Background-The ubiquitin proteasome system maintains a dynamic equilibrium of proteins and prevents accumulation of damaged and misfolded proteins, yet its role in human cardiac dysfunction is not well understood. The present study evaluated ubiquitin proteasome system function in human heart failure and hypertrophic cardiomyopathy (HCM). Methods and Results-Proteasome function was studied in human nonfailing donor hearts, explanted failing hearts, and myectomy samples from patients with HCM. Proteasome proteolytic activities were markedly reduced in failing and HCM hearts compared with nonfailing hearts (PϽ0.01). This activity was partially restored after mechanical unloading in failing hearts (PϽ0.01) and was significantly lower in HCM hearts with pathogenic sarcomere mutations than in those lacking these mutations (PϽ0.05). There were no changes in the protein content of ubiquitin proteasome system subunits (ie, 11S, 20S, and 19S) or in active-site labeling of the 20S proteolytic subunit ␤-5 among groups to explain decreased ubiquitin proteasome system activity in HCM and failing hearts. Examination of protein oxidation revealed that total protein carbonyls, 4-hydroxynonenylated proteins, and oxidative modification to 19S ATPase subunit Rpt 5 were increased in failing compared with nonfailing hearts. Conclusions-Proteasome activity in HCM and failing human hearts is impaired in the absence of changes in proteasome protein content or availability of proteolytic active sites. These data provide strong evidence that posttranslational modifications to the proteasome may account for defective protein degradation in human cardiomyopathies. Key Words: apoptosis Ⅲ cardiomyopathy Ⅲ heart failure Ⅲ hypertrophy Ⅲ myocardium Ⅲ proteins P roteolytic degradation is critical for maintaining a dynamic equilibrium of proteins and destroying damaged or misfolded proteins. As the major pathway for intracellular protein degradation, the ubiquitin proteasome system (UPS) requires precise control to sustain most biological processes. Regulation of proteasome function may occur by altered proteasome composition (ie, association of the 20S proteolytic core with different regulatory complexes such as the 19S or 11S) 1,2 or by posttranslational modifications (ie, phosphorylation, oxidation) that affect proteasome assembly, stability, and activity. [3][4][5][6] Proteasome regulation thus has the potential to provide highly dynamic responses to cellular signals and stresses. Clinical Perspective on p 1004Despite recognition that UPS function is dysregulated in many diseases, 7-11 the importance of UPS function in cardiac diseases is only beginning to gain attention. Desmin-related cardiomyopathy mouse models provide compelling data for UPS dysfunction, in which cardiomyocyte accumulation of protein aggregates is postulated to inhibit proteasome function by restricting entry of ubiquitinated proteins into the proteasome. 12,13 Another notable example is acute cardiac ischemia, in which proteasome inhibition is thought to occur as a resul...

Plants respond to insect feeding with a number of defense mechanisms. Using maize genotypes derived from Antiquan germ plasm that are resistant to Lepidoptera, we have demonstrated that a unique 33-kD cysteine proteinase accumulates in the whorl in response to larval feeding. The abundance of the proteinase increased dramatically at the site of larval feeding after 1 hr of infestation and continued to accumulate for as long as 7 days. The 33-kD cysteine proteinase was most abundant in the yellow-green portion of the whorl-the normal site of larval feeding and the tissue that has the greatest inhibitory effect on larval growth in bioassays. The proteinase was expressed in response to wounding and was found in senescent leaves. It may be a marker of programmed cell death. The gene coding for the proteinase, mir1 , has been transformed into Black Mexican Sweet callus. When larvae were reared on callus expressing the proteinase, their growth was inhibited ‫ف‬ 60 to 80%. The expression of a cysteine proteinase, instead of a cysteine proteinase inhibitor, may be a novel insect defense mechanism in plants. INTRODUCTIONOver the past 25 years, maize inbreds resistant to feeding by larvae of numerous lepidopteran species have been developed from Antiguan germ plasm (Williams and Davis, 1982;Williams et al., 1990a). Inbreds derived from this germ plasm (Mp704 and Mp708) are resistant to feeding by fall armyworm ( Spodoptera frugiperda ), southwestern corn borer ( Diatraea grandiosella), European corn borer ( Ostinia nubilalis ), sugarcane borer ( D. saccharalis), tobacco budworm ( Heliothis virescens ), corn earworm ( Helicoverpa zea ), and other Lepidoptera. Fall armyworm larvae feed extensively on whorl leaf tissue, often resulting in crop losses. Genetic and quantitative trait loci analyses indicate that resistance to these Lepidoptera is a quantitative trait regulated by several genes (Williams et al., 1989;Khairallah et al., 1998). Traits such as high hemicellulose content, low protein content, and leaf toughness appear to be correlated with reduced larval growth (Williams et al., 1998). No studies have indicated conclusively that secondary products contribute to the resistance, but two-dimensional gel electrophoresis has indicated that the presence of 36-and 21-kD proteins in the whorl may be predictive of resistance (Callahan et al., 1992).Bioassays in which fall armyworm larvae are reared on lyophilized whorl tissues indicate that larvae reared on resistant material weigh ‫ف‬ 50% less than those reared on susceptible material (Williams et al., 1990b). Larvae reared on lyophilized whorl tissue from resistant genotypes are smaller, grow more slowly, and pupate later than those reared on similar material from susceptible genotypes (Chang et al., 2000). The major effect of this germplasm is to slow larval growth and development and to increase the amount of time larvae are vulnerable to predators and parasites.The same phenotype, a 50% reduction in larval growth, is apparent when larvae are reared on nonfriable callus ...

From a molecular perspective, DFUs exhibit a chronic inflammatory predisposition. In addition, increased local hypoxic conditions and impaired cellular responses to hypoxia are pathogenic factors that contribute to delayed wound healing. Finally, recent evidence suggests a role for epigenetic alterations, including microRNAs, in delayed DFU healing due to the complex interplay between genes and the environment. In this regard, notable progress has been made in the molecular and genetic understanding of DFU formation. However, further studies are needed to translate preclinical investigations into clinical therapies.

Background Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain-of-function for missense mutations or loss-of-function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. Methods and Results Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ~1:5, in contrast to ~1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by ~9 fold in HCM samples with MYBPC3 mutations compared to control hearts and to HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples and no truncated proteins were detected. By absolute quantification of abundance (AQUA) with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild-type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30–84%. Conclusions These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable, or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes.

A series of laboratory assays were performed to compare the relative impact of commercial and experimental cultivars of cotton, Gossypium hirsutum (L.), expressing zero, one, or two insecticidal proteins of Bacillus thuringiensis Berliner, on several lepidopteran pests. Assays in which larvae were fed fresh plant tissue indicated that dual-toxin B. thuringiensis (Bt) cultivars, expressing both Cry1Ac and Cry2Ab endotoxins of B. thuringiensis, were more toxic to bollworms, Helicoverpa zea (Boddie), fall armyworms, Spodoptera frugiperda (J. E. Smith), and beet armyworms, Spodoptera exigua (Hubner), than single-toxin cultivars expressing Cry1Ac. Assays in which lyophilized plant tissue was incorporated into artificial diet also indicated improved activity of the dual-toxin Bt cultivar compared with single-toxin plants. Both bollworm and tobacco budworm, Heliothis virescens (F.), growth was reduced by Bt cotton, particularly the dual-toxin cultivar. Although assays with lyophilized tissues were done using largely sublethal doses, bollworm survival was reduced by the dual-toxin cultivar. It appears that this newly developed Bt cotton expressing two toxins will be more effective and have a wider range of activity on these lepidopteran pests.

Objective Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that are critical for proper healing, however, in diabetes, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood-monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown. Approach and Results Here, we characterized the kinetics and function of Ly6CHi[Lin− (CD3−CD19−NK1.1−Ter-119−)Ly6G−CD11b+] and Ly6CLo[Lin− (CD3−CD19−NK1.1−Ter-119−)Ly6G−CD11b+] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato-labeled Ly6CHi monocyte/macrophages, we show Ly6CHi cells transition to a Ly6CLo- phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6CHi cells that fail transition to Ly6CLo. The second wave of Ly6CHi cells in diabetic wounds corresponded to a spike in MCP-1 and selective administration of anti-MCP-1 reversed the second Ly6CHi influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq-based transcriptome profiling was performed on flow-sorted Ly6CHi[Lin−Ly6G−CD11b+] and Ly6CLo[Lin−Ly6G−CD11b+] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6CHi cells demonstrated differences in pro-inflammatory and pro-fibrotic genes compared to controls. Conclusions Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11b+Ly6CHi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.

Angiotensin II (Ang II) promotes development of ascending aortic aneurysms (AAs), but progression of this pathology is undefined. We evaluated factors potentially involved in progression, and determined the temporal sequence of tissue changes during development of Ang II-induced ascending AAs. Ang II infusion into C57BL/6J mice promoted rapid expansion of the ascending aorta, with significant increases within 5 days, as determined by both in vivo ultrasonography and ex vivo sequential acquisition of tissues. Rates of expansion were not significantly different in LDL receptor-null mice fed a saturated fat-enriched diet, demonstrating a lack of effect of hypercholesterolemia. Augmenting systolic blood pressure with norepinephrine infusion had no significant effect on ascending aortic expansion. Pathological changes observed within 5 days of Ang II infusion included increased medial thickness and intramural hemorrhage characterized by erythrocyte extravasation in outer lamellar layers of the media. Intramedial hemorrhage was not observed after prolonged Ang II infusion, although partial medial disruption was present. Elastin fragmentation and transmural medial breaks of the ascending aorta were observed with continued Ang II infusion, which were restricted to anterior aspects. CD45(+) cells accumulated in adventitia but were minimal in media. Similar pathology was observed in tissues obtained from patients with ascending AAs. In conclusion, Ang II promotes ascending AAs through region-specific changes that are independent of hypercholesterolemia or systolic blood pressure.

Impaired wound healing is a major secondary complication of type 2 diabetes that often results in limb loss and disability. Normal tissue repair progresses through discrete phases including hemostasis, inflammation, proliferation, and remodeling. In diabetes, normal progression through these phases is impaired resulting in a sustained inflammatory state and dysfunctional epithelialization in the wound. Due to their plasticity, macrophages play a critical role in the transition from the inflammation phase to the proliferation phase. Diabetes disrupts macrophage function by impairing monocyte recruitment to the wound, reducing phagocytosis, and prohibiting the transition of inflammatory macrophages to an anti-inflammatory state. Diabetes also impedes keratinocyte and fibroblast function during the later phases resulting in impaired epithelialization of the wound. Several recent studies suggest that altered epigenetic regulation of both immune and structural cells in wounds may influence cell phenotypes and healing, particularly in pathologic states, such as diabetes. Specifically, it has been shown that macrophage plasticity during wound repair is partly regulated epigenetically and that diabetes alters this epigenetic regulation and contributes to a sustained inflammatory state. Epigenetic regulation is also known to regulate keratinocyte and fibroblast function during wound repair. In this review, we provide an introduction to the epigenetic mechanisms that regulate tissue repair and highlight recent findings that demonstrate how epigenetic events are altered during the course of diabetic wound healing.