BBA - General Subjects (v.1860, #10)

Targeted delivery of quercetin loaded mesoporous silica nanoparticles to the breast cancer cells by Abhijit Sarkar; Shatadal Ghosh; Sayantani Chowdhury; Bhawna Pandey; Parames C. Sil (2065-2075).
Mesoporous silica nanoparticles (MSNs) have been promising vehicles for drug delivery. Quercetin (Q), a natural flavonoid, has been reported to have many useful effects. However, poor water solubility as well as less bioavailability has confined its use as a suitable anti-cancer drug. Therefore, profound approach is required to overcome these drawbacks.We have synthesized folic acid (FA) armed mesoporous silica nanoparticles (MSN-FA-Q) loaded with quercetin and then characterized it by DLS, SEM, TEM and FTIR. MTT, confocal microscopy, flow cytometry, scratch assay and immunoblotting were employed to assess the cell viability, cellular uptake, cell cycle arrest, apoptosis, wound healing and the expression levels of different signalling molecules in breast adenocarcinoma cells. Nanoparticle distribution was investigated by using ex vivo optical imaging and CAM assay was employed to assess tumor regression.MSN-FA-Q facilitates higher cellular uptake and allows more drug bioavailability to the breast cancer cells with over-expressed folate receptors. Our experimental results suggest that the newly synthesized MSN-FA-Q nanostructure caused cell cycle arrest and apoptosis in breast cancer cells through the regulation of Akt & Bax signalling pathways. Besides, we also observed that MSN-FA-Q has a concurrent anti-migratory role as well.This uniquely engineered quercetin loaded mesoporous silica nanoparticle ensures a targeted delivery with enhanced bioavailability.Effective targeted therapeutic strategy against breast cancer cells.
Keywords: Nanoparticle; Breast cancer; Folic acid; Biodistribution; Ex-ovo; Apoptosis;

Tideglusib, a chemical inhibitor of GSK3β, attenuates hypoxic-ischemic brain injury in neonatal mice by Haitao Wang; Sammen Huang; Kuipo Yan; Xiaoyan Fang; Ahmed Abussaud; Ana Martinez; Hong-Shuo Sun; Zhong-Ping Feng (2076-2085).
Hypoxia-ischemia is an important cause of brain injury and neurological morbidity in the newborn infants. The activity of glycogen synthase kinase-3β (GSK-3β) is up-regulated following neonatal stroke. Tideglusib is a GSK-3β inhibitor which has neuroprotective effects against neurodegenerative diseases in clinical trials. However, the effect of tideglusib on hypoxic-ischemic (HI) brain injury in neonates is still unknown.Postnatal day 7 (P7) mouse pups subjected to unilateral common carotid artery ligation followed by 1 h of hypoxia or sham surgery was performed. HI animals were administered tideglusib (5 mg/kg) or vehicle intraperitoneally 20 min prior to the onset of ischemia. The brain infarct volume and whole brain images, were used in conjunction with Nissl staining to evaluate the protective effects of tideglusib. Protein levels of glial fibrillary acidic protein (GFAP), Notch1, cleaved caspase-3/9, phosphorylated signal transducer and activator of transcription 3 (STAT3), GSK-3β and protein kinase B (Akt) were detected to identify potentially involved molecules.Tideglusib significantly reduced cerebral infarct volume at both 24 h and 7 days after HI injury. Tideglusib also increased phosphorylated GSK-3β(Ser9) and Akt(Ser473), and reduced the expression of GFAP and p-STAT3(Tyr705). In addition, pretreatment with tideglusib also enhanced the protein level of Notch1. Moreover, tideglusib reduced the cleavage of pro-apoptotic signal caspase proteins, including caspase 3 and caspase 9 following HI.These results indicate that tideglusib shows neuroprotection against hypoxic-ischemic brain injury in neonatal mice.Tideglusib is a potential compound for the prevention or treatment of hypoxic-ischemic brain injury in neonates.
Keywords: Tideglusib; GSK-3β; Neonatal hypoxia–ischemia; Apoptosis; Neuroprotection;

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters by Olga N. Koroleva; Evgeniy V. Dubrovin; Igor V. Yaminsky; Valeriy L. Drutsa (2086-2096).
Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood.The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM).In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance.The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one.The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.Display Omitted
Keywords: Convergent transcription; Transcription interference; E. coli RNA polymerase; DNA structure; Open promoter complexes; Atomic force microscopy;

Alteramide B is a microtubule antagonist of inhibiting Candida albicans by Yanjiao Ding; Yaoyao Li; Zhenyu Li; Juanli Zhang; Chunhua Lu; Haoxin Wang; Yuemao Shen; Liangcheng Du (2097-2106).
Alteramide B (ATB), isolated from Lysobacter enzymogenes C3, was a new polycyclic tetramate macrolactam (PTM). ATB exhibited potent inhibitory activity against several yeasts, particularly Candida albicans SC5314, but its antifungal mechanism is unknown.The structure of ATB was established by extensive spectroscopic analyses, including high-resolution mass spectrometry, 1D- and 2D-NMR, and CD spectra. Flow cytometry, fluorescence microscope, transmission electron microscope, molecular modeling, overexpression and site-directed mutation studies were employed to delineate the anti-Candida molecular mechanism of ATB.ATB induced apoptosis in C. albicans through inducing reactive oxygen species (ROS) production by disrupting microtubules. Molecular dynamics studies revealed the binding patterns of ATB to the β-tubulin subunit. Overexpression of the wild type and site-directed mutants of the β-tubulin gene (TUBB) changed the sensitivity of C. albicans to ATB, confirming the binding of ATB to β-tubulin, and indicating that the binding sites are L215, L217, L273, L274 and R282. In vivo, ATB significantly improved the survival of the candidiasis mice and reduced fungal burden.The molecular mechanism underlying the ATB-induced apoptosis in C. albicans is through inhibiting tubulin polymerization that leads to cell cycle arrest at the G2/M phase. The identification of ATB and the study of its activity provide novel mechanistic insights into the mode of action of PTMs against the human pathogen.This study shows that ATB is a new microtubule inhibitor and a promising anti-Candida lead compound. The results also support β-tubulin as a potential target for anti-Candida drug discovery.ATB, a new polycyclic tetramate macrolactam (PTM) compound, was isolated and shown to have potent activity against Candida albicans SC5314 in vitro and in vivo. ATB induced apoptosis of C. albicans through inducing the production of reactive oxygen species (ROS) by disrupting microtubules.Display Omitted
Keywords: Alteramide B; Candida albicans SC5314; Reactive oxygen species; Apoptosis; β-Tubulin;

Polyphenol compounds and PKC signaling by Joydip Das; Rashmi Ramani; M. Olufemi Suraju (2107-2121).
Naturally occurring polyphenols found in food sources provide huge health benefits. Several polyphenolic compounds are implicated in the prevention of disease states, such as cancer. One of the mechanisms by which polyphenols exert their biological actions is by interfering in the protein kinase C (PKC) signaling pathways. PKC belongs to a superfamily of serine-threonine kinase and are primarily involved in phosphorylation of target proteins controlling activation and inhibition of many cellular processes directly or indirectly.Despite the availability of substantial literature data on polyphenols' regulation of PKC, no comprehensive review article is currently available on this subject. This article reviews PKC-polyphenol interactions and its relevance to various disease states. In particular, salient features of polyphenols, PKC, interactions of naturally occurring polyphenols with PKC, and future perspective of research on this subject are discussed.Some polyphenols exert their antioxidant properties by regulating the transcription of the antioxidant enzyme genes through PKC signaling. Regulation of PKC by polyphenols is isoform dependent. The activation or inhibition of PKC by polyphenols has been found to be dependent on the presence of membrane, Ca2 + ion, cofactors, cell and tissue types etc. Two polyphenols, curcumin and resveratrol are in clinical trials for the treatment of colon cancer.The fact that 74% of the cancer drugs are derived from natural sources, naturally occurring polyphenols or its simple analogs with improved bioavailability may have the potential to be cancer drugs in the future.
Keywords: Polyphenol; Protein kinase C; Signal transduction; Cancer; Antioxidant;

Organization of the native ribosome–translocon complex at the mammalian endoplasmic reticulum membrane by Stefan Pfeffer; Johanna Dudek; Richard Zimmermann; Friedrich Förster (2122-2129).
In eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex.The aim of this review is to summarize the current structural knowledge about protein translocon components in mammals.Various structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation.The protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.Display Omitted
Keywords: Ribosome; Endoplasmic reticulum; Protein translocon; Membrane protein biogenesis; Structural biology;

Lung cancer is the leading cause of cancer-related death worldwide. In addition to surgical resection, which is considered first-line treatment at early stages of the disease, chemotherapy and radiation are widely used when the disease is advanced. Of multiple responses that may occur in the tumor cells in response to cancer therapy, the functional importance of autophagy remains equivocal; this is likely to restrict current efforts to sensitize this malignancy to chemotherapy and/or radiation by pharmacological interference with the autophagic response.In this review, we attempt to summarize the current state of knowledge based on studies that evaluated the function of autophagy in non-small cell lung cancer (NSCLC) cells in response to radiation and the most commonly used chemotherapeutic agents.In addition to the expected prosurvival function of autophagy, where autophagy inhibition enhances the response to therapy, autophagy appears also to have a “non-cytoprotective” function, where autophagy blockade does not affect cell viability, clonogenicity or tumor volume in response to therapy. In other cases, autophagy may actually mediate drug action via expression of its cytotoxic function.These observations emphasize the complexity of autophagy function when examined in different tumor cell lines and in response to different chemotherapeutic agents. A more in-depth understanding of the conditions that promote the unique functions of autophagy is required in order to translate preclinical findings of autophagy inhibition to the clinic for the purpose of improving patient response to chemotherapy and radiation.
Keywords: Autophagy; Non-Small cell lung cancer; Chemotherapy; Radiation; Sensitization; Apoptosis; Non-cytoprotective;

Tat PTD-Endostatin-RGD: A novel protein with anti-angiogenesis effect in retina via eye drops by Yan Li; Lian Li; Zhiwei Li; Juzheng Sheng; Xinke Zhang; Danyang Feng; Xu Zhang; Fengxin Yin; Aijun Wang; Fengshan Wang (2137-2147).
Diabetic retinopathy is a leading cause of blindness. The objective was to design a novel fusion protein, Tat PTD-Endostatin-RGD, to treat retinal neovascularization via eye drops instead of traditional intravitreal injection trepapeutical methods.The anti-angiogenesis ability was evaluated in vitro by chick embryo chorioallantoic membrane assay, wound healing assay and tube formation assay. Corneal barrier and blood-retina barrier were constructed in vitro to investigate the penetration ability of Tat PTD-Endostatin-RGD. Western blot was used to detect the integrin αvβ3 expression level in rat retina microvascular endothelial cells which was stimulated by S-nitroso-N-acetylpenicillamine. The binding affinity of Tat PTD-Endostatin-RGD to integrin αvβ3 was investigated by evaluating the penetration ability on blood-retina barriers treated with S-nitroso-N-acetylpenicillamine. The pharmacodynamics and efficacy analysis were further carried out in the oxygen-induced retinopathy model in vivo. In addition, the pharmacokinetic profile via eye drops was studied on a C57BL/6 mice model.Tat PTD-Endostatin-RGD showed high anti-angiogenesis activity and high ability to penetrate these two barriers in vitro. The Western blot results indicated S-nitroso-N-acetylpenicillamine upregulated the expression level of integrin αvβ3 in a dose-dependent manner. Tat PTD-Endostatin-RGD showed a high affinity to rat retina microvascular endothelial cells treated with S-nitroso-N-acetylpenicillamine. The results showed that Tat PTD-Endostatin-RGD could inhibit abnormal angiogenesis in retina via eye drops.Tat PTD-Endostatin-RGD showed high penetration ability through ocular barriers, bound specifically to integrin αvβ3 and effectively inhibited the abnormal angiogenesis.Tat PTD-Endostatin-RGD represents a potent novel drug applied via eye drops for fundus oculi neovascularization diseases.Display Omitted
Keywords: Neovascularization; Oxygen-induced retinopathy; Corneal barriers; Blood-retina barrier;

Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice by Jin-Hong Chang; Ilham Putra; Yu-hui Huang; Michael Chang; Kyuyeon Han; Wei Zhong; Xinbo Gao; Shuangyong Wang; Jennifer Dugas-Ford; Tara Nguyen; Young-Kwon Hong; Dimitri T. Azar (2148-2156).
Immunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo.The eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea + bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated.Neither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal + bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal + bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9 ± 0.8%, 7.14 ± 2.4%, 2.29 ± 1%, and 15.05 ± 2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45 ± 1.51%, 4.85 ± 0.95%, 2.95 ± 1.27%, and 4.15 ± 3.85%, respectively.Substantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity.Our study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.
Keywords: Cornea; Bulbar conjunctiva; Corneal neovascularization (NV); Hemangiogenesis (HA); Lymphangiogenesis (LA); In vivo imaging;

Does chemistry govern biology or it is the other way around - that is a broad connotation of the question that this study attempted to answer.Comparison was made between the solubility and osteoclastic resorbability of four fundamentally different monophasic calcium phosphate (CP) powders with monodisperse particle size distributions: alkaline hydroxyapatite (HAP), acidic monetite (DCP), β-calcium pyrophosphate (CPP), and amorphous CP (ACP). Results With the exception of CPP, the difference in solubility between different CP phases became neither mitigated nor reversed, but augmented in the resorptive osteoclastic milieu. Thus, DCP, a phase with the highest solubility, was also resorbed more intensely than any other CP phase, whereas HAP, a phase with the lowest solubility, was resorbed least. CPP becomes retained inside the cells for the longest period of time, indicating hindered digestion of only this particular type of CP. Osteoclastogenesis was mildly hindered in the presence of HAP, ACP and DCP, but not in the presence of CPP. The most viable CP powder with respect to the mitochondrial succinic dehydrogenase activity was the one present in natural biological bone tissues: HAP.Chemistry in this case does have a direct effect on biology. Biology neither overrides nor reverses the chemical propensities of inorganics with which it interacts, but rather augments and takes a direct advantage of them.These findings set the fundamental basis for designing the chemical makeup of CP and other biosoluble components of tissue engineering constructs for their most optimal resorption and tissue regeneration response.Display Omitted
Keywords: Calcium phosphate; Nanoparticles; RAW264.7; Resorbability; Solubility;

Prolyl hydroxylation in elastin is not random by Christian E.H. Schmelzer; Marcus B.M. Nagel; Szymon Dziomba; Yulia Merkher; Sarit S. Sivan; Andrea Heinz (2169-2177).
This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues.Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools.It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%–24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied.The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues.The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.Display Omitted
Keywords: Mass spectrometry; Prolyl 4-hydroxylase; Hydroxyproline; Extracellular matrix; Elastic fiber; Hydroxylysine;

Epidermal growth factor receptor inhibitor cancer drug gefitinib modulates cell growth and differentiation of acute myeloid leukemia cells via histamine receptors by Manisha Yadav; Abhishek Kumar Singh; Harish Kumar; Geeta Rao; Bandana Chakravarti; Anagha Gurjar; Shalini Dogra; Sapana Kushwaha; Achchhe Lal Vishwakarma; Prem Narayan Yadav; Dipak Datta; Anil Kumar Tripathi; Naibedya Chattopadhyay; Arun Kumar Trivedi; Sabyasachi Sanyal (2178-2190).
Epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) is used for treating non-small cell lung cancer. Gefitinib also induces differentiation in acute myeloid leukemia (AML) cell lines and patient samples lacking EGFR by an unknown mechanism. Here we dissected the mechanism of gefitinib action responsible for its EGFR-independent effects.Signaling events were analyzed by homogenous time-resolved fluorescence and immunoblotting. Cellular proliferation and differentiation were assessed by ATP measurement, trypan blue exclusion, 5-bromo-2′-deoxyuridine incorporation and flow-cytometry. Gefitinib and G protein-coupled receptor (GPCR) interactions were assessed by β-arrestin recruitment, luciferase and radioligand competition assays. Role of histamine receptors (HR) in gefitinib actions were assessed by HR knockdown or pharmacological modulation. EGFR and HR interaction was assessed by co-immunoprecipitation.Gefitinib reduced cyclic AMP content in both AML and EGFR-expressing cells and induced ERK phosphorylation in AML cells. Dibutyryl-cAMP or PD98059 suppressed gefitinib-induced AML cell cytostasis and differentiation. Gefitinib bound to and modulated HRs with subtype selectivity. Pharmacological or genetic modulations of H2 and H4 HRs (H2R and H4R) not only suppressed gefitinib-induced cytostasis and differentiation of AML cells but also blocked EGFR and ERK1/2 inhibition in MDA-MB-231 cells. Moreover, in MDA-MB-231 cells gefitinib enhanced EGFR interaction with H4R that was blocked by H4R agonist 4-methyl histamine (4MH).HRs play critical roles in anti-cancer effects of gefitinib in both EGFR-deficient and EGFR-rich environments.We furnish fresh insights into gefitinib functions which may provide new molecular clues to its efficacy and safety issues.
Keywords: Cancer; Gefitinib; EGFR; Tyrosine kinase; GPCR; Histamine receptor;

T-2 toxin induces the expression of porcine CYP3A22 via the upregulation of the transcription factor, NF-Y by Xin Liu; Jikai Wen; Ruohong Chen; Tingting Zhang; Jun Jiang; Yiqun Deng (2191-2201).
T-2 toxin is one of the major pollutants in crops and feedstuffs. CYP3A22, one of hCYP3A4 homologs, detoxifies T-2 toxin in pigs. We investigated the mechanisms of expression activation of CYP3A22 under basal and induced conditions.Based on MatInspector analysis, several mutations in the CYP3A22 promoter were assayed by dual luciferase reporter to identify the function of cis elements in the region. EMSA experiments were used to assess the binding of transcription factors to the cis elements. The mRNA and protein levels of CYP3A22 and the transcription factors were measured by RT-qPCR and Western blot. The enhancement of NF-Y binding to the CYP3A22 promoter was assayed by ChIP.As predicted, two cis DNA elements in the CYP3A22 promoter, a CCAAT box and GC box, were confirmed to be crucial in the activation of CYP3A22 transcription. These two DNA motifs recruited two transcription factors, NF-Y and Sp1, which are involved in the activation of the basal transcription of CYP3A22. More interestingly, CYP3A22 expression was induced in porcine primary hepatocytes by the treatment with 0.1 μg/mL T-2 toxin. This induction of transcription by T-2 toxin was dominantly regulated by the binding of NF-Y to the CCAAT box, rather than GC box, which recruits Sp1 and functions only in the constitutive expression of CYP3A22.Our study reveals the regulatory mechanisms of both basal and inducible transactivation of CYP3A22 in pigs. In particular, we identified that the mechanism by which T-2 toxin induces CYP3A22 expression is mediated by the upregulation of NF-YA.Although porcine CYP3A22 is homologous to hCYP3A4, the regulation of basal and induced expression of CYP3A22 occurred via distinct mechanisms. This may account for the variety of CYP3A expression in animals and humans.
Keywords: CYP3A22; T-2 toxin; Sp1; NF-Y; Transcription regulation;

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin by Massimiliano Magro; Luca Fasolato; Emanuela Bonaiuto; Nadia Andrea Andreani; Davide Baratella; Vittorino Corraducci; Giovanni Miotto; Barbara Cardazzo; Fabio Vianello (2202-2210).
Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe3 + sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens.SAMNs were applied for mimicking Fe3 + ions in solution, acting as magnetically drivable probes to evaluate putative Fe3 + recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV–Vis spectroscopy and mass spectrometry.The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores.In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation.The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms.
Keywords: Maghemite nanoparticles; Iron sensing; Oprf porin; Siderophores; Membrane proteins; Bacteria;

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway by Liqiang Zhang; Wenjia Liu; Jiangdong Zhao; Xiaojie Ma; Lin Shen; Yongjie Zhang; Fang Jin; Yan Jin (2211-2219).
The balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood.Here, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100 kpa static pressure for 1 h or 12 h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells. In vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1 h relative to 12 h. In contrast, the expression ratio of RANKL/OPG was reduced at 1 h and significantly increased at 12 h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1 h.Our findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity.We describe a novel potential mechanism related to tooth movement.
Keywords: Wnt/β-catenin pathway; Mechanical stress; PDLSCs; Osteogenic differentiation; RANKL/OPG; Orthodontic tooth movement;

The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 play important links between the innate and adaptive immune systems. Due to the importance of C3d–CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to investigate C3d–CR2 interaction. Many studies have indicated C3d–CR2 interactions are ionic strength-dependent.To investigate the molecular mechanism of C3d–CR2 interaction and the origin of effects of ionic strength, molecular dynamics simulations for C3d–CR2 complex together with the energetic and structural analysis were performed.Our results revealed the increased interactions between charged protein and ions weaken C3d–CR2 association, as ionic strengths increase. Moreover, ion strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths.Our results reveal the origins of the effects of ionic strengths on C3d–CR2 interactions are due to the changes of water, ion occupancies and distributions.This study uncovers the origin of the effect of ionic strength on C3d–CR2 interaction and deepens the understanding of the molecular mechanism of their interaction, which is valuable for the design of vaccines and small molecule inhibitors.Display Omitted
Keywords: C3d; Complement; CR2; Molecular dynamics simulation; MM-GBSA;

Haem, an essential cofactor in aerobic organisms, can cause oxidative stress and impose toxic effects on tissues and organs. It can induce aggregation of proteins via dityrosine cross-linking and cause neurodegenerative diseases. Although dityrosine cross-linking in many proteins induced by haem has been reported, not all the proteins have the same effect or the efficiency of cross-linking varies, while the reason has not been clarified.The correlation of protein structure/conformation with its aggregation tendency via dityrosine induced by hematin (oxidized form of haem) in the presence of hydrogen peroxide (H2O2) was studied through reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence and circular dichroism (CD) measurements, and the mechanism was investigated by performing UV–Vis absorbance, Raman spectroscopy and low-temperature electron spin resonance (ESR) experiments.It was found that proteins in unstructured state are more readily to be cross-linked via dityrosine formation by hematin-H2O2. The unstructured protein without steric effect can coordinate with hematin to form six-coordinated protein-hematin complex, in which the produced tyrosyl radicals by H2O2 are with high tendency to dimerize to form dityrosine.Our results demonstrate that protein structure/conformation can affect its coordination state with haem, and the tendency of reaction of two tyrosyl radicals, further influencing the yield and efficiency of dityrosine cross-linking in the presence of H2O2.This research can help to deepen our understanding of the protein aggregation and inactivation mechanisms in varied sophisticated conditions, and especially give us the new insight into the toxic effects under haem stress.Display Omitted
Keywords: Protein structure; Dityrosine; Cross-linking; Hematin; Structural effect;

Interactions of GFAP with ceftriaxone and phenytoin: SRCD and molecular docking and dynamic simulation by Paolo Ruzza; Rosa Maria Vitale; Rohanah Hussain; Barbara Biondi; Pietro Amodeo; GianPietro Sechi; Giuliano Siligardi (2239-2248).
GFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld–Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood–brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine.In this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results.We found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin.SRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP.This result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases.Display Omitted
Keywords: Ceftriaxone; Glial fibrillary acidic protein; Neurodegenerative diseases; Synchrotron radiation circular dichroism spectroscopy;

Yeast trehalases: Two enzymes, one catalytic mission by Sergi Maicas; José P. Guirao-Abad; Juan-Carlos Argüelles (2249-2254).
Trehalose is a non-reducing disaccharide highly conserved throughout evolution. In yeasts, trehalose hydrolysis is confined to the enzyme trehalase, an α-glucosidase specific for trehalose as sole substrate. Two kinds of trehalase activity exist in yeasts: neutral and acid enzymes.This review makes a comparative survey of the main biochemical and genetic parameters, regulatory systems, tridimensional structure and catalytic mechanism of the two yeast trehalases.The yeast neutral and acid trehalases display sharp differences in biochemical features (optimum pH, Mr or amino acid sequence) physiological roles, subcellular location (cytosol vs vacuoles or cell wall) and regulatory control (phosphorylation vs catabolite repression). However, their identical specificity for trehalose is based on the presence of an (α/α)6 toroid folding structure in the active centre and a catalytic mechanism of anomeric inversion.This review expands our knowledge of the homology, functional features and catalytic mechanisms of α-glucosidases in yeasts. It provides a further analysis of the correlation between structures and predicted biological roles of macromolecules.
Keywords: Yeasts; Trehalase; Trehalose; Neutral and acid enzymes; Anomeric inversion;

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells by Marisa M. Fernández; Fátima Ferragut; Víctor M. Cárdenas Delgado; Candelaria Bracalente; Alicia I. Bravo; Alejandro J. Cagnoni; Myriam Nuñez; Luciano G. Morosi; Héctor R. Quinta; María V. Espelt; María F. Troncoso; Carlota Wolfenstein-Todel; Karina V. Mariño; Emilio L. Malchiodi; Gabriel A. Rabinovich; María T. Elola (2255-2268).
We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell–cell interactions. Gal-8 is a “tandem-repeat”-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon.We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization.We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells.Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms.A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.
Keywords: ALCAM/CD166; Breast cancer cell; Galectin-8; Physical interaction; Receptor segregation;

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease by Alexey E. Alekseev; Rita Guzun; Santiago Reyes; Christophe Pison; Uwe Schlattner; Vitaly A. Selivanov; Marta Cascante (2269-2278).
Chronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility.Myosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres.The developed reaction–diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles.ATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients.This study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.
Keywords: Myosin ATPase; Rigor tension; Intracellular compartmentation; Oxygen consumption; Diffusion coefficient;

Protein antigens are degraded by endosomal protease in antigen presentation cell. T cells recognize peptides derived from antigen proteins bound to class II major histocompatibility complex molecules. We previously reported that an increase in the conformational stability of an antigen depressed its immunogenicity. However, there is little information on antigens with differences in molecular properties such as net charges and molecular weight.Denaturation experiments against guanidine hydrochloride. The serum IgE levels to protein antigens at 35 days after the first immunization analyzed using ELISA.The Der p 2 mutations in which Ile13 is mutated to Ala (I13A) and Ala122 is mutated to Ile (A122I) were shown to have lower and higher conformational stability than the wild-type, respectively, by denaturation experiments. The amount of IgE production by the less stable I13A mutant was higher and that of the stable A122I mutant was lower than that of the wild-type.Our results suggest that the increased conformational stability of Der p 2 depressed the IgE production in mice.These findings should provide a milestone for the engineering of allergen vaccines.
Keywords: ADAs (anti-drug antibodies); Allergen; Conformational stability; Der p 2; Immunogenicity;

Galectin-related protein: An integral member of the network of chicken galectins 1. From strong sequence conservation of the gene confined to vertebrates to biochemical characteristics of the chicken protein and its crystal structure by Gabriel García Caballero; Andrea Flores-Ibarra; Malwina Michalak; Nailya Khasbiullina; Nicolai V. Bovin; Sabine André; Joachim C. Manning; Sabine Vértesy; Federico M. Ruiz; Herbert Kaltner; Jürgen Kopitz; Antonio Romero; Hans-Joachim Gabius (2285-2297).
Endogenous lectins are multifunctional effectors in cell physiology. Adding the sixth member of the galectin family in chicken, a model organism for systematic profiling of these adhesion/growth-regulatory proteins, is a step toward comprehensive network monitoring.Database mining and computational data processing are applied for gene detection, chromosomal location and sequence alignments. Cloning, recombinant production and fusion-protein technology gain access to the protein, mass spectrometry and gel electrophoresis/filtration provide analytical data. Haemagglutination, glycan microarray and cell assays assess binding capacity, and crystallography of a shortened variant (also analyzed by ultracentrifugation and small angle X-ray scattering) determines its structure.The gene for the galectin-related protein (GRP) is present exclusively in vertebrates with high-level sequence conservation and similar chromosomal positioning. The chicken protein is monomeric and has lost the canonical galectin property of binding lactose. The crystal structure of the variant without the 36-amino-acid extension at the start provides explanations for this lack of binding.Chicken GRP is special within this family of six proteins by being unable to bind lactose. The documented high degree of sequence conservation among vertebrate orthologues confers the status of a model for delineating an assumedly shared functionality to this GRP.Biochemical characterization of a product of a gene under strong positive selection is a prerequisite for functional characterization. It is also essential for network monitoring by adding a new member to this lectin family.Display Omitted
Keywords: Adhesion; Crystallography; Lectin; Phylogenesis; Proliferation;

Galectin-related protein: An integral member of the network of chicken galectins by Herbert Kaltner; Gabriel García Caballero; Fred Sinowatz; Sebastian Schmidt; Joachim C. Manning; Sabine André; Hans-Joachim Gabius (2298-2312).
Galectin-related protein (GRP), present in vertebrates, is special within this family of adhesion/growth-regulatory proteins due to its strong positive selection and loss of canonical lectin activity.RT-PCR and Western blotting together with flow cytofluorimetry and immunocyto- and histochemistry monitor expression and localization of chicken GRP. The promoter sequence of the GRP gene is processed computationally to detect putative sites for binding transcription factors. The labeled protein is applied as probe to detect binding sites on cells and in sections, along with glycocompounds to test inhibition of the association.Expression of GRP in chicken is limited to bursa of Fabricius, immunohistochemically found in B cells, also in bursal epithelium and vessels. Presence in B cells is shared with only one canonical galectin, i.e. CG-8. Binding to a chicken lymphoma line was specific and saturable, not affected by lactose but completely blocked by heparin, as also seen in sections.Expression monitoring initiated for GRP reveals a distinct site of localization in chicken, much more restricted than for any of its canonical galectins.Display Omitted
Keywords: B cells; Bursa; Lectin; Lymphocytes; Promoter;

Lid closure dynamics of porcine pancreatic lipase in aqueous solution by Neshatul Haque; N. Prakash Prabhu (2313-2325).
Pancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water.Molecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated.The lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around β5- and β9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation.PPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure.Conformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.
Keywords: Pancreatic lipase; Lid closure; Molecular simulation; Water; Colipase;

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents by Neshatul Haque; N. Prakash Prabhu (2326-2334).
Understanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood.Dynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPLmut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed.PPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPLmut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents.Only the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid.This study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue–residue interactions during lid opening movement in water and in octanol.
Keywords: Pancreatic lipase; Lid opening; Molecular simulation; Water; Octanol;