Small Molecular Mass Research Articles

Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

Inducible tolerance to dietary protease inhibitors inDaphnia magna

Daphnia has been shown to acquire tolerance to cyanobacterial toxins within an animals' lifetime and to transfer this tolerance to the next generation. Here we used a strain of the cyanobacterium Microcystis aeruginosa, which contained two chymotrypsin inhibitors (BN920 and CP954), the green alga Scenedesmus obliquus as reference food and a clone of D. magna to investigate the physiological mechanism of acquired tolerance to these cyanobacterial toxins. The intracellular concentrations of CP954 and BN920 were 1550 and 120 μmol l(-1), respectively. When food suspensions of the green alga contained >60% M. aeruginosa, growth rates of D. magna were reduced. When grown on the green alga, three chymotrypsins ranging in mass from 16 to 22 kDa were distinguished in D. magna. Purified BN920 and CP954 specifically inhibited D. magna chymotrypsins. Feeding with encapsulated BN920 resulted in growth depression in D. magna and replacement of the chymotrypsins by three chymotrypsins with smaller molecular mass. With just 20% M. aeruginosa, the same changes in the chymotrypsin pattern as with the pure inhibitor were observed. IC(50) values for inhibition of chymotrypsins of D. magna growing on the green alga were 5.4 nmol l(-1) (BN920) and 7.4 nmol l(-1) (CP954). When D. magna was grown on 20% M. aeruginosa, 2.2-fold higher IC(50) values were observed. This indicated that increased tolerance to these dietary inhibitors was acquired within an animal's lifetime by remodelling the digestive chymotrypsins, which in turn serves as an intra-generational defence against these cyanobacterial inhibitors. This mechanism might be relevant for the transfer of tolerance to the next generation through maternal effects.

Discovery and Optimization of New Benzimidazole- and Benzoxazole-Pyrimidone Selective PI3Kβ Inhibitors for the Treatment of Phosphatase and TENsin homologue (PTEN)-Deficient Cancers

Most of the phosphoinositide-3 kinase (PI3K) kinase inhibitors currently in clinical trials for cancer treatment exhibit pan PI3K isoform profiles. Single PI3K isoforms differentially control tumorigenesis, and PI3Kβ has emerged as the isoform involved in the tumorigenicity of PTEN-deficient tumors. Herein we describe the discovery and optimization of a new series of benzimidazole- and benzoxazole-pyrimidones as small molecular mass PI3Kβ-selective inhibitors. Starting with compound 5 obtained from a one-pot reaction via a novel intermediate 1, medicinal chemistry optimization led to the discovery of compound 8, which showed a significant activity and selectivity for PI3Kβ and adequate in vitro pharmacokinetic properties. The X-ray costructure of compound 8 in PI3Kδ showed key interactions and structural features supporting the observed PI3Kβ isoform selectivity. Compound 8 achieved sustained target modulation and tumor growth delay at well tolerated doses when administered orally to SCID mice implanted with PTEN-deficient human tumor xenografts.

Abstract 479: The effects of sarcophine-diol (SD) on melanoma B16F10 cell line

Abstract Sarcophine diol (SD) is a non-toxic semi-synthetic derivative of sarcophine, a marine natural product. Previously, we studied the chemopreventive effects of SD on non-melanoma tumor development using both animal and cellular models. In this current study, we used the mouse melanoma B16F10 cell line to investigate the possible effects of SD on melanoma cell development. We found that SD at a 250 µM concentrations inhibits cell duplication. Melanoma cells treated with SD do not form the contractile ring, a multi-protein complex involved in cell division. As a result of cytoskeleton modification, cells treated with SD do not show the ability to carry out de novo synthesis of DNA, and their DNA show a high degree of fragmentation compared to untreated controls. To explore the molecular mechanism(s) involved in the disassembly of the contractile machinery, our studies show that SD treatment inhibits cell membrane permeability for small molecular mass compounds (e.g. ethidium bromide which is used as an indicator for cell permeability to Ca2+ ions). These findings are consistent with the observation that SD increases cleavage of phospholipase A2 (PLA2), resulting in low PLA2 enzymatic activity. These findings are also consistent with the observation that SD, in a time- and concentration-dependent manner, inhibits the expression level of the cyclooxygenase-2 (Cox-2). As a result of the decline in membrane permeability in SD treated cells and the negative effect of SD on the protein level of phospholipase C (PLC), which both affect the activity of contractile machinery, the non-dividing cells undergo apoptosis. The latter notion is supported by findings that SD inhibits the expression of STAT-3 (signal transducer and activator of transcription protein), and cyclin D1 (an activator of cyclin-dependent kinase 4, Cdk4). SD treatment also enhances the cellular level of the tumor suppressing protein 53 (p53) and stimulates cleavage of the nuclear poly(ADP-ribose) polymerase (cleaved-PARP). SD also enhances the cellular level of cleaved-Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. All these findings, in addition to inhibition of cell viability, suggest that SD most likely inhibits melanoma cell growth by arresting the cell-division cycle in a Go quiescent phase and simultaneously activates apoptosis via extrinsic and intrinsic pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 479. doi:1538-7445.AM2012-479

Characteristics of water vapor turbocompressors applied in refrigeration and heat pump systems

Interdependence between centrifugal stage pressure ratio, peripheral Mach number and impeller peripheral speed, as well the influence of the refrigerating capacity on the dimensions of the centrifugal compressor are determined to obtain the range of reasonable usage of R718 refrigerating/heat pump systems. The deep vacuum operating conditions, the low specific volumetric cooling capacity of the R718 and the small molecular mass of the water are the peculiarities which determined the main parameters of the centrifugal compressor and the range of rational application of the R718 refrigerating/heat pump systems. The high peripheral impeller speed, high Mach number and high pressure ratio cause separated jet-wake flow and transonic flow phenomena in the compressor flow field. Estimation of the flow characteristics and of the performance characteristics of the water vapor centrifugal compressors is presented, using results of CFD simulations and experimental investigations for high pressure ratio centrifugal compressors.

The novel, orally bioavailable HSP90 inhibitor NVP‐HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases

Heat shock protein 90 (HSP90) binds and stabilizes numerous proteins and kinases essential for myeloma cell survival and proliferation. We and others have recently demonstrated that inhibition of HSP90 by small molecular mass inhibitors induces cell death in multiple myeloma (MM). However, some of the HSP90 inhibitors involved in early clinical trials have shown limited antitumor activity and unfavorable toxicity profiles. Here, we analyzed the effects of the novel, orally bioavailable HSP90 inhibitor NVP-HSP990 on MM cell proliferation and survival. The inhibitor led to a significant reduction in myeloma cell viability and induced G2 cell cycle arrest, degradation of caspase-8 and caspase-3, and induction of apoptosis. Inhibition of the HSP90 ATPase activity was accompanied by the degradation of MM phospho-Akt and phospho-ERK1/2 and upregulation of Hsp70. Exposure of MM cells to a combination of NVP-HSP990 and either melphalan or histone deacetylase (HDAC) inhibitors caused synergistic inhibition of viability, increased induction of apoptosis, and was able to overcome the primary resistance of the cell line RPMI-8226 to HSP90 inhibition. Combined incubation with melphalan and NVP-HSP990 led to synergistically increased cleavage of caspase-2, caspase-9, and caspase-3. These data demonstrate promising activity for NVP-HSP990 as single agent or combination treatment in MM and provide a rationale for clinical trials.

Effect of H2O on the density of silicate melts at high pressures: Static experiments and the application of a modified hard-sphere model of equation of state

Density of ultramafic silicate melts was determined using the sink/float technique at high pressures. Seven melt compositions were studied, among which three were dry compositions with different Mg#’s (molar MgO/(MgO+FeO)×100) and the other four were hydrous compositions synthesized by adding 2–7wt.% H2O to the anhydrous ones. Experimental conditions range from 9 to 15GPa and from 2173 to 2473K. The sinking and floatation of density markers were observed for all melt compositions. Melt density data were analyzed by applying the Birch–Murnaghan equation of state and a newly developed equation of state for silicate melts based on the model of hard sphere mixtures. The presence of water can significantly reduce the density of melts due to its small molecular mass. On the other hand, water makes hydrous silicate melts more compressible than anhydrous melts and therefore the effect of H2O on melt density is less significant at high pressures. The density of hydrous melts was then calculated as a function of H2O content at the conditions of the bottom of the upper mantle, and was compared with the density of the dominant upper mantle minerals. Results show that the conditions for a negatively buoyant melt that coexists with a pyrolite mantle atop the 410km discontinuity are marginally satisfied if H2O is the only volatile component to facilitate melting, but such conditions will be satisfied by a broader range of conditions when other heavier volatile elements (C, K, etc.) are also present.

TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses (∼35kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

A large cryogenic magnetocaloric effect exhibited at low field by a 3D ferromagnetically coupled Mn(ii)–Gd(iii) framework material

The large cryogenic magnetocaloric effect of a 3D oxydiacetate-bridged gadolinium-manganese MOF material, [Mn(H(2)O)(6)][MnGd(oda)(3)](2)·6H(2)O (1), was evaluated by magnetization and heat capacity measurements. A maximum -ΔS(m) of 50.1 J kg(-1) K(-1) for ΔH = 70 kG along with significant entropy change at lower field was found on account of the weak Mn···Gd ferromagnetic interactions and the small molecular mass. This suggests that 1 could be considered as a potential coolant for liquid helium temperature applications.

The Øie–Tozer model of drug distribution and its suitability for drugs with different pharmacokinetic behavior

Introduction: Drug distribution is a major pharmacokinetic process that affects the time course of drug concentrations in tissues, biological fluids and the resulting pharmacological activities. Drug distribution may follow different pathways and patterns, and is governed by the drug's physicochemical properties and the body's physiology. The classical Øie–Tozer model is frequently used for predicting volume of drug distribution and for pharmacokinetic calculations.Areas covered: In this review, the suitability of the Øie–Tozer model for drugs that exhibit different distribution patterns is critically analyzed and illustrated. The method used is a pharmacokinetic modeling and simulation approach. It is demonstrated that the major limitation of the Øie–Tozer model stems from its focus on the total drug concentrations and not on the active (unbound) concentrations. Moreover, the Øie–Tozer model may be inappropriate for drugs with nonlinear or complex pharmacokinetic behavior, such as biopharmaceuticals, drug conjugates or for drugs incorporated into drug delivery systems. Distribution mechanisms and alternative distribution models for these drugs are discussed.Expert opinion: The Øie–Tozer model can serve for predicting unbound volume of drug distribution for 'classical' small molecular mass drugs with linear pharmacokinetics. However, more detailed mechanism-based distribution models should be used in preclinical and clinical settings for drugs that exhibit more complex pharmacokinetic behavior.

Cryptococcus Neoformans Galactoxylomannan is a Potent Negative Immunomodulator, Inspiring New Approaches in Anti-Inflammatory Immunotherapy

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The fungus is endowed with several virulence factors, including capsular polysaccharides that play a key role in virulence. The capsule is composed of 90-95% glucuronoxylomannan (GXM), 5-8% galactoxylomannan (GalXM) and <1% mannoproteins. Capsular polysaccharides are shed into tissue where they produce many deleterious effects. Since GalXM has a smaller molecular mass, the molar concentration of GalXM in polysaccharide that is shed could exceed that of GXM in C. neoformans exopolysaccharides. Moreover, GalXM exhibits a number of unusual biologic properties both in vitro and in vivo. Here, we summarize the principal immunomodulatory effects of GalXM described during the last 20 years, particularly the mechanisms leading to induction of apoptosis in T lymphocytes, B lymphocytes and macrophages. Since the capacity of GalXM to induce widespread immune suppression is believed to contribute to the virulence of C. neoformans, this property might be exploited therapeutically to dampen the aberrant activation of immune cells during autoimmune disorders.

An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Second, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one-third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides.

The small molecular mass antifungal protein of Penicillium chrysogenum – a mechanism of action oriented review

The β-lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine-tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two-component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca(2+) -homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review.

Human T-lymphotropic Virus Type 1 p30 Interacts with REGγ and Modulates ATM (Ataxia Telangiectasia Mutated) to Promote Cell Survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Identification of novel α-gliadin genes

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

Thermal analysis of a sodium salt of the maleic acid-acrylic acid copolymer used as a polymeric binder

Samples of sodium salt of the maleic acid-acrylic acid copolymer used as a binding agent in molding sands were investigated. Methods of thermal analysis (DTG, TG, DSC) were applied to assess the thermal stability of the investigated polymer sample by estimation of temperature and thermal effects of transformations occurring during its heating. Examinations were performed at the temperature range -100-1000 °C. It was found that the total degradation process starts at the temperature about 400 °C. In addition, the analysis of volatile decomposition products was carried out by means of the Fourier transform infrared spectroscopy (FT-IR) and the thermogravimetric (TG) method coupled "on-line" with the mass spectrometry (MS) method. At the temperature range 300-400 °C the signals for small molecular masses were found, which indicates that the degradation process and the fragmentation of polymer chains occurred as well as small particles were formed (molecular weight: 15-55), including, first of all, H2O and CO2. Probably during the degradation radicals are also formed, which is indicated by the MS signals.

Dual purpose contraceptives: targeting fertility and sexually transmitted disease

There have been no radically new forms of contraception since the pill was introduced 1960 and even this form of fertility regulation can be traced back to endocrine advances that were made in the 1920s. Whatever new forms of fertility control we introduce for the future, they should exploit the significant advances that have been made in our understanding of the reproductive system in recent years and be tailored to the needs of the 21st century. In this context, there is an urgent need to develop novel, safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases (STDs). To achieve this aim we have researched a class of a topical contraceptive agent that selectively and instantaneously immobilizes millions of spermatozoa, while suppressing the infectivity of pathogenic microbes, such as Chlamydia, in the ejaculate. This approach is based upon the ability of small molecular mass organic compounds to selectively and covalently adduct key proteins in spermatozoa and pathogenic organisms and disrupt their biological function. We have also successfully developed strategies for the preparation of latent formulations that would only become activated on contact with seminal plasma. The further development and refinement of these molecules should permit a radical rethink in the way that safe, effective topical protection is provided to control both fertility and the world-wide spread of STDs.

In vitro expression of NGN3 identifies RAB3B as the predominant Ras-associated GTP-binding protein 3 family member in human islets.

Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.

Combination of high-performance refractometry and infrared spectroscopy as a probe for chemically induced gelation and vitrification of epoxies

A combination of infrared spectroscopy and high-performance refractometry was used to investigate the chemically induced sol–gel and glass transition during the polymerization of epoxies. Representations of the refractive index versus chemical conversion reveal an interesting insight into the optical properties accompanying gelation and vitrification. Whereas the electronic polarizability of the liquid state of small average molecular mass and the glassy state is dominated by the mass density, an unexpected excess polarizability observed during the gelation is attributed to cooperative dipole–dipole interactions.

Zeptomole Electrochemical Detection of Metallothioneins

BackgroundThiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT.Methodology/Principal FindingsIn this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals.Conclusions/SignificanceThis paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab–on-chip instrument.