Classical Biochemical Assays Research Articles

Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model

BackgroundHepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options.ResultsIn this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size.ConclusionThese results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids.

Characterization of non-tuberculosis mycobacteria by neutron radiography

The genus Mycobacterium shares many characteristics with Corynebacterium and Actinomyces genera, among which the genomic guanine plus cytosine content and the production of long branched-chain fatty acids, known as mycolic acids are enhanced. Growth rate and optimal temperature of mycobacteria are variable. The genus comprises more than 140 known species; however Mycobacterium fortuitum, a fast growing nontuberculous mycobacterium, is clinically significant, because it has been associated to several lesions following surgery procedures such as liposuction, silicone breast and pacemaker implants, exposure to prosthetic materials besides sporadic lesions in the skin, soft tissues and rarely lungs. The objective of the present study is to reduce the time necessary for M. fortuitum characterization based on its morphology and the use of the neutron radiography technique substituting the classical biochemical assays. We also aim to confirm the utility of dendrimers as boron carriers. The samples were sterilized through conventional protocols using 10% formaldehyde. In the incubation process, two solutions with different molar ratios (10:1 and 20:1) of sodium borate and PAMAM G4 dendrimer and also pure sodium borate were used. After doping and sterilization procedures, the samples were deposited on CR-39 sheets, irradiated with a 4.6×105n/cm2s thermal neutron flux for 30min, from the J-9 irradiation channel of the Argonauta IEN/CNEN reactor. The images registered in the CR-39 were visualized in a Nikon E400 optical transmission microscope and captured by a Nikon Coolpix 995 digital camera. Developing the nuclear tracks registered in the CR-39 allowed a 1000× enlargement of mycobacterium images, facilitating their characterization, the use of more sophisticated equipment not being necessary. The use of neutron radiography technique reduced the time necessary for characterization. Doping with PAMAM dendrimer improved the visualization of NTM in neutron radiography images.

Xanthomonas campestris pv. campestris race 1 is the main causal agent of black rot of Brassicas in Southern Mozambique

Severe outbreaks of bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) were observed in Brassica production fields of Southern Mozambique. The causal agent of the disease in the Mahotas and Chokwe districts was identified and characterised. In total, 83 Xanthomonas -like strains were isolated from seed samples and leaves of cabbage and tronchuda cole with typical symptoms of the disease. Forty-six out of the 83 strains were found to be putative Xcc in at least one of the tests used: Classical biochemical assays, enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies, Biolog identification system, polymerase chain reaction (PCR) with specific primers and pathogenicity tests. The ELISA tests were positive for 43 strains. Biolog identified 43 strains as Xanthomonas , but only 32 as Xcc. PCR tests with primers targeting a fragment of the hrpF gene were positive for all 46 strains tested. Three strains were not pathogenic or weakly pathogenic and all other strains caused typical black rot symptoms in brassicas. Race type differentiation tests revealed the Xcc strains from Mozambique as members of race 1. The prevalence of this pathogenic race of the Xcc pathogen in Mozambique should be considered when black rot resistant cultivars are evaluated or introduced into the production regions of this country. Keywords : Cole crops, cabbage, tronchuda, landraces, seeds, black rot, enzyme-linked immunosorbent assay (ELISA), Biolog, polymerase chain reaction (PCR), race-type African Journal of Biotechnology Vol. 12(6), pp. 602-610

Comparison of the metabolic profiling of hepatitis B virus‐infected cirrhosis and alcoholic cirrhosis patients by using 1H NMR‐based metabonomics

The aims of the present study were to depict the serum metabolic characteristics of hepatitis B virus (HBV)-infected cirrhosis and alcoholic cirrhosis patients, and to find the specific serum biomarkers associated with the diseases. A pilot metabolic profiling study was conducted using three groups: HBV-infected cirrhosis patients (n = 21), alcoholic cirrhosis patients (n = 20) and healthy controls (n = 20). (1) H nuclear magnetic resonance (NMR)-based metabonomics was used to obtain the serum metabolic profiles of the samples. The acquired data were processed by multivariate principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA). The discriminatory metabolites between HBV-infected cirrhosis and alcoholic cirrhosis were further validated by classical biochemical assays. The OPLS-DA model was capable of distinguishing between HBV-infected and alcoholic cirrhosis patients. Five metabolites, creatine, acetoacetate, isobutyrate, glutamine and glutamate, were identified as the most influential factors to compare HBV-infected cirrhosis and alcoholic cirrhosis. The validation tests showed that the changes of the five metabolites were well coincident with the results of NMR. NMR spectra combined with pattern recognition analysis techniques may provide a new way to explore the pathogenesis of HBV-infected and alcoholic cirrhosis patients.

JAK's SOCS: A Mechanism of Inhibition

SOCS1 and SOCS3 are specific inhibitors for JAK tyrosine kinases. In this issue of Immunity, Babon et al. (2012) discovered the inhibition mechanism of SOCS3 by employing nuclear magnetic resonance and classical enzyme kinetics.

Nonspecific Bacterial Flora Isolated from the Body Surface and Inside Ixodes ricinus Ticks

Ixodes ricinus and other representatives of the order Ixodida are vectors of typical pathogens: Borrelia burgdorferi sensu lato, Anaplasma phagocytophilium, Babesia spp., a tick-borne encephalitis virus, and other microorganisms which are important from a medical and veterinary point of view. The presented study focuses on the verification of nonspecific bacterial flora of I. ricinus. We analyzed ticks collected in a forest region in Silesia, an industrial district in Poland. Methods of classical microbiology and biochemical assays (API 20 NE test, API Staph test and MICRONAUT System) were used for isolation and identification of microorganisms living on the body surface of I. ricinus and inside ticks. The results show the presence of various bacteria on the surface and inside ticks' bodies. During the study, we isolated Acinetobacter lwoffi, Pseudomonas fluorescens, Aeromonas hydrophila, Achromobacter denitrificans, Alcaligenes faecalis, Stenotrophomonas maltophilia, Pseudomonas oryzihabitans, Micrococcus spp., Kocuria varians, Staphylococcus lentus, Kocuria kristinae, Streptococcus pneumoniae, Rhizobium radiobacter, Staphylococcus xylosus. Majority of the isolated species are non-pathogenic environmental microorganisms, but some of the isolated bacterial strains could cause severe infections.

Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition.

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.

CMOS based capacitive sensor laboratory-on-chip: a multidisciplinary approach

In this paper, we review the recent advances of CMOS-based capacitive sensors for Lab-on-chip (LoC) applications. LoC design is a multidisciplinary approach of adapting classical biochemical assays to a miniaturized platform by exploiting advances in microelectronic and microfluidic technologies. By offering low cost and integrated devices, CMOS based LoCs could be amenable to a large number of biological and biochemical assays for disease diagnostics and biotechnology in the near future. While an exhaustive, all-encompassing review of CMOS-based LoCs is beyond the scope of this review, we have focused on the design and implementation of CMOS-based capacitive sensor LoCs for the most important biochemical applications. For each application, the corresponding biochemical sensing layer, interface circuit and microfluidic packaging technique are discussed based on the recent literature studies.

Marked Defects in the Expression and Glycosylation of α2-HS Glycoprotein/Fetuin-A in Plasma from Neonates with Intrauterine Growth Restriction: Proteomics Screening and Potential Clinical Implications

Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the alpha2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates.

Fragment-Based Approach to Drug??Lead Discovery

In the last 20 years the advent of new technologies, such as high-throughput screening (HTS) and combinatorial chemistry, has produced new tools for the discovery of biologically active molecules. In the past decade, fragment-based drug discovery has emerged as a more rational and focused approach that concentrates on the quality, rather than the quantity, of hits and leads. The principles behind this strategy are different from those that represented the basis of conventional HTS. The starting point of this approach is always a small chemical entity (typically MW 150-200), a fragment, with low affinity for the selected target. Fragments should satisfy key features such as diversity, reduced structural complexity, aqueous solubility and availability. Because of their small size, they occupy a smaller region of chemical space if compared with classical HTS compounds; hence, fragment libraries provide a good diversity with a relatively low number of compounds. Classical biochemical assays are often not suitable to detect the low binding affinities involved, so some well known biophysical techniques, such as nuclear magnetic resonance and x-ray, have been opportunely modified in order to render them able to perform the task. When selecting fragments suitable for subsequent optimization, a useful parameter has been introduced, the ligand efficiency, which is defined as the free energy of binding divided by the non-hydrogen atom count. Once selected, a fragment must undergo a heavy elaboration to improve binding affinity, at the same time acquiring drug-like properties. There are two main ways to go on at this point. The most common one is the so-called 'fragment evolution', consisting of a stepwise and systematic addition of chemical functionalities to the starting fragment core, together with a continuous feedback for pharmacological and physicochemical properties. The second one, less common but with great potential, is 'fragment linking': when two or more fragment hits are found to bind in adjacent regions of the target protein, they can be linked through appropriate spacers to rapidly produce a single molecule with much higher binding affinity. Two representative case histories are described: Abbott's ABT 518, an MMP (matrix metalloproteinase) inhibitor, and Eli-Lilly's LY-517717, an inhibitor of factor Xa serine protease. In addition, a list of molecules claimed to be derived from fragment approach and currently undergoing clinical trials is presented.

Ligand-induced interactions of PR-isoforms and cofactors

Progesterone is a key modulator of normal reproductive functions, amongst them being ovulation, uterine and mammary gland development, decidualization, implantation and maintenance of pregnancy. The diverse effects of progesterone on the female reproductive target tissues are mediated by two specific progesterone receptor isoforms (PR-A and PR-B), members of the nuclear receptor (NR) family of ligand-dependent transcription factors, which are derived from a single gene via alternative promoters. Synthetic ligands modulating PR activity, that differ in chemical structure, potency and efficacy, are used for oral contraception, hormone therapy and for the treatment of reproductive disorders. Especially PR-antagonists and selective progesterone receptor modulators (SPRMs) have been shown to be efficacious in treatment of gynecological disorders like endometriosis and uterine leiomyoma. However, they can display diverse characteristics in vivo, often hard to subsume in in vitro experiments. As the recruitment of cofactors has the potential to affect gene transcription in a cell- and tissue-specific manner, we developed in vitro assays to analyze ligand-induced cofactor interactions to define differences and further understand PR-mediated mechanism of antagonists / SPRMs action. In mammalian two hybrid assays PR-isoforms (PR-A; PR-B) display ligand- and dose-dependent association with the family of p160 coactivators as well as the corepressors NCOR and SMRT. Different antagonists induce an interaction of the PR isoforms with both, corepressors and coactivators, although binding of coactivators was much less pronounced. Furthermore, we implemented the specific interaction of PR-B with c-src-kinase to include the non-genomic action of the progesterone receptor. Interestingly, the PR antagonists display divergent effects for this PR signalling, too. In summary, we show that progesterone receptor ligands with similar profiles in classical biochemical assays can be differentiated in vitro via recruitment of cofactors and non-genomic signalling.

Leukotriene receptors: state of the art.

The leukotrienes (cysLTs) are lipid mediators derived from the ubiquitous membrane component arachidonic acid. The plethora of biological activities suggested that they activate different receptors. A number of elegant genetic and biochemical studies have been performed to elucidate the nature of these receptors. Results from classical pharmacological and molecular studies indicate that there are two main classes of leukotriene receptors. One based on the biological activities of leukotriene B4 and related hydroxyacids, referred to as BLT receptors, and a second class identified by the cysteinyl- leukotrienes (cysLTs). Activation of the BLT receptors initially was shown to produce potent chemotactic activities on leukocytes whereas the latter class (CysLT receptors) stimulated smooth muscle as well as other cells. Furthermore, there is now sufficient information from classical pharmacological and biochemical assays as well as molecular investigations to divide both categories into receptor sub-classes, namely, BLT1 and BLT2 as well as CysLT1 and CysLT2 (Table 1).KeywordsAirway Smooth Muscle CellVascular Smooth Muscle Cell ProliferationHuman Airway Smooth MuscleHuman Airway Smooth Muscle CellCysLT ReceptorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

ULTRASTRUCTURAL EVIDENCE THAT APOPTOSIS IS THE MECHANISM BY WHICH HUMAN AMYLIN EVOKES DEATH IN RINm5F PANCREATIC ISLET β-CELLS

A view is emerging that human amylin (HA) kills pancreatic islet β-cells by apoptosis. This study strengthens this view by documenting time-dependent morphological and ultrastructural changes in 10μm HA-treated cultured RINm5F islet β-cells. Membrane blebbing and microvilli loss were the earliest detectable apoptosis-related phenomena, already evident 1h after HA exposure. Following 6–12h of HA-treatment, chromatin margination became evident, consistent with detecting DNA laddering about the same time. Nuclear shrinkage, nuclear membrane convolution and prominent cytoplasmic vacuolization were clearly recognized at 22h post-treatment. Together, these cellular changes constitute a strong case for HA-induced apoptosis, and further demonstrates that electron microscopy is a more sensitive tool for early apoptosis detection in cultured cells than classical biochemical assays like visualizing DNA laddering. The ultrastructural changes reported here contribute further evidence to be included in the ongoing dissection of molecular mechanisms underlying HA-induced apoptosis, as may occur in type-2 diabetes mellitus.

Membrane lipid a-crystallin interaction and membrane Ca2+-ATPase activities

Purpose. To determine the effect of a-crystallin binding on lens membrane lipid characteristics and the stability of Ca2+-ATPase activity when challenged with H2O2 or elevated temperatures. Methods. a-Crystallin binding to muscle sarcoplasmic reticulum membranes was quantified using a centrifugation protocol. a-Crystallin binding to lens epithelial lipids was measured by a fluorescence energy transfer technique. Lipid phase transition temperature and lipid order was measured using fluorescence spectroscopy. Ca2+-ATPase activity was measured using classical biochemical assays. Results. The main phase transition temperatures of multilamellar vesicles composed of sphingomyelin or lipids extracted from bovine lens were 40°C and 20°C, respectively. In the presence of saturating amounts of a-crystallin, the phase transition temperature and lipid order of both sphingomyelin and lens lipid membranes remained almost the same as that without a-crystallin. The interaction of a-crystallin and lipid is likely to be restricted to the membrane surface. The binding of a-crystallin did not influence the oxidative or thermal inactivation of the Ca2+-ATPase pump. Conclusion. a-Crystallin-lens membrane binding does not protect the Ca2+-ATPase pump from thermal derangement or oxidation by H2O2.

Quantitative Immunological Detection of Total Estrogen Receptor (Cytosolic and Nuclear) in Term Decidua of Preeclampsia: a Preliminary Study

Although progesterone and estrogens are essential for maintaining human pregnancy after implantation, few reports have investigated the localization of their specific receptors in different uterine cell types throughout pregnancy (1)(2). Wu et al. detected estrogen receptors (ER) in low concentrations in decidua in early pregnancy but not in term decidua (2). ER concentrations in term decidua of pregnancies with complications (in particular, preeclampsia) have not been investigated. Classical biochemical assay methods estimate ER either in cytosol fraction or in the high-salt extracted nuclei (3)(4)(5). These assays need two subcellular fractionation steps and two ER assays in two fractions: cytosol and nuclear extract. To measure total ER (cytosolic and nuclear) in one fraction using one single assay in decidual samples, we modified the assay method by using a homogenization buffer containing KCl. The effective KCl concentration for maximum ER extraction without inhibition of the ER enzyme immunoassay (EIA) ranged …

Le syndrome nerveux des ovins en Côte-d'Ivoire. I. Etude épidémiologique et clinique, méthodes de diagnostic et traitement

The ovine nervous syndrome in Côte-d'Ivoire is similar to the cerebrocortical necrosis (CCN) due to vitamin B1 deficiency. All classical symptoms of CCN were observed (locomotor ataxia with subsequent paralysis) and histological evidence for polioencephalomalacia was given. However, the circumstances for occurrence of the disease are very different in the two cases, i.e. CCN is a disease encountered in young fattening ruminants in developed countries while the ovine nervous syndrome is mainly observed in Côte-d'Ivoire during the dry season when pastures become sparse and dry and when the feed supply is insufficient. Thus, the main cause, which is rather univocal, is a sudden decrease in the nutritive value of the diet, but the accurate etiopathogenesis of the disease has not yet been determined. In a flock where 10-30% of the animals are ill, the mortality may reach 80-90%. No classical biochemical assays were specific enough to establish a precise diagnosis of the nervous syndrome. However, it should be pointed out that the CK (creatinine kinase) values very regularly rose and that the ASAT (aspartate aminotransferase) values were high in 75% of the cases. In the present African field conditions, the precise diagnosis is based on the efficiency of the vitamin B1 treatment and, for the dead animals, on the histological analysis of the brain.