Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated(3). Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by Xray diffraction. We formulate a general catalytic mechanism for all retaining beta -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate

XRCC1 Arg194Trp polymorphism, risk of nonmelanoma skin cancer and extramammary Paget’s disease in a Japanese population

The X-ray repair cross-complementing groups 1 gene plays an important role in base excision repair. At least three common single nucleotide polymorphisms frequently occur in this gene (Arg399Gln, Arg194Trp and Arg280His). Recent studies reported that these polymorphisms were associated with not only risk of visceral malignancy but also that of skin cancer such as basal cell carcinoma and squamous cell carcinoma, whereas the results of previous study vary among races. In this case–control study, we investigated whether these single nucleotide polymorphisms were associated with the risk of skin cancer in a Japanese population. The study population was composed of 197 patients with skin cancer (27 actinic keratoses, 47 basal cell carcinomas, 27 squamous cell carcinomas, 29 Bowen’s diseases, 46 malignant melanomas and 21 extramammary Paget’s diseases) and 93 control subjects. We genotyped two single nucleotide polymorphisms (Arg194Trp and Arg399Gln) using polymerase chain reaction-restriction fragments length polymorphism analysis. We found a significantly increased risk for basal cell carcinoma, squamous cell carcinoma and extramammary Paget’s disease associated with Arg194Trp [adjusted odds ratio (AOR) = 2.347, 3.587, 3.741, 95 % confidence interval (CI) 1.02–5.39, 1.19–10.8, 1.15–12.2, respectively]. We also found a significantly decreased risk for basal cell carcinoma associated with Gln399Gln (AOR = 0.259, 95 % CI 0.07–0.96). Our data suggest that the Arg194Trp polymorphism could be associated with nonmelanoma skin cancer and extramammary Paget’s disease risk in a Japanese population

Chronic Exposure to Low Frequency Noise at Moderate Levels Causes Impaired Balance in Mice

We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60–70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10–20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance

Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis

Expression of CCN family of genes in human skin in vivo and alterations by solar-simulated ultraviolet irradiation

The CCN family of proteins is involved in diverse biological functions such as cell growth, adhesion, migration, angiogenesis, and regulation of extracellular matrix. We have investigated expression of CCN family genes and alternations induced by solar-simulated ultraviolet irradiation in human skin in vivo. Transcripts of all six CCN genes were expressed in human skin in vivo. CCN5 was most abundantly expressed followed by CCN2>CCN3>CCN1>CCN4>CCN6. Solar-simulated ultraviolet irradiation increased mRNA expression of CCN1 and CCN2. In contrast, mRNA levels of CCN3, CCN4, CCN5, and CCN6, were reduced. Knowledge gained from this study provides the foundation to explore the functional roles of CCN gene products in cutaneous biology and responses to solar ultraviolet irradiation

Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog’s phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5′ donor splice site of intron 50 in the CKCS breed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-016-9675-2) contains supplementary material, which is available to authorized users

Doxorubicin loaded Polymeric Nanoparticulate Delivery System to overcome drug resistance in osteosarcoma

<p>Abstract</p> <p>Background</p> <p>Drug resistance is a primary hindrance for the efficiency of chemotherapy against osteosarcoma. Although chemotherapy has improved the prognosis of osteosarcoma patients dramatically after introduction of neo-adjuvant therapy in the early 1980's, the outcome has since reached plateau at approximately 70% for 5 year survival. The remaining 30% of the patients eventually develop resistance to multiple types of chemotherapy. In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure incurred from multidrug resistant (MDR) tumor cells, we explored the possibility of loading doxorubicin onto biocompatible, lipid-modified dextran-based polymeric nanoparticles and evaluated the efficacy.</p> <p>Methods</p> <p>Doxorubicin was loaded onto a lipid-modified dextran based polymeric nano-system. The effect of various concentrations of doxorubicin alone or nanoparticle loaded doxorubicin on KHOS, KHOS_R2, U-2OS, and U-2OS_R2cells was analyzed. Effects on drug retention, immunofluorescence, Pgp expression, and induction of apoptosis were also analyzed.</p> <p>Results</p> <p>Dextran nanoparticles loaded with doxorubicin had a curative effect on multidrug resistant osteosarcoma cell lines by increasing the amount of drug accumulation in the nucleus via Pgp independent pathway. Nanoparticles loaded with doxorubicin also showed increased apoptosis in osteosarcoma cells as compared with doxorubicin alone.</p> <p>Conclusion</p> <p>Lipid-modified dextran nanoparticles loaded with doxorubicin showed pronounced anti-proliferative effects against osteosarcoma cell lines. These findings may lead to new treatment options for MDR osteosarcoma.</p

p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1

Novel strategies in tendon and ligament tissue engineering: Advanced biomaterials and regeneration motifs

Tendon and ligaments have poor healing capacity and when injured often require surgical intervention. Tissue replacement via autografts and allografts are non-ideal strategies that can lead to future problems. As an alternative, scaffold-based tissue engineering strategies are being pursued. In this review, we describe design considerations and major recent advancements of scaffolds for tendon/ligament engineering. Specifically, we outline native tendon/ligament characteristics critical for design parameters and outcome measures, and introduce synthetic and naturally-derived biomaterials used in tendon/ligament scaffolds. We will describe applications of these biomaterials in advanced tendon/ligament engineering strategies including the utility of scaffold functionalization, cyclic strain, growth factors, and interface considerations. The goal of this review is to compile and interpret the important findings of recent tendon/ligament engineering research in an effort towards the advancement of regenerative strategies