Homologous spectrin-like repeats can mediate specific protein interaction. The underlying mechanism is poorly understood. Dystrophin contains 24 spectrin-like repeats. However, only repeats 16 and 17 (R16/17) are required for anchoring neuronal NOS (nNOS) to the sarcolemma. Through an adeno-associated virus-based in vivo binding assay, we found that membrane expression of correctly phased R16/17 was sufficient to recruit nNOS to the sarcolemma in mouse muscle. Utrophin R15/16 is homologous to dystrophin R16/17. Substitution of dystrophin R16/17 microdomains with the corresponding regions of utrophin R15/16 suggests that the nNOS binding site is located in a 10-residue fragment in dystrophin R17 α1 helix. Interestingly, swapping this microdomain back into utrophin did not convey the nNOS binding activity. To identify other structural features that are required for nNOS interaction, we replaced an individual α-helix of dystrophin R16/17 with an equivalent α-helix from another dystrophin repeat. In vitro study with yeast two-hybrid suggests that most α-helices of R16/17, except for the R17 α1 helix, were dispensable for nNOS interaction. Surprisingly, in vivo binding assay showed that α2 and α3 helices of both R16 and R17 were essential for nNOS binding in muscle. We concluded that a microdomain in the α1 helix of dystrophin R17 binds to nNOS in a way uniquely defined by two pairs of the flanking helices. Our results provide an explanation for how structurally similar spectrin-like repeats in dystrophin display selective interaction with nNOS. The results also open new therapeutic avenues to restore defective nNOS homeostasis in dystrophin-null Duchenne muscular dystrophy.Becker muscular dystrophy | BMD | DMD | gene therapy | microdystrophin S pectrin-type repeat (STR) is a common structural element in a variety of proteins, especially cytoskeletal proteins. STR is composed of 106-122 amino acids folded in a triple α-helical unit. STR exists either as a single-copy or tandem repeats. STR-containing proteins play a fundamental role in maintaining the cytoskeletal architecture and organizing protein complexes (1, 2). Dystrophin is a vital STR-containing protein in striated muscles that links the cytoskeleton with the extracellular matrix and, hence, preserves sarcolemmal integrity during muscle contraction. Besides mechanical support, dystrophin also scaffolds neuronal nitric oxide synthase (nNOS) and several other signaling proteins to the sarcolemma.Absence of dystrophin results in Duchenne muscular dystrophy (DMD), an X-linked lethal muscle disease (3). Although increased membrane fragility has been considered as a primary pathogenic mechanism of DMD, accumulated evidence suggests that the loss of sarcolemmal nNOS also contributes to the dystrophic process (4-7). A clear understanding of how nNOS is localized to the membrane may thus offer insight to our understanding of the disease and open new therapeutic avenues.Dystrophin has four functional domains including the N-terminal (NT), middle rod, cysteine-rich (CR)...
Alkaline condition (especially pH 10) has been demonstrated to be a promising method for short-chain fatty acid (SCFA) production from waste activated sludge anaerobic fermentation, because it can effectively inhibit the activities of methanogens. However, due to the limit of sludge solubilization rate, long fermentation time is required but SCFA yield is still limited. This paper reports a new pretreatment method for alkaline fermentation, i.e., using free nitrous acid (FNA) to pretreat sludge for 2 d, by which the fermentation time is remarkably shortened and meanwhile the SCFA production is significantly enhanced. Experimental results showed the highest SCFA production of 370.1 mg COD/g VSS (volatile suspended solids) was achieved at 1.54 mg FNA/L pretreatment integration with 2 d of pH 10 fermentation, which was 4.7- and 1.5-fold of that in the blank (uncontrolled) and sole pH 10 systems, respectively. The total time of this integration system was only 4 d, whereas the corresponding time was 15 d in the blank and 8 d in the sole pH 10 systems. The mechanism study showed that compared with pH 10, FNA pretreatment accelerated disruption of both extracellular polymeric substances and cell envelope. After FNA pretreatment, pH 10 treatment (1 d) caused 38.0% higher substrate solubilization than the sole FNA, which indicated that FNA integration with pH 10 could cause positive synergy on sludge solubilization. It was also observed that this integration method benefited hydrolysis and acidification processes. Therefore, more SCFA was produced, but less fermentation time was required in the integrated system.
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease characterized by inflammatory cell activation and the release of inflammatory mediators. Interleukin-33 (IL-33) plays a critical role in various inflammatory and immunological pathologies, but evidence for its role in COPD is lacking. This study aimed to investigate the expression of IL-33 in COPD and to determine whether IL-33 participates in the initiation and progression of COPD. Levels of serum IL-33 and its receptors were measured by ELISA, and serum levels of IL-33, ST2, and IL-1 receptor accessory protein were elevated in patients with COPD compared with control subjects. Flow cytometry analysis further demonstrated an increase in peripheral blood lymphocytes (PBLs) expressing IL-33 in patients with COPD. Immunofluorescence analysis revealed that the main cellular source of IL-33 in lung tissue was human bronchial epithelial cells (HBEs). Cigarette smoke extract and lipopolysaccharide could enhance the ability of PBLs and HBEs to express IL-33. Furthermore, PBLs from patients with COPD showed greater IL-33 release in response to the stimulus. Collectively, these findings suggest that IL-33 expression levels are increased in COPD and related to airway and systemic inflammation. Therefore, IL-33 might contribute to the pathogenesis and progression of this disease.
Background
A multitude of epidemiological studies have shown that ambient fine particulate matter 2.5 (diameter < 2.5um; PM
2.5
) was associated with increased morbidity and mortality of chronic obstructive pulmonary disease (COPD). However, the underlying associated mechanisms have not yet been elucidated. We conducted this study to investigate the role of PM
2.5
in the development of COPD and associated mechanisms.
Methods
We firstly conducted a cross-sectional study in Chinese han population to observe PM
2.5
effects on COPD morbidity. Then, in vitro, we incubated human bronchial epithelial cells to different concentrations of PM
2.5
for 24 h. The expression levels of IL-6 and IL-8 were detected by ELISA and the levels of MMPs, TGF-β1, fibronectin and collagen was determined by immunoblotting. In vivo, we subjected C57BL/6 mice to chronic prolonged exposure to PM
2.5
for 48 weeks to study the influence of PM
2.5
exposure on lung function, pulmonary structure and inflammation.
Results
We found that the effect of PM
2.5
on COPD morbidity was associated with its levels and that PM
2.5
and cigarette smoke could have a synergistic impact on COPD development and progression. Both vitro and vivo studies demonstrated that PM
2.5
exposure could induce pulmonary inflammation, decrease lung function, and cause emphysematous changes. Furthermore, PM
2.5
could markedly aggravated cigarette smoke-induced changes.
Conclusions
In short, we found that prolonged chronic exposure to PM
2.5
resulted in decreased lung function, emphysematous lesions and airway inflammation. Most importantly, long-term PM
2.5
exposure exacerbateed cigarette smoke-induced changes in COPD.
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