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Results for “virtual protein profiling”

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PubMed1984

Molecular cloning and expression of the phenoxazinone synthase gene from Streptomyces antibioticus.

DNA fragments from the Streptomyces antibioticus genome have been cloned in Streptomyces lividans using the SphI site of the vector, pIJ702. Using a sib selection procedure, transformants were screened for their ability to produce levels of the enzyme, phenoxazinone synthase, which were detectable by enzyme assay. Three clones were obtained by this procedure. The recombinant plasmids from these clones contained inserts of 1.77, 2.45, and 4.28 kilobases in size. The clones and the corresponding recombinant plasmids were, therefore, referred to as PHS 1.8, PHS 2.4 and PHS 4.3. PHS 2.4 produced transformants with significantly higher phenoxazinone synthase specific activities than those observed in comparably grown S. antibioticus. Southern blotting showed that the cloned 2.45-kilobase insert hybridized to a fragment of exactly the same size in an SphI digest of S. antibioticus total DNA. Using a streptomycete-coupled transcription-translation system, PHS 2.4 was shown to code for the synthesis of a protein with the electrophoretic and immunological properties of the phenoxazinone synthase subunit. Thus, the 2.45-kilobase insert represents the structural gene for that subunit. The protein produced by S. lividans transformed with PHS 2.4 was shown to be virtually identical to the S. antibioticus subunit in terms of its electrophoretic mobility, peptide profile, and amino acid composition. It was further observed that cloning of the 2.45-kilobase insert in the orientation opposite to that obtained originally still resulted in high levels of phenoxazinone synthase expression in transformed S. lividans. However, the repression of phenoxazinone synthase synthesis by glucose which has been observed in S. antibioticus was not found when PHS 2.4 was used to transform S. lividans.

Amino Acids↗

PubMed1984

A new model-independent physiological approach to study hepatic drug clearance and its applications.

After entering the liver a drug molecule is removed by two competitive processes: (a) blood flow to carry it away intact from liver and (b) elimination by hepatic enzyme(s). The relative magnitude of the overall force or efficiency of these two processes will statistically decide its probability for elimination, i.e., extraction ratio (E). Blood flow rate is chosen to represent the force due to blood flow, and the force due to hepatic elimination can then be simply calculated in terms of blood flow per unit time. Under linear conditions any changes in either force can be used to predict new E. Binding in blood, permeability across hepatocellular membrane and intrinsic enzyme activity will affect the hepatic elimination force. The approach employed has virtually made no prior assumptions regarding distribution profiles of drug, enzymes, sinusoids and intracellular bindings as well as drug permeability. Bioavailability predictions upon changes in hepatic flow or protein binding are consistent with most reported data. It can explain potential differences in the fate of drug pre-equilibrated or not pre-equilibrated with blood before entering the liver and also that of metabolite performed or not performed in liver. Importance of erythrocytes as potential elimination barriers in hepatic first-pass effect and existence of both transverse and longitudinal (not considered in previous hepatic models) drug concentration gradients in the liver are emphasized. Applications to studies of instantaneous, time-averaged, beta and intrinsic clearances, as well as hepatic transit time and nonlinear kinetics are discussed. The present simple dynamic approach can also successfully predict the quantitative correlation between renal clearance and changes in urine flow and/or pH for many drugs in humans and animals. Using steady-state input conditions the present derived equations can be obtained thus adding the mathematical validity of the approach.

Animals↗

PubMed1983

Mechanisms of blood-brain barrier breakdown after microembolization of the cat's brain.

Unilateral microembolization of the cat brain with carbonized microspheres 15 microns in diameter ten minutes (min) before death induced multifocal disruption of the blood-brain barrier (BBB) to horseradish peroxidase (HRP) in nine adult cats. The gross pattern of HRP extravasates was studied: (a) after five min of in vivo circulation, (b) following infusion of HRP immediately before chemical fixation, and (c) following infusion of HRP after 60 min of aldehyde fixation. Examination of the material from the three different experimental groups revealed no qualitative differences at the light microscopic level; specific features such as ring-shaped extravasations of HRP occurred irrespective of the mode of tracer injection. Tracer-filled pinocytotic vesicles and tubular profiles were abundant in the vascular endothelium after in vivo circulation of HRP, but were virtually absent after supravital and postmortem HRP administration. The results suggest that BBB breakdown for proteins after microembolization is not an energy-dependent process mediated by either pinocytosis or tubular-endothelial channel formation.

Animals↗

PubMed2000

Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs.

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Adenylyl Cyclase Inhibitors↗

PubMed2005

Overexpression of fatty acid synthase in chemically and hormonally induced hepatocarcinogenesis of the rat.

Fatty acid synthase (FAS) is the key enzyme of de novo fatty acid synthesis and has been shown to be involved in carcinogenesis of numerous human malignancies, including breast, colorectal, and prostate carcinomas, often associated with a worse prognosis. Although FAS is mainly expressed in the liver, an implication of FAS in hepatocarcinogenesis, has not yet been investigated. FAS expression is stimulated by insulin and glucose, and insulin is also the primary trigger of hepatocarcinogenesis in an endocrine experimental model, which is induced by low-number transplantation of islets of Langerhans into the livers of diabetic rats. We therefore investigated, whether FAS is implicated in hepatocarcinogenesis in this model and in comparison to chemically induced hepatocarcinogenesis after N-nitrosomorpholine (NNM) treatment in diabetic and normoglycemic rats. Preneoplastic clear-cell foci of altered hepatocytes (FAH), harvested after laser-microdissection of kryostat sections, showed an overexpression of FAS messenger RNA in gene expression profiles, done by array-hybridization, and in quantitative RT-PCR (Light-Cycler). Virtually, all (96-98%) of the subsequently investigated FAH and the glycogenotic hepatocellular adenomas and carcinomas showed an additional strong FAS protein overexpression. In the NNM-model, FAS protein was also overexpressed in the vast majority (87%) of glycogenotic FAH and neoplasms, in particular in the diabetic animals. Also two spontaneous glycogenotic FAH in control animals displayed strong FAS overexpression. Basophilic lesions and neoplasms, which occasionally develop out of the primary clear-cell FAH at later stages of carcinogenesis, however, lost FAS overexpression. In conclusion, FAS overexpression is an early phenonemon in spontaneous, hormonally and chemically induced rat hepatocarcinogenesis, demonstrable in early clear-cell (glycogenotic) FAH and hepatocellular neoplasms. FAS overexpression can be attributed to the local hyperinsulinemia in the transplantation model and belongs to cellular and metabolic alterations in the chemical model, which are induced by an insulinomimetic but yet unknown mechanism.

Adenoma↗

PubMed1991

Predominant cultivable flora in pericoronitis.

The predominant cultivable flora in pericoronitis was investigated by culturing pus from affected sites in 20 patients. Twenty colonies were picked at random from non-selective plates and identified using conventional biochemical and physiological tests, analysis of metabolic end-products by gas chromatography and protein profile analysis. The most frequently isolated organisms were Prevotella (Bacteroides) intermedia, Peptostreptococcus micros, Veillonella species, Fusobacterium nucleatum and Streptococcus mitis. Porphyromonas (Bacteroides) gingivalis was not isolated and asaccharolytic Eubacterium species were virtually absent. The predominant cultivable microflora in pericoronitis was found to be highly anaerobic in nature and superficially similar to that found in chronic periodontitis, although proposed marker organisms of severe periodontitis were absent.

Adolescent↗

PubMed2002

Functional cloning, sorting, and expression profiling of nucleic acid-binding proteins.

A major challenge in the post-sequencing era is to elucidate the activity and biological function of genes that reside in the human genome. An important subset includes genes that encode proteins that regulate gene expression or maintain the structural integrity of the genome. Using a novel oligonucleotide-binding substrate as bait, we show the feasibility of a modified functional expression-cloning strategy to identify human cDNAs that encode a spectrum of nucleic acid-binding proteins (NBPs). Approximately 170 cDNAs were identified from screening phage libraries derived from a human colorectal adenocarcinoma cell line and from noncancerous fetal lung tissue. Sequence analysis confirmed that virtually every clone contained a known DNA- or RNA-binding motif. We also report on a complementary sorting strategy that, in the absence of subcloning and protein purification, can distinguish different classes of NBPs according to their particular binding properties. To extend our functional annotation of NBPs, we have used GeneChip expression profiling of 14 different breast-derived cell lines to examine the relative transcriptional activity of genes identified in our screen and cluster analysis to discover other genes that have similar expression patterns. Finally, we present strategies to analyze the upstream regulatory region of each gene within a cluster group and select unique combinations of transcription factor binding sites that may be responsible for dictating the observed synexpression.

Adenocarcinoma↗

PubMed2001

Amino acid substitutions in Gag protein at non-cleavage sites are indispensable for the development of a high multitude of HIV-1 resistance against protease inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

Amino Acid Sequence↗

PubMed1996

A review of current clinical findings with fluvastatin.

Fluvastatin, the newest member of the class of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, is structurally different from the fungal metabolites (lovastatin, pravastatin, and simvastatin) and is wholly synthetic. Fluvastatin has a distinct biopharmaceutical profile, including a short systemic exposure time (half-life of 1.2 hours) and virtually no active circulating metabolites. Fluvastatin is targeted to the liver, where it is rapidly metabolized; 98% of fluvastatin is protein bound. Double-blind, placebo-controlled studies have demonstrated that fluvastatin at daily dosages of 20-40 mg produces significant decreases from baseline in low-density lipoprotein (LDL) cholesterol on the order of 22-31% in patients with severe primary hypercholesterolemia (mean baseline LDL cholesterol 227 mg/dL) and decreases of 19-25% in patients with familial hypercholesterolemia (mean baseline LDL cholesterol 270 mg/dL). Interim results of a titrate-to-goal, 20-week study in patients with moderate hypercholesterolemia (LDL cholesterol >= 160 mg/dL and triglycerides <= 350 mg/dL) demonstrate that fluvastatin, 20 mg/day, lowers LDL cholesterol by 21% within 6 weeks. Long-term results indicate that the lipid-lowering effects of fluvastatin are sustained for 96 weeks. Further, 1 study has shown that the combination of low-dose fluvastatin plus niacin decreased LDL cholesterol levels 40% without untoward adverse events, suggesting that this combination is effective and safe for patients needing intensive lipid-lowering therapy. Asymptomatic, reversible increases in hepatic transaminase levels occur in fluvastatin-treated patients at a frequency comparable to that reported for other HMG-CoA reductase inhibitors. The 20-30% reduction in LDL cholesterol required by the majority of patients with hypercholesterolemia can be achieved with fluvastatin at 20 or 40 mg/day as well as with the other available HMG-CoA reductase inhibitors at their most commonly prescribed doses. Fluvastatin, priced 40% lower than other statins, provides the most cost-effective means of safely achieving goal LDL cholesterol levels in these patients.

Fatty Acids, Monounsaturated↗

PubMed1999

Specific binding of the E2 subunit of pyruvate dehydrogenase to the upstream region of Bacillus thuringiensis protoxin genes.

During sporulation, Bacillus thuringiensis produces inclusions comprised of different amounts of several related protoxins, each with a unique specificity profile for insect larvae. A major class of these genes designated cry1 have virtually identical dual overlapping promoters, but the upstream sequences differ. A gel retardation assay was used to purify a potential regulatory protein which bound with different affinities to these sequences in three cry1 genes. It was identified as the E2 subunit of pyruvate dehydrogenase. There was specific competition for binding by homologous gene sequences but not by pUC nor Bacillus subtilis DNA; calf thymus DNA competed at higher concentrations. The B. thuringiensis gene encoding E2 was cloned, and the purified glutathione S-transferase-E2 fusion protein footprinted to a consensus binding sequence within an inverted repeat and to a potential bend region, both sites 200-300 base pairs upstream of the promoters. Mutations of these sites in the cry1A gene resulted in decreased binding of the E2 protein and altered kinetics of expression of a fusion of this regulatory region with the lacZ gene. Recruitment of the E2 subunit as a transcription factor could couple the change in post exponential catabolism to the initiation of protoxin synthesis.

Amino Acid Sequence↗

PubMed2006

Antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide, fumarate (LF22-0542), a novel nonpeptidic bradykinin B1 receptor antagonist.

The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

Acrylamides↗

PubMed2007

Global structure analysis of acid-unfolded myoglobin with consideration to effects of intermolecular coulomb repulsion on solution X-ray scattering.

To obtain information on the global structure of protein in the acid-unfolded (AU) state, the structure of apomyoglobin (apoMb) was analyzed by using the solution X-ray scattering (SXS) method. SXS profiles were obtained over a wide range of protein concentrations, 1-18 mg mL-1, under strongly acidic conditions. From analysis of the SXS profile extrapolated to a zero protein concentration, the mean square radius, Rsq, of AU-apoMb at 20 mM HCl was estimated to be 4.81 +/- 0.31 nm. This estimate is more than 1.3 nm larger than those of 3.0-3.5 nm reported thus far. The difference originates from the fact that effects of Coulomb repulsive forces acting between AU-apoMb molecules have not been correctly taken into account in the conventional analysis. In fact, even at a low protein concentration of 1 mg mL-1 close to the limit of measurement in the present SXS method, the solution condition applicable to estimating accurately structural parameters of AU-apoMb is very limited. At HCl concentrations lower than 10 mM, the scattering intensity at a small scattering vector decreases remarkably through the effect of intermolecular repulsive forces and the forward scattering intensity is significantly lower than the estimate from the partial specific volume of protein. On the other hand, at HCl concentrations higher than 50 mM, some compact molten-globule-like structures emerge. As a result, the intermediate concentration of 20 mM HCl is the best choice of the solution condition for determining Rsq of AU-apoMb. The effect of intermolecular Coulomb repulsion on the SXS profile of AU-apoMb is at its maximum for forward scattering and decreases monotonously with an increase in the scattering angle to be virtually negligible at K approximately 0.63 nm(-1). Whereas urea-denatured apoMb shows a SXS profile typical of Gaussian chains, the intrinsic SXS profile of AU-apoMb differs significantly from those of Gaussian chains.

Apoproteins↗

PubMed1991

Reversed-phase high-performance liquid chromatographic characterization of acetic acid extracts of the normal and the diabetic human pancreas.

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. greater than 6000 dalton (Peak I) or with mol. wt. less than or equal to 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscured chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifluoroacetic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗

PubMed2001

Fibroblast growth factor-2 and transforming growth factor-beta1 immunostaining in rat brain after cerebral postischemic reperfusion.

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.

Animals↗

PubMed2006

Development of a three-dimensional multiscale agent-based tumor model: simulating gene-protein interaction profiles, cell phenotypes and multicellular patterns in brain cancer.

Experimental evidence suggests that epidermal growth factor receptor (EGFR)-mediated activation of the signaling protein phospholipase Cgamma plays a critical role in a cancer cell's phenotypic decision to either proliferate or to migrate at a given point in time. Here, we present a novel three-dimensional multiscale agent-based model to simulate this cellular decision process in the context of a virtual brain tumor. Each tumor cell is equipped with an EGFR gene-protein interaction network module that also connects to a simplified cell cycle description. The simulation results show that over time proliferative and migratory cell populations not only oscillate but also directly impact the spatio-temporal expansion patterns of the entire cancer system. The percentage change in the concentration of the sub-cellular interaction network's molecular components fluctuates, and, for the 'proliferation-to-migration' switch we find that the phenotype triggering molecular profile to some degree varies as the tumor system grows and the microenvironment changes. We discuss potential implications of these findings for experimental and clinical cancer research.

Apoptosis↗

PubMed2005

The global transcriptional effects of the human papillomavirus E6 protein in cervical carcinoma cell lines are mediated by the E6AP ubiquitin ligase.

The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of approximately 31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.

Cell Line, Tumor↗

PubMed2003

Biosensor profiling of molecular interactions in pharmacology.

Techniques employed to profile the pharmacological properties of a molecule in vitro normally require some type of radio-, enzymatic- or fluorescent-labeling of the ligand and/or the receptor. In contrast, biosensor techniques do not require labeling, and they allow virtually any complex to be screened with minimal assay development. Scientists in both academia and industry are now using biosensors in areas that encompass almost all sectors of drug discovery, diagnostics and the life sciences. Assays have been developed for the analysis of small molecules, proteins, oligonucleotides, bacteriophage, viruses, bacteria and cells. In addition, novel biosensor applications are being developed for the predictive profiling of key pharmacokinetic parameters of a molecule (adsorption, distribution, metabolism, excretion and toxicity).

Animals↗

PubMed1991

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase.

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Acetates↗