T95 nucleophosmin phosphorylation as a novel mediator and marker of regulated cell death in acute kidney injury.

The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated.

Cancer cells resist hyperthermia due to its obstructed activation of caspase 3.

Aim: It is well known that inducing hyperthermia is a type of cancer treatment but some research groups indicate that this treatment is not effective. This article finds and explains the mechanism of this treatment and its possible problems.

Background: Hyperthermia is commonly known as a state when the temperature of the body rises to a level that can threaten one's health.

Ex-Vivo Model Systems of Cancer-Bone Cell Interactions.

This chapter elaborates on the state-of-the-art experimental procedures utilized in ex-vivo model systems of cancer-bone cell interactions under "static and dynamic" culture conditions and their potential use to understand cellular and molecular mechanisms as well as drug testing and discovery. An additional focus of this chapter is to provide details of how to incorporate varying oxygen tension, viz., hypoxic, normoxic, and hyperoxic, in such studies and regulate the bone biology toward dissociation of the bone remodeling stages to achieve only "bone resorption" or "bone formation" individually.

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI.

Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target. Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury.

The involvement of Kav001 in inhibition of LPS/P. gingivalis-induced.

TNF-a is an important cytokine mediator of inflammation which suggests that inhibition of TNF activity may provide potential for clinical application. Recent data indicated that treatment of both human and mouse cells with Kavain significantly modulates P. gingivalis- and LPS-induced TNF-α expression.

A method for high transfection efficiency in THP-1 suspension cells without PMA treatment.

Adherent cells such as mouse RAW cells or human cancer U2OS cells are beneficial to DNA transfection, with 20%-60% transfection efficiency. However, this DNA transfection is rarely used on suspension cells due to its low transfection efficiency (≤5%). We recently found a new DNA transfection method to increase the efficiency up to 13.

Qualitative and Quantitative Proteome Analysis of Oral Fluids in Health and Periodontal Disease by Mass Spectrometry.

The significance of protein identification and characterization by classical protein chemistry approaches is clearly highlighted by our detailed understanding of the biological systems assembled over a time period of almost a century. The advent of state-of-the-art mass spectrometry (MS) with sensitivity, speed, and global protein analysis capacity without individual protein purification has transformed the classical protein chemistry with premise to accelerate discovery. These combined with the ability of the oral fluids such as whole saliva (WS) and gingival crevicular fluid (GCF) to reflect both systemic and locally derived proteins have generated significant interest to characterize these fluids more extensively by MS technology.

Bone microenvironment-mediated resistance of cancer cells to bisphosphonates and impact on bone osteocytes/stem cells.

Anti-resorptive bisphosphonates (BPs) have been clinically used to prevent cancer-bone metastasis and cancer-induced bone pathologies despite the fact that the phenotypic response of the cancer-bone interactions to BP exposure is "uncharted territory". This study offers unique insights into the interplay between cancer stem cells and osteocytes/osteoblasts and mesenchymal stem cells using a three-dimensional (3D) live cancer-bone interactive model. We provide extraordinary cryptic details of the biological events that occur as a result of alendronate (ALN) treatment using 3D live cancer-bone model systems under specific bone remodeling stages.

Effect of Corticotomies with Different Instruments on Cranial Bone Biology Using an Ex Vivo Calvarial Bone Organ Culture Model System.

Different corticotomy surgical procedures have been developed to shorten orthodontic treatment times by stimulating bone remodeling. Although all corticotomy procedures involve physical injury to the bone, the clinical outcomes can vary. Using an ex vivo calvarial bone organ culture model system, the authors evaluated the biologic response of bone to different corticotomies.

Quantitative gingival crevicular fluid proteome in health and periodontal disease using stable isotope chemistries and mass spectrometry.

Aim: Application of quantitative stable isotope-labelling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease.

Material And Methods: Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable isotope-labelling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods.

Results: We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients.

Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin.

Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry.

Unlabelled: Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively.

Three-dimensional cancer-bone metastasis model using ex-vivo co-cultures of live calvarial bones and cancer cells.

One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of cancer cells in a roller tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz.

Quantitative analysis of cytokine-induced hepatocyte nuclear factor-4α phosphorylation by mass spectrometry.

Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored.

Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry.

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5.

The isolation and characterization of glycosylated phosphoproteins from herring fish bones.

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones.

Mass spectrometric identification of key proteolytic cleavage sites in statherin affecting mineral homeostasis and bacterial binding domains.

Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events.

Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry.

To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).

Characterization of recombinant lysyl oxidase propeptide.

Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP.

Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva.

Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50-75 kDa region.

Modulation of bone resorption by phosphorylation state of bone sialoprotein.

We have determined transmembrane protein tyrosine phosphorylation (outside-in signaling) in cultured osteoclasts and macrophages in response to added native purified bone sialoprotein (nBSP) and its dephosphorylated form (dBSP). There were selective/differential and potent inhibitory effects by dBSP and minimal effect by nBSP on intracellular tyrosine phosphorylation in macrophages and osteoclasts. Further studies on the downstream gene expression effects led to identification of a large number of differentially expressed genes in response to nBSP relative to dBSP in both macrophages and osteoclasts.

Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall V(max)/K(m) ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1.

Identification of Lys-Pro-Gln as a novel cleavage site specificity of saliva-associated proteases.

The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins.

Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification.

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Salivary proteome and its genetic polymorphisms.

Salivary diagnostics for oral as well as systemic diseases is dependent on the identification of biomolecules reflecting a characteristic change in presence, absence, composition, or structure of saliva components found under healthy conditions. Most of the biomarkers suitable for diagnostics comprise proteins and peptides. The usefulness of salivary proteins for diagnostics requires the recognition of typical features, which make saliva as a body fluid unique.