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The selection of technological parameters for nanoparticle formulation represents a complicated development phase. Therefore, the statistical analysis based on Box–Behnken methodology is widely used to optimize technological processes, including poly(lactic-co-glycolic acid) nanoparticle formulation. In this study, we applied a two-level three-factor design to optimize the preparation of nanoparticles loaded with cobalt (CoTPP), manganese (MnClTPP), and nickel (NiTPP) metalloporphyrins (MeP). The resulting nanoparticles were examined by dynamic light scattering, X-ray diffraction, Fourier transform infrared spectroscopy, MTT test, and hemolytic activity assay. The optimized model of nanoparticle formulation was validated, and the obtained nanoparticles possessed a spherical shape and physicochemical characteristics enabling them to deliver MeP in cancer cells. In vitro hemolysis assay revealed high safety of the formulated MeP-loaded nanoparticles. The MeP release demonstrated a biphasic profile and release mechanism via Fick diffusion, according to release exponent values. Formulated MeP-loaded nanoparticles revealed significant antitumor activity and ability to generate reactive oxygen species. MnClTPP- and CoTPP-nanoparticles specifically accumulated in tissues, preventing wide tissue distribution caused by long-term circulation of the hydrophobic drug. Our results suggest that MnClTPP- and CoTPP-nanoparticles represent the greatest potential for utilization in in anticancer therapy due to their effectiveness and safety.

C-Raf is a member of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf–MEK interaction is enhanced due to the down-regulation of RKIP and MST2.

Both maspin and glutathione S-transferase pi (GSTp) are implicated as tumor suppressors and downregulated in human prostate cancer. It is well established that GSTp downregulation is through DNA methylation–based silencing. We report here that maspin expression in prostate cancer cell line DU145 reversed GSTp DNA methylation, as measured by methylation- specific PCR, MethyLight assay, and bisulfite sequencing. The effect of maspin on GSTp expression was similar to that of the combination of a synthetic histone deacetylase (HDAC) inhibitor and DNA methylation inhibitor 5-aza-2′-deoxycytidine. Maspin expression also led to an increased level of acetylated histone 3, decreased level of methyl transferase, and methyl-CpG–binding domain proteins at the site of demethylated GSTp promoter DNA. Earlier, we have shown that maspin inhibits HDAC1. In PC3 cells, where both maspin and GSTp are expressed at a reduced level, maspin knockdown led to a significant reduction in GSTp expression, whereas dual knockdown of maspin and HDAC1 barely increased the level of GSTp expression. Thus, HDAC1 may play an essential role in cellular response to maspin-mediated GSTp desilencing. Maspin has been shown to increase tumor cell sensitivity to drug-induced apoptosis. Interestingly, GSTp reexpression in the absence of maspin expression perturbation blocked the phosphorylation of histone 2A.X, the induction of hypoxia-induced factor 1α (HIF-1α), and cell death of LNCaP cells under oxidative stress. Because DNA hypermethylation–based silencing may couple with and depend on histone deacetylation, our study suggests that endogenous HDAC inhibition by maspin may prevent pathologic gene silencing in prostate tumor progression. Mol Cancer Res; 9(6); 733–45. ©2011 AACR.

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

Gram-negative bacteria can bind complement protein C1q in an antibody-independent manner and activate classical pathway via their lipopolysaccharides (LPS). Earlier studies have implicated the collagen-like region of human C1q in binding LPS. In recent years, a number of C1q target molecules, previously considered to interact with collagen-like region of C1q, have been shown to bind via the globular domain (gC1q). Here we report, using recombinant forms of the globular head regions of C1q A, B and C chains, that LPS derived from Salmonella typhimurium interact specifically with the B-chain of the gC1q domain in a calcium-dependent manner. LPS and IgG-binding sites on the gC1q domain appear to be overlapping and this interaction can be inhibited by a synthetic C1q inhibitor, suggesting common interacting mechanisms.

Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.

The Ras-Raf-MAPK pathway regulates diverse physiological processes by transmitting signals from membrane based receptors to various nuclear, cytoplasmic and membrane-bound targets, coordinating a large variety of cellular responses. Function of Raf family kinases has been shown to play a role during organism development, cell cycle regulation, cell proliferation and differentiation, cell survival and apoptosis and many other cellular and physiological processes. Aberrations along the Ras-Raf-MAPK pathway play an integral role in various biological processes concerning human health and disease. Overexpression or activation of the pathway components is a common indicator in proliferative diseases such as cancer and contributes to tumor initiation, progression and metastasis. In this review, we focus on the physiological roles of Raf kinases in normal and disease conditions, specifically cancer, and the current thoughts on Raf regulation.

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Delta(5)-3beta-hydroxycholenic, Delta(5)-3beta-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin–CDK complexes Cln1–Cdk1 and Cln2–Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin–CDK complexes are not involved. It has been proposed that the meiosis-specific protein kinase Ime2 functionally replaces the G1 cyclin–CDK complexes to promote Sic1 destruction. To investigate this possibility, we compared Cln2–Cdk1 and Ime2 protein kinase activities in vitro. Both enzyme preparations were capable of catalyzing phosphorylation of a GST–Sic1 fusion protein, but the phosphoisomers generated by the two activities had significantly different electrophoretic mobilities. Furthermore, mutation of consensus CDK phosphorylation sites in Sic1 affected Cln2–Cdk1- but not Ime2-dependent phosphorylation. Phosphoamino acid analysis and phosphopeptide mapping provided additional evidence that Cln2–Cdk1 and Ime2 targeted different residues within Sic1. Examination of other substrates both in vitro and in vivo also revealed differing specificities. These results indicate that Ime2 does not simply replace G1 cyclin–CDK complexes in promoting Sic1 degradation during meiosis.

Classical complement pathway is an important innate immune mechanism, which is usually triggered by binding of C1q to immunoglobulins, pentraxins and other target molecules. Although the activation of the classical pathway is crucial in the host defence, its undesirable and uncontrolled activation can lead to tissue damage. Thus, understanding the molecular basis of complement activation and its inhibition are of great biomedical importance. Recently, we proposed a mechanism for target recognition and classical pathway activation by C1q, which is likely governed by calcium-controlled reorientation of macromolecular electric moment vectors. Here we sought to define the mechanism of C1q inhibition by low molecular weight disulphate compounds that bind to the globular (gC1q) domain, using experimental, computational docking and theoretical modelling approaches. Our experimental results demonstrate that betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of C1q and its recombinant globular modules with target molecules IgG1, C-reactive protein (CRP) and long pentraxin 3 (PTX3). In most C1q-inhibitor docked complexes, there is a reduction of electric moment scalar values and similarly altered direction of electric/dipole moment vectors. This could explain the inhibitory effect by impaired electrostatic steering, lacking optimal target recognition and formation of functional complex. In the presence of the inhibitor, the tilt of gC1q domains is likely to be blocked by the altered direction of the electric moment vector. Thus, the transition from the inactive (closed) towards the active (open) conformation of C1q (i.e. the complement activation signal transmission) will be impaired and the cascade initiation disrupted. These results could serve as a starting point for the exploration of a new form of ‘electric moment inhibitors/effectors’. Copyright © 2007 John Wiley & Sons, Ltd.

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.

Maspin, a class II tumor suppressor, is often downregulated during tumor progression and its depletion from the nucleus is associated with poor prognosis. Recently, we reported that reintroduction of maspin is sufficient for redifferentiation of prostate cancer cells to epithelial phenotype, a reversal of epithelial-to-mesenchymal transition. We have linked this effect of maspin with its ability to directly inhibit HDAC1, thereby influencing the acetylation state of transcription factors and other proteins. Maspin overexpression leads to changes in the expression level of a large number of proteins and these changes are often microenvironment specific. In this review, we summarize the epigenetic effects of maspin and provide comprehensive bioinformatic analysis of microarray-derived gene expression changes caused by maspin in different microenvironments. The analysis was performed on multiple levels, including identification of statistically enriched gene ontology groups, detection of overreprepresented transcription factors binding sites in promoters of differentially expressed genes, followed by searching for key nodes of regulatory networks controlling these transcription factors. The results are consistent with our hypothesis that maspin serves as an endogenous regulator of HDAC activity and suggest that the effect of maspin is primarily mediated by TGFβ, β-catenin/E-cadherin pathways, and network key nodes such as Abl kinase, p62, IL1, and caspases 6 and 8.

The Ras–Raf–mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.

Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C 1 r ¯ / C 1 s ¯ components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.