyme-linked immunosorbent assay (ELISA) was conducted to measure the content of interleukin-1 beta (IL-1ß) and IL-6. RESULTS: Immunohistochemistry showed that the positive expression of mTOR increased remarkably in the model group and inhibitor group when compared with that of the normal group (p<0.05). However, it decreased notably in the inhibitor group when compared with the model group (p<0.05). Western blotting indicated that the protein expressions of P85, P110 and p-Akt in model group and inhibitor group were significantly higher than the ones of the normal group (p<0.05). However, the inhibitor group showed markedly lower relative protein expressions of P85, P110 and p-Akt than the ones of the model group (p<0.05). Compared with the normal group, the expression level of miR-223 was significantly elevated in model group and inhibitor group (p<0.05). However, there was no significant difference in the mRNA expression of miR-233 between the model group and the inhibitor group (p>0.05). The apoptosis rate of the cells increased prominently in the model group and in the inhibitor group when compared with the normal group (p<0.05). However, it was remarkably reduced in the inhibitor group than the model group (p<0.05). In comparison with the normal group, the content of IL-1ß and IL-6 was significantly up-regulated in the model group and in the inhibitor group (p<0.05). However, it declined notably in the inhibitor group compared with the model group (p<0.05). CONCLUSIONS: MiR-223 can trigger cell apoptosis and inflammation in UC by up-regulating the PI3K/Akt-mTOR signaling pathway.

[Association between nuclear factor kappa-light-chain-enhancer of activated B cells genetic polymorphisms and HCV susceptibility among the Chinese population under high-risk].

Objective: To explore the association between nuclear factor kappa-light-chain-enhancer of activated genetic polymorphisms in B cells (NF-κB) and the HCV susceptibility, among the Chinese population. Methods: A total of 1 679 participants were enrolled; including 503 drug users and 1 176 other participants at risk under the exposure for blood. By using the logistic regression analysis, related risk factors for HCV infection among subjects were analyzed. Two NF-κB pathway variants, NF-κB1 rs72696119 and REL rs13031237 were then genotyped by TaqMan assay method. Logistic regression analysis was performed to analyze the association between gene polymorphisms and the susceptibility on HCV. Results: Among the drug users, women (OR=0.408, 95%CI: 0.308-0.767) appeared to be associated with the decreased risk for HCV infection, while factors as drug injection (OR=8.817, 95%CI: 5.577-13.937) and the duration of drug-intake >5.5 years (OR=2.891, 95%CI: 1.824-4.583) were associated with the increased risk for HCV infection. Among the participants who had been exposed to blood, women (OR=3.431, 95%CI: 2.360-4.988) were associated with the increased risk for HCV infection, while the levels of education beyond elementary school (OR=0.613, 95%CI: 0.429-0.876) were associated with the decreased risk for HCV infection. Compared to the reference NF-κB1 rs72696119 CC genotype, the carriage of GG genotype was associated with an increased risk of susceptibility on HCV (OR=1.412, 95%CI: 1.035-1.927) among the total study population. Results from the interaction analysis showed that there was no interactive effects appeared between rs72696119 and route of infection, or between rs72696119 and gender among the total population under study (all P>0.05). Conclusion: NF-κB1 polymorphism rs72696119 and related factors seemed associated with the susceptibility to HCV infection among high-risk Chinese populations.

[Changing trends in etiologies of hospitalized patients with liver disease during 2006 -2014].

Objective: To analyze the changing trends in etiologies of hospitalized patients with liver disease and provide clinical basis for the formulation of medical policy. Methods: Patients who were hospitalized in the Department of Infectious Diseases from 2006 to 2014 were selected as the research subjects. Data of patients with liver diseases were retrospectively analyzed to determine the proportion of main causes of infection, the proportion of different viral infections in viral hepatitis, and the changing trends in proportion of hepatitis B in different age groups. Kruskal-Wallis test was used for statistical analysis. Results: During 9 consecutive years, the overall proportion of inpatients with liver disease decreased continuously, but the number of patients increased. The top five etiologies of liver diseases were viral hepatitis, drug-induced liver disease, autoimmune liver disease, alcoholic and non-alcoholic fatty liver disease. The proportion of viral hepatitis decreased gradually, and the proportion of drug-induced liver disease and autoimmune liver disease increased markedly. Among viral hepatitis patients, hepatitis B, hepatitis C and hepatitis E were in the top three, with hepatitis B stabilized at around 70%, and the proportion of hepatitis C showed an upward trend. The hospitalization time of hepatitis B patients was gradually shortened, the difference was statistically significant (χ (2) = 205.31, P < 0.001), and the hepatitis B patients were mainly distributed in age groups 31-40, 41-50, and 51-60, the total proportion was above 60%. The difference between the different years of the same age group was not evident, but the proportion of hepatitis B patients decreased gradually in the 14-23 -year- old age group, the difference was statistically significant (χ (2) = 19.51, P = 0.01). Conclusion: Liver disease still holds a principal position in the distribution of infectious diseases, and especially the cause of non-infectious liver disease require sufficient attention and concern. The use of hepatitis B vaccine has effectively diminished the infection rate, but the prevention and control of chronic hepatitis B infection is still facing challenges.

Pathway crosstalk analysis in prostate cancer based on protein-protein network data.

Prostate cancer (PCa) is one of the major leading cause in men and no effective biomarkers or therapy have been approved for it to date. This study aimed to explore the molecular mechanisms and identify the potential molecular biomarkers of PCa. The microarray profile GSE38241 including 18 prostate cancer metastasis and 21 normal prostate samples was retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified by Limma. DEGs functions were investigated by Gene Ontology (GO) and pathway enrichment analysis. Moreover, protein-protein interaction (PPI) network of DEGs was constructed, followed by functional analysis of modules. Additionally, pathway crosstalk network was constructed by integrating PPI network and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Totally, 334 up - and 703 down-regulated DEGs were identified. The functions of up-regulated DEGs were significantly enriched in GO terms of cell cycle phase and cell cycle process. While down-regulated DEGs mainly participated in actin filament-based process. Among these pathways in the pathway crosstalk network, T cell receptor signaling pathway, chemokine signaling pathways, endometrial cancer and glioma were found to play critical roles during PC progression. Cell division cycle 45 (CDC45), baculoviral IAP repeat containing 5 (BIRC5) and cell division cycle associated 5 (CDCA5) may be useful markers for predicting tumor metastasis and therapeutic targets for the treatment of PCa patients. Moreover, the pathway crosstalk network provides the groundwork that targeting multiple pathways might be more effective than targeting one pathway alone.

[Optimization of the method to cultivate NK cells from abandoned white cells].

OBJECTIVE: Using abandoned white cells separated from preparation of blood products to cultivate NK cells in vitro, and to optimize the method of cultivation of allogeneic NK cells for clinical application. METHODS: Abandoned white cells separated from blood production were collected from 15 healthy donors. PBMCs were isolated from the abandoned white cells and cultured for 17 days using culture bottles as previously coated antibodies (group CD3 mAb was coated with CD3 mAb, group CD 16mAb was coated with CD16mAb, and group CD3 mAb+ CD16 mAb was coated with CD3 mAb and CD16 mAb). Flow cytometry was used to determine the ratio of CD3(-)CD56(+) cells, expression of activated cell surface receptors, and secretion of IFN-γ. The anti-tumor cytotoxicity against K562 and Raji cells was determined using LDH cytotoxicity assay and flow cytometry. RESULTS: After expansion for 17 days, the proportions of CD3(-)CD56(+) cells was (15.19±12.22)% in the group CD3 mAb, (83.63±10.63)% in the group CD16 mAb, (49.40±12.64)% in the group CD3 mAb+ CD16 mAb, and it was (16.34±10.51)% before expansion. The total number of NK cells was more than 10(9). The expression ratios of NK cell surface activated receptors NKp30 and NKp46 were significantly increased, while that of the NKG2D was not significantly changed. The NK cells after expansion showed high cytotoxicity activity against K562 cells, reaching up to(76.97±3.16)% when effector-cell-to-target-cell ratio (E∶T ratio) was 40∶1. CONCLUSIONS: NK cells can be obtained from abandoned white cells after cultivation for 17 days, with a purity up to 90% and total cell number of more than 10(9). Their activity was reinforced, the anti-tumor cytotoxicity activity was increased, and may meet the standard of clinical therapeutic application.

Novel representation of RNA secondary structure used to improve prediction algorithms.

We propose a novel representation of RNA secondary structure for a quick comparison of different structures. Secondary structure was viewed as a set of stems and each stem was represented by two values according to its position. Using this representation, we improved the comparative sequence analysis method results and the minimum free-energy model. In the comparative sequence analysis method, a novel algorithm independent of multiple sequence alignment was developed to improve performance. When dealing with a single-RNA sequence, the minimum free-energy model is improved by combining it with RNA class information. Secondary structure prediction experiments were done on tRNA and RNAse P RNA; sensitivity and specificity were both improved. Furthermore, software programs were developed for non-commercial use.

Study of scattering from a sphere with an eccentrically located spherical inclusion by generalized Lorenz-Mie theory: internal and external field distribution.

Based on the recent results in the generalized Lorenz-Mie theory, solutions for scattering problems of a sphere with an eccentrically located spherical inclusion illuminated by an arbitrary shaped electromagnetic beam in an arbitrary orientation are obtained. Particular attention is paid to the description and application of an arbitrary shaped beam in an arbitrary orientation to the scattering problem under study. The theoretical formalism is implemented in a homemade computer program written in FORTRAN. Numerical results concerning spatial distributions of both internal and external fields are displayed in different formats in order to properly display exemplifying results. More specifically, as an example, we consider the case of a focused fundamental Gaussian beam (TEM(00) mode) illuminating a glass sphere (having a real refractive index equal to 1.50) with an eccentrically located spherical water inclusion (having a real refractive index equal to 1.33). Displayed results are for various parameters of the incident electromagnetic beam (incident orientation, beam waist radius, location of the beam waist center) and of the scatterer system (location of the inclusion inside the host sphere and relative diameter of the inclusion to the host sphere).

DNA mismatch repair related gene expression as potential biomarkers to assess cadmium exposure in Arabidopsis seedlings.

In the current study, Arabidopsis seedlings were hydroponically grown on MS media containing cadmium (Cd) of 0-2.0 mg L(-1) for 60 h of treatment. Gene expression profiles were used to relate exposure to Cd with some altered biological responses and/or specific growth effects. RT-PCR analysis was used to quantitate mRNA expression for seven genes known to be involved in DNA mismatch repair (MMR) system and cell division. Results indicated that Cd concentrations of 0.25-2.0 mg L(-1) cause increased total soluble protein levels in shoots of Arabidopsis seedlings in an inverted U-shaped dose-response manner. Exposure to 0.25 and 0.5 mg L(-1) of Cd dramatically induced expression of four genes (i.e. proliferating cell nuclear antigen 2 (atPCNA 2), MutL1 homolog (atMLH1), MutS 2 homolog (atMSH2) and atMSH3) and five genes (i.e. atPCNA1,2, atMLH1 and atMSH2,7), respectively, in shoots of Arabidopsis seedlings; Exposure to 1.0 mg L(-1) of Cd significantly elevated expression of only two genes (atMSH6,7), but caused prominent inhibition in expression of three genes (atPCNA2, atMLH1 and atMSH3) in shoots of Arabidopsis seedlings. The expression alterations of the above genes were independent of any biological effects such as survival, fresh weight and chlorophyll level of shoots. However, shoots of Arabidopsis seedlings exposed to 2.0 mg L(-1) of Cd exhibited statistically prominent repression in expression of these seven genes, and showed incipient reduction of fresh weight and chlorophyll level. This research provides data concerning sensitivity of expression profiles of atMLH1, atMSH2,3,6,7 and atPCNA1,2 genes in Arabidopsis seedlings to Cd exposure, as well as the potential use of these gene expression patterns as representative molecular biomarkers indicative of Cd exposure and related biological effects.

Risk assessment of cadmium-contaminated soil on plant DNA damage using RAPD and physiological indices.

Impact assessment of contaminants in soil is an important issue in environmental quality study and remediation of contaminated land. A random amplified polymorphic DNA (RAPD) 'fingerprinting' technique was exhibited to detect genotoxin-induced DNA damage of plants from heavy metal contaminated soil. This study compared the effects occurring at molecular and population levels in barley seedlings exposed to cadmium (Cd) contamination in soil. Results indicate that reduction of root growth and increase of total soluble protein level in the root tips of barley seedlings occurred with the ascending Cd concentrations. For the RAPD analyses, nine 10-base pair (bp) random RAPD primers (decamers) with 60-70% GC content were found to produce unique polymorphic band patterns and subsequently were used to produce a total of 129 RAPD fragments of 144-2639 base pair in molecular size in the root tips of control seedlings. Results produced from nine primers indicate that the changes occurring in RAPD profiles of the root tips following Cd treatment included alterations in band intensity as well as gain or loss of bands compared with the control seedlings. New amplified fragments at molecular size from approximately 154 to 2245 bp appeared almost for 10, 20 and 40 mg L(-1) Cd with 9 primers (one-four new polymerase chain reaction, (PCR) products), and the number of missing bands enhanced with the increasing Cd concentration for nine primers. These results suggest that genomic template stability reflecting changes in RAPD profiles were significantly affected and it compared favourably with the traditional indices such as growth and soluble protein level at the above Cd concentrations. The DNA polymorphisms detected by RAPD can be applied as a suitable biomarker assay for detection of the genotoxic effects of Cd stress in soil on plants. As a tool in risk assessment the RAPD assay can be used in characterisation of Cd hazard in soil.

Clinical usefulness of magnetic resonance cisternography in patients having hemifacial spasm.

To evaluate the usefulness of MR cisternography fourteen patients that had hemifacial spasm and 20 control patients underwent MR cisternography. All the patients with hemifacial spasm had a confirmed vascular compression after surgery. MR cisternography was performed using a 1.5-tesla superconducting MR magnet in which a 3D (dimensional) heavily T2-weighted turbo spin-echo sequence was used. In 34 randomly selected individuals, we retrospectively determined whether MR cisternography images could be used to evaluate symptoms, and what the benefits of obtaining this image was. The results were correlated with the surgical findings. The sensitivity was 100% and the specificity was 94% in all patients having a hemifacial spasm. The offending vessels were the anterior inferior cerebellar artery (AICA) in six patients cases, the posterior inferior cerebellar artery (PICA) in six, both the vertebral artery and PICA in one, and the vertebral artery in one. All the images showed good resolution and contrast, and also showed the exact correlation between the facial nerve and intracranial vessels in the multiplaner image. The findings of neurovascular compression were well correlated with the surgical findings. We believe that high-resolution 3D MR cisternography is a very useful method for evaluating the neurovascular compression in patients that have hemifacial spasm.

Transforming growth factor-beta - and tumor necrosis factor-alpha -mediated induction and proteolytic activation of MMP-9 in human skin.

Both cytokines and matrix metalloproteinases (MMPs) are active during physiologic and pathologic processes such as cancer metastasis and wound repair. We have systematically studied cytokine-mediated MMP regulation. Cytokine-mediated proteinase induction and activation were initially investigated in organ-cultured human skin followed by determination of underlying cellular and molecular mechanisms using isolated skin cells. In this report we demonstrate that tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) synergistically induce pro-MMP-9 in human skin as well as isolated dermal fibroblasts and epidermal keratinocytes. Furthermore, TNF-alpha promotes proteolytic activation of pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active enzyme. This activation occurred only in skin organ culture and not by either isolated fibroblasts or keratinocyte, although the pro-MMP-9 activation could be measured in a cell-free system derived from TNF-alpha-activated skin. The cytokine-mediated induction of pro-MMP-9 in dermal fibroblasts was evident by increased mRNA. At the transcription level, we examined the cytokine-mediated transactivation of the 5'-region promoter of the human MMP-9 in dermal fibroblasts. The results demonstrated that TNF-alpha and TGF-beta could independently stimulate the 5'-flanking 670-base pair promoter. A TGF-beta-response element (-474) and an NF-kappaB-binding site (-601) were identified to be the cis-elements for TGF-beta or TNF-alpha activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the cells of human skin. These results imply roles for these cytokines in the regulation of MMP-9 in physiologic and pathologic tissue remodeling.

TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP.

Tumor necrosis factor-alpha (TNF-(alpha)) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-(alpha) are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro-inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-(alpha) stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing. To investigate this observation at the cellular and molecular levels, we examined TNF-(alpha) mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-(alpha) substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-(alpha) individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-(alpha) significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-(alpha) and collagen activate the NF-(kappa)B pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-(kappa)B is essential to induce MT1-MMP expression in fibroblasts after TNF-(alpha) exposure. First, SN50, a peptide inhibitor for NF-(kappa)B nuclear translocation, simultaneously blocked the TNF-(alpha) and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-(alpha) induced I(kappa)B to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-(kappa)B activation. Lastly, a consensus binding site for p65 NF-(kappa)B (TGGAGCTTCC) was found in the 5'-flanking region of human mt1-mmp gene. Based on these results and previous reports, we propose a model to explain TNF-(alpha) activation of MMP-2 in human skin. Activation of NF(kappa)B signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-(alpha) may affect matrix remodeling during wound healing and other physiological and pathological processes.

[Ultrastructural observation of E. coli K12 treated with antibacterial peptide CM4].

The effect of antibacterial peptide CM4 of Bombyx mori against E. coli K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrinkled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.

Conformational changes in the activation loop of the insulin receptor's kinase domain.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4-0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses > or =90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.

Phenylarsine oxide inhibits insulin activation of phosphatidylinositol 3'-kinase.

Two early events downstream of insulin receptor autophosphorylation that are necessary for activation of glucose transport in adipocytes appear to be: (1) The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) which (2) recruits and activates phosphatidylinositol 3'-kinase (PI3'-K). Phenylarsine oxide (PAO) has long been known to inhibit glucose transport, without inhibiting insulin receptor auto- or substrate phosphorylation. However, the PAO-sensitive site downstream of these early regulatory eventshas not been identified. Here we provide evidence that exposure of 3T3-L1 adipocytes to PAO inhibits PI3'-K activation, but it does not decrease either IRS-1 tyrosine-phosphorylation or the recruitment of PI3'-K to IRS-1 after insulin stimulation. PAO is also shown to inhibit PI3'-K activity in vitro. Therefore, since PI3'-K activation is essential for insulin stimulation of glucose transport, our results demonstrate that PI3'-K is a PAO-sensitive target of the insulin signaling pathway regulating glucose transport.