Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.

Spore-forming Bacillus strains that produce extracellular poly-␥-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.The Bacillus subtilis species complex is a tight assemblage of related species that includes the laboratory strain B. subtilis subsp. subtilis 168, which has been used as a model organism for 32). B. subtilis subsp. subtilis BEST195, the starter strain used for natto (fermented soybean food) production, is thought to have been isolated about 100 years ago. Since then, it has been distributed to major commercial manufacturers (28). Before the isolation of the starter strain, dried rice straw had been used to initiate natto fermentation; rice straw is the natural habitat of the spore-forming B. subtilis strains that facilitate soybean fermentation. Recently, the entire genome information of the BEST195 strain was published (21). Completion of this project has facilitated the creation of a genomically directed starter cultivation program for natto. Natto starter cultivation targets genes regulating the metabolic pathways of secondary metabolites that affect flavor, sensitivity to bacteriophages, spore formation, and germination.The genome structure of B. subtilis subsp. subtilis BEST195 is very similar to that of the B. subtilis laboratory strain B. subtilis subsp. subtilis 168 (15, 21, 32), but it is not possible to produce natto with the latter (21), apparently due to mutations in the genes degQ and swrA, which regulate poly-␥-glutamic acid (␥PGA) synthesis (27). In contrast, BEST195 actively produces ␥PGA via the pgs operon (1, 2, 4), which provides a sticky texture to natto. However, ␥PGA production alone is not a predictor of the ability to ferment natto, as many ␥PGA-positive strains cannot be used for natto production. These observations have spurred great interest in the genetic basis of natto fermentation ability in Bacillus. Reports on the diversity of natto-fermenting strains are limited to elucidating the genetic background required for natto fermentation. To clarify the taxonomic positions of such strains within the genus Bacillus and to increase genetic sources for cultivating starters, we screened ␥PGA-positive strains from rice straw to determine their abilities to ferment natto.The commercial natto starter strain BEST195 requires biotin for growth. It is sensitive to specific bacteriophages (10, 25)...

Objective-The response-to-tissue-injury theory is currently the favorite paradigm to investigate valve pathology. To the best of our knowledge, there are currently no in vivo valve injury models. There are few calcific aortic valve stenosis (AVS) models that develop hemodynamically significant stenosis. Here, we investigated the effect of direct mechanical injury on aortic valves in vivo and developed a novel mouse model of calcific AVS. Approach and Results-Aortic valve injury was created by inserting and moving a spring guidewire under echocardiographic guidance into the left ventricle of male C57/BL6 mice via right common carotid artery. Serial echocardiographic measurements revealed that aortic velocity was increased 1 week after injury and persistently increased until 16 weeks after injury. AVS mice showed a higher heart weight/body weight ratio and decreased left ventricular fractioning shortening 4 weeks after injury, compared with sham mice. We found remarkable proliferation of valve leaflets 4 weeks after injury. Proliferative valves showed increased production of reactive oxygen species and expression of inflammatory cytokines and osteochondrogenic factors. Alizarin red staining showed valvular calcification 12 weeks after injury. Conclusions-We report a novel calcific AVS model to support the response-to-tissue-injury theory. This model may be a valuable tool for analyzing the mechanism of AVS and assessing therapeutic options. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Aortic Valve InjuryThe surgical procedure time, including anesthesia, needed for aortic valve injury was 21.0±5.1 minutes. Seven of 132 mice died during the operation due to sudden cardiac arrest (5 mice) and bleeding (2 mice). In the sham group, 1 of 63 mice died as a result of bleeding. After aortic valve injury, 25 of 125 mice died during the next 16 weeks. Survival curves showed that 18.7% of mice with aortic valve injury died <4 weeks, whereas none in the sham-operated mice died ( Figure 2A). The causes of death after aortic valve injury were congestive heart failure with pleural effusion at autopsy (5) and unknown causes (20), most likely attributable to cardiac arrhythmia or heart failure without obvious effusion. Valve and Ventricular FunctionImmediately after injury, aortic regurgitation was not observed on 2-dimensional color Doppler and pulse-wave Doppler imaging, and aortic velocity did not increase. Mice with aortic valve injury had significantly higher aortic velocity and smaller aortic valve area compared with sham-operated mice 1 week after surgery. The elevated velocity persisted for 16 weeks without improvement. Left ventricular outflow tract velocity was not significantly increased at all time points. Left ventricular fractional shortening was significantly decreased 4 weeks after surgery, and left ventricular end-diastolic diameter was increased 8 weeks after injury (see Table). Heart weight/body weight ratio gradually increased ( Figure 2C). Additionally, real-t...

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We report the first in vivo and in vitro evidence that cardiac HMGB1 increases HSPB1 expression and attenuates cardiomyocyte apoptosis associated with doxorubicin-induced cardiomyopathy. Cardiac HMGB1 increases HSPB1 expression in cardiomyocytes in a heat shock factor 2-dependent manner.

The online-only Data Supplement is available at http://circheartfailure.ahajournals.org/lookup/suppl

BackgroundThe homologous to the E6‐AP carboxyl terminus (HECT)–type ubiquitin E3 ligase ITCH is an enzyme that plays a pivotal role in posttranslational modification by ubiquitin proteasomal protein degradation. Thioredoxin‐interacting protein (TXNIP) is a negative regulator of the thioredoxin system and an endogenous reactive oxygen species scavenger. In the present study, we focused on the functional role of ubiquitin E3 ligase ITCH and its interaction with TXNIP to elucidate the mechanism of cardiotoxicity induced by reactive oxygen species, such as doxorubicin and hydrogen peroxide.Methods and ResultsProtein interaction between TXNIP and ITCH in cardiomyocyte was confirmed by immunoprecipitation assays. Overexpression of ITCH increased proteasomal TXNIP degradation and augmented thioredoxin activity, leading to inhibition of reactive oxygen species generation, p38 MAPK, p53, and subsequent intrinsic pathway cardiomyocyte apoptosis in reactive oxygen species–induced cardiotoxicity. Conversely, knockdown of ITCH using small interfering RNA inhibited TXNIP degradation and resulted in a subsequent increase in cardiomyocyte apoptosis. Next, we generated a transgenic mouse with cardiac‐specific overexpression of ITCH, called the ITCH‐Tg mouse. The expression level of TXNIP in the myocardium in ITCH‐Tg mice was significantly lower than WT littermates. In ITCH‐Tg mice, cardiac dysfunction and remodeling were restored compared with WT littermates after doxorubicin injection and myocardial infarction surgery. Kaplan–Meier analysis revealed that ITCH‐Tg mice had a higher survival rate than WT littermates after doxorubicin injection and myocardial infarction surgery.ConclusionWe demonstrated, for the first time, that ITCH targets TXNIP for ubiquitin‐proteasome degradation in cardiomyocytes and ameliorates reactive oxygen species–induced cardiotoxicity through the thioredoxin system.

ObjectiveOral lichen planus (OLP) characterized by interface mucositis frequently shows hyper-keratinization. To clarify mechanisms of excess keratinization, we investigated key molecules for cornified cell envelope (CE).MethodsInvolucrin (IVL), loricrin (LOR), transglutaminase 1 (TGase 1) and transglutaminase 3 (TGase 3) were immunohistochemically examined in 20 specimens of OLP; five specimens of buccal mucosa served as controls. Subsequently, the data were statistically analyzed.ResultsIVL in OLP was localized in the cell membrane, in contrast to its localization in the cytoplasm in controls. No positive reaction indicative of LOR was noted in any specimens. Although the TGase 1 localization in controls was restricted to the upper three-quarters of the membrane, the localization in OLP was in both membrane and in the cytoplasm of full thickness mucosal layers. The TGase 3 localization pattern was dramatically altered from cytoplasmic to membranous in OLP.ConclusionOur data suggest that aberrant TGase 1 and TGase 3 localization and distribution are closely related to hyper-keratinization in OLP. This is the first report of ectopic transglutaminase localization in OLP.