Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Computational prediction”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 973 records · Page 54Linked to original sources
PubMed2004

Cellular models for ADMET predictions and evaluation of drug-drug interactions.

Deficiencies in ADMET (absorption, distribution, metabolism, excretion and toxicity) properties and drug-drug interactions are collectively the major causes of attrition during drug development. As such, assays have been developed with which to study and optimize these key properties in early dug discovery. While screening using systems expressing discrete proteins have provided valuable insight, quantitative structure-activity relationships (QSARs) and predictive computational models, the ability to study several processes in tandem is paramount to in vivo projection. In particular, the key role of transporter proteins in controlling access to drug metabolizing enzymes and other intracellular processes cannot be overlooked. In this respect, cellular models provide a key platform to study the complex interplay between xenobiotic transport and metabolism, which underlie many ADMET issues. In addition, uptake and accumulation in tissues may provide a mechanistic insight into false negatives arising from simple, primary screens, for example, cytochrome P450 (CYP) inhibition analysis. Qualitative and quantitative interspecies differences in the regulation, expression and functional activity of key ADMET processes confound extrapolation from animals to man. However, complementary screens using animal and human material may assist the interpretation of safety assessment findings and help project the risk for early human studies.

Animals↗
PubMed2003

Hepatitis C virus (HCV)--a review molecular biology of the virus, immunodiagnostics, genomic heterogeneity and the role of virus in hepatocellular carcinoma.

Hepatitis C virus (HCV), an RNA and a hepatotropic virus, is the leading cause of viral hepatitis worldwide. Infection with this virus causes a repertoire of liver diseases that include acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), in addition to a number of extra-hepatic manifestations such as lichen planus, oral cancer, etc. At present, patients infected with this virus are treated with interferon either alone or in combination with ribavirin, a guanosine-like nucleoside analog. However, response to this treatment has been rather disappointing. For about a decade, lack of an alternative animal model other than chimpanzee, and an efficient cell culture system that could support long-term replication of the virus, hampered research on HCV. Despite this, a significant amount of information with regard to the molecular biology of the virus is available using bacterial cloning-expression systems, and based on computer predictions and analysis. Recent discovery of a cellular receptor to which the virus binds, identification of efficient cell culture/cell-free systems, HCV replicons and the development of a chimeric mouse model, provide a platform to verify the existing knowledge about this virus in the coming years. Additionally these developments aid the researchers in identifying novel therapeutic agents, apart from allowing us to reassess the efficiency of the currently available therapeutics. Presented in this article are a review of existing information with regard to the molecular biology of the virus, immunodiagnostic assays, genomic heterogeneity and the role of the virus in hepatocellular carcinoma. Likely therapeutic strategies other than those currently available are also introduced.

Carcinoma, Hepatocellular↗
PubMed2004

A mutation in signal peptide of rat resistin gene inhibits differentiation of 3T3-L1 preadipocytes.

AIM: To detect the resistin expression of white adipose tissue in diet-induced obese (DIO) versus diet-resistant (DR) rats, and to investigate the relationship of mutated resistin and 3T3-L1 preadipocytes differentiation. METHODS: RT-PCR and Western Blot were used to detect gene /protein expression. 3T3-L1 cells were cultured, transfected, and induced to differentiation using 0.5 mmol/L 3-isobutyl-1-methylxanthine (MIX), 1 mg/L insulin, and 1 micromol/L dexamethasone. Oil red O staining was applied to detect the degree of preadipocytes differentiation. RESULTS: Expression of resistin mRNA was upregulated in DIO rats and downregulated in DR rats. However, the expression levels varied greatly within the groups. Sequencing of the resistin genes from DIO and DR rats revealed a Leu9Val (C25G) missense mutation within the signal peptide in one DR rat. The mutant resistin inhibited preadipocyte differentiation. Local experiments and Western blotting with tagged resistin fusion proteins identified both mutant and wild type proteins in the cytoplasm and secreted into the culture medium. Computer predictions using the Proscan and Subloc programs revealed four putative phosphorylation sites and a possible leucine zipper motif within the rat resistin protein. CONCLUSION: Resistin-increased differentiation may be inhibited by the mutation-containing precursor protein, or by the mutant non-secretory resistin isoform.

3T3-L1 Cells↗
PubMed2004

Inhibition of TF gene expression by antisense oligonucleotides in different cancer cell lines.

Human tissue factor (TF) is the initiator of blood coagulation. Beside this function it is involved in tumor angiogenesis and metastasis. In the study we have evaluated the efficiency of antisense oligonucleotides (AS-ODNs) against TF selected from computational prediction of TF mRNA structure. Fourteen different AS-ODNs were tested in three cell lines of different origin with a high TF content. In cell line MCF-7 expression of TF gene was inhibited up to 50% by the AS-ODN AS-7 in comparison to reference. To investigate the dependence of inhibition efficiency on the AS-ODN position inside a potential target motive we designed further AS-ODNs shifted 2-3 nt among AS-7. One AS-ODN was found as more effective than AS-7. In cell line T508 were obtained moderate effects in inhibition of TF gene expression of 30% by AS-4. In J82 cells TF protein was inhibited up to 68% by two AS-ODNs. In conclusion, we compared inhibition of TF gene expression in different cancer cell lines and found that all effective AS-ODNs were located in the translated region of TF mRNA. Suitability of a target region of an AS-ODN is relatively independent on cell line. In contrast, optimal transfection conditions are dependent on cell line.

Cell Line, Tumor↗
PubMed2005

Synthetic peptide analogs derived from bcr/abl fusion proteins and the induction of heteroclitic human T-cell responses.

BACKGROUND AND OBJECTIVES: Chronic myelogenous leukemia (CML) presents a unique opportunity to develop therapeutic strategies using vaccination against a truly tumor-specific antigen that is also the oncogenic protein required for neoplasia. We have shown in phase I and II trials that a tumor-specific, bcr-abl-derived peptide vaccine can be safely administered to patients with chronic phase CML and can elicit a reliable specific CD4 immune response. However, variable CD8 responses and no HLA A0201-restricted responses were found. One strategy to circumvent this poor immunogenicity is to design synthetic immunogenic analog peptides that cross-react with the native peptides (a heteroclitic response). The aim of this study was to design such peptides. DESIGN AND METHODS: By using computer prediction analysis. We designed a number of synthetic peptides derived from the junctional sequences of CML (p210/b3a2 or p210/b2a2) in which single and double amino acid substitutions were introduced at key HLA A0201 binding positions. The binding of these peptides was tested by a thermostabilization assay using a T2 cell line. RESULTS: We found three peptides that predicted good binding to HLA A0201 molecules and stabilized MHC class I A0201 molecules on the surface of T2 cell lines. These peptides were screened for eliciting HLA restricted, peptide-specific cytotoxic T lymphocyte responses using CD3+ T cells from several A0201 donors and CML patients. The CD8+ cytotoxic T lymphocytes lines were assessed by either interferon-g ELISPOT or a chromium release assay using pulsed, HLA-matched leukemic cell lines. The analog peptides generated larger immune responses (increased CD8 T-cell precursor frequency) than did the native peptides. Importantly, CD8+ T cells stimulated with the new synthetic peptides cross-reacted with the native bcr-abl peptides. INTERPRETATION AND CONCLUSIONS: In conclusion, analog CML fusion peptides with increased immunogenicity and heteroclitic properties can be synthesized and may be useful in vaccination strategies.

Cell Line, Tumor↗
PubMed2005

Resolving T-cell receptor clonality in two and genotype in four multiplex polymerase chain reactions.

BACKGROUND AND OBJECTIVES: The diagnosis of T-cell neoplasia requires the use of immunohistochemistry on tumor sections or molecular genetic analysis of T-cell receptor (TCR) clonality. Multiplex polymerase chain reactions (PCR) offer a sensitive and expeditious approach to determining clonality early in the diagnostic work-up. We determined the sensitivity and specificity of four multiple PCR for genotyping lymphoid neoplasms at the TCR loci gamma (TCRG), delta (TCRD) and beta (TCRB, including complete [Vbeta-Jbeta] and incomplete [Dbeta-Jbeta] rearrangements). DESIGN AND METHODS: Template DNA was derived from frozen or formalin-fixed tissue and from imprints of aspirates or cut tissue surface on a FTA MicroCard. Each multiplex PCR was performed for 36 cycles in a single tube with multiple previously reported fluorescently labeled primers (TCRG and TCRD) or novel homologous primers (TCRB) and analyzed on electropherograms (Genescan), applying stringent criteria for interpreting clonal peaks. Two hundred and eleven clinically and immunohistochemically well-characterized benign and malignant non-T-cell lymphoid proliferations, including 138 B-cell lymphomas, were analyzed to determine specificity. The results were compared with those of 28 peripheral and immature T-cell neoplasms and two NK/T-cell lymphomas to determine sensitivity and compute predictive values. RESULTS: In all T-cell tumors, one or more TCR loci showed clonal rearrangement, which was not evident in two NK/T-cell lymphomas. TCRG was the single most informative locus (clonal rearrangement in 89%), followed by TCRB (79%) and TCRD (39%). Multiplex PCR targeting of TCRG and TCRD together resolved clonality in all T-cell neoplasms, whereas the TCRB locus was clonal in two of three cases with polyclonal TCRG. Unexpectedly, in B-cell lymphomas single clonal incomplete TCRB (Dbeta-Jbeta) peaks were 20 times more likely to occur than clonal TCRG. INTERPRETATION AND CONCLUSIONS: Clonality can be accurately determined in nodal T-cell lymphoma with two single-tube multiplex PCR targeting TCRG and TCRD. TCRB analysis should be considered in equivocal cases in which a polyclonal background may obscure clonal TCRD, but clonal incomplete TCRB rearrangement alone is insufficient for presuming T-cell lineage. In the absence of objective evidence of B-cell neoplasia, multiplex PCR of T-cell receptor genes may be used early in the diagnostic work-up, including for fine needle aspirates.

Adolescent↗
PubMed1992

Mutagenesis of the iron-regulatory element further defines a role for RNA secondary structure in the regulation of ferritin and transferrin receptor expression.

Within the 5'-untranslated region of ferritin mRNAs, there is a conserved region of 28 nucleotides (nt) (the iron regulatory element (IRE)) that binds a protein (the IRE-binding protein (IRE-BP)) involved in the iron regulation of ferritin mRNA translation. We have examined the role of RNA secondary structure on the interaction of the IRE with the IRE-BP. First, the rat light ferritin IRE possesses a structure similar to that of the bullfrog heavy ferritin IRE (Wang, Y.-H., Sczekan, S. R., and Theil, E. C. (1990) Nucleic Acids Res. 18, 4463-4468). This includes an extended stem, interrupted at various points by bulge nucleotides and a 6-nt single-stranded loop (CAGUGU) at its top. Computer predictions and mapping results suggest the presence of a 3-nt (UGC) bulge 5 bases 5' of the loop in the rat IRE. Second, disruption of the base pairing in the upper stem alters IRE secondary structure and reduces the affinity with which the IRE-BP binds the IRE. Third, increasing the size of the loop or the distance between the UGC bulge and the loop reduces the IRE/IRE-BP interaction. Our results indicate that several aspects of IRE secondary structure are important for its high affinity binding to the IRE-BP.

Animals↗
PubMedDate not supplied

A practical approach to the balance between acid production and renal acid excretion in humans.

Precise measurements of net endogenous acid production (NEAP) to determine net acid balance require labor and laboratory intensive steady-state measurements of dietary nutrient intakes and urine and stool composition. In an effort to simplify the task, investigators have devised several alternative methodologies, especially computational predictive models based on diet composition. This paper describes the so-called gold standard, and the details of each alternative methodology, discussing their strengths and potential pitfalls. We also briefly discuss what we believe the optimal NEAP for adult humans, and how to achieve that through diet.

Acid-Base Equilibrium↗
PubMed2006

In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.

The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.

Animals↗
PubMed1991

Functional domains of the 67-kDa laminin receptor precursor.

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.

Amino Acid Sequence↗
PubMed1991

Effect of sub-anaesthetic infusions of propofol on peak velocity of saccadic eye movements.

Peak velocity of saccadic eye movements was studied in six healthy volunteers who received either 0.9% sodium chloride (as a control) or a stepwise rising propofol infusion regime of 0.24, 0.6 and 1.5 mg kg-1 h-1, which produced arterialized venous concentrations equivalent to 1%, 6% and 13% of the estimated EC50 for propofol. The infusion lasted for 75 min (25 min at each infusion rate) and was followed by a 2-h post-infusion recovery period. Peak saccadic velocity was highly significantly depressed during the 15-25-min period after the start of the second and third propofol infusions compared with saline. The relationship between arterialized venous blood concentration and peak saccadic velocity was similar during recovery when concentrations were falling to that during increasing blood propofol concentrations. Peak saccadic velocity decreased linearly with increasing log10 propofol concentration in the range of 25-800 ng ml-1 (14% of control per decade). Measured arterialized venous-blood propofol concentrations were variable, and systematically greater than computer predictions at the two higher infusion rates. In only half the subjects were the subjective assessments significantly correlated with log10 propofol concentrations or with percentage reduction in peak saccadic velocity.

Adult↗
PubMed1989

Sequestration and degradation of alpha 2u-globulin in rat kidney lysosomes.

alpha 2u-Globulin (M.W. = 18,709) is the major protein synthesized in the liver of adult male rats. alpha 2u-Globulin, secreted from the liver, is filtered by the kidneys where about a half of the protein is reabsorbed and the remainder is excreted in the urine. Highly purified lysosomes from rat kidneys were subjected to immunoblot analysis using antiserum against alpha 2u-globulin. A major immuno-reactive band was observed at the position about 1 k dalton smaller than that of the authentic alpha 2u-globulin. The immuno-reactive band in SDS-polyacrylamide gel was excised and the protein, eluted from the gel, was subjected to amino acid sequence analysis by micro Edman method. The N-terminal amino acid sequence of the intralysosomal alpha 2u-globulin was found to be leu-asp-val-ala-lys-leu-asn-gly-... which exactly corresponded to the sequence from the 10th leucine residue of the authentic urinary alpha 2u-globulin. This result suggests that alpha 2u-globulin, sequestered into the kidney lysosomes, is cleaved intralysosomally between the 9th asparagine and the 10th leucine, and the degradative intermediate, thus produced, is accumulated in the lysosomes. The immuno-blot assay gave the value of 210 +/- 70 mg alpha 2u-globulin/g kidney lysosomal proteins. The molecular basis for the resistance of the degradative intermediate of alpha 2u-globulin against further degradation in the kidney lysosomes was discussed in the light of the computer-predicted secondary structure of alpha 2u-globulin.

Alpha-Globulins↗
PubMed1989

Defining agonist peptides of human interleukin-1 beta.

The structure-function relationship in the human IL-1 beta protein has been analyzed by means of computer prediction, synthesis of peptides and use of monoclonal antibodies of predetermined specificity. A nine amino acid-long fragment (VQGEESNDK), corresponding to the human IL-1 beta sequence in position 163-171, was shown to possess only some of the IL-1 activities, i.e. those directed to the immune system, while being devoid of IL-1-like inflammatory effects. It is thus proposed that discrete domains within the IL-1 beta protein might be responsible for the different biological activities of the molecule and that the 163-171 fragment may represent one of the immunostimulatory sites of IL-1 beta.

Adjuvants, Immunologic↗
PubMed1987

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit.

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.

Amino Acid Sequence↗
PubMed1982

Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha.

The effect of the DNA sequence of a template on the progress of DNA polymerase alpha was determined at the resolution of single nucleotides by using single-stranded, circular phi X174 DNA as a template and unique phi X 174 ONA fragments terminally labeled at their 3'-ends as primers. Therefore, the amount of radioactivity observed was always proportional to the number and not the length of nascent DNA chains. Extension of a primer by alpha-polymerase revealed that 3'-ends of nascent DNA chains accumulated at specific arrest sites consisting of GC-rich sequences of 1-8 bases distributed nonuniformly along the template with intervening sequences of 0-140 bases. The precise location and composition of these sites were determined by DNA sequencing methods, but a consensus sequence was not detected. However, the same pattern of arrest sites recognized by DNA polymerase alpha from CV-1 cells also arrested alpha-polymerase from HeLa cells, calf thymus tissue, and phage T4 DNA polymerase. An exception was DNA polymerase I from Escherichia coli which was insensitive to arrest signals. Analysis of recombinant DNA templates containing phi X174 DNA inserted into M13 DNA revealed that arrest sites were defined exclusively by those sequences within 24 bases upstream and 140 bases downstream of the arrest point; long range interactions between template sequences were not involved. One of the strongest arrest sites was located in the first two nucleotides at the base of the stem on the primer-proximal side of a stable hairpin structure, suggesting that such structures arrest the polymerase. However, only 28% of all arrest site nucleotides were found in this position; the rest were up to 25 bases upstream from any computer-predicted hairpin. Therefore, all arrest sites cannot be defined by secondary structure alone, although proximity to secondary structures may amplify normal variations in the rate of DNA elongation caused by primary sequences.

Animals↗
PubMed1995

Structure and function of the selenium translation element in the 3'-untranslated region of human cellular glutathione peroxidase mRNA.

In eukaryotes, incorporation of selenocysteine into the polypeptide chain at a UGA codon requires a unique sequence motif, or "selenium translation element" (STE), located in the 3'-untranslated region of the mRNA. The present study examines structure-function relationships of conserved sequence elements and of the putative stem-loop secondary structure in the STE of human GPX1 mRNA, which encodes the important antioxidant enzyme cellular glutathione peroxidase (EC 1.11.1.9). Deletion of the basal stem, upper stem, or apical loop of the stem-loop structure eliminated the ability of the STE to direct selenocysteine incorporation at the UGA codon of an epitope-tagged GPX1 reporter construct transfected into COS1 cells. However, mutations that change the primary nucleotide sequence of nonconserved portions of the stem-loop, but preserve its overall secondary structure, by inversion of apical loop sequences or exchange of 5' and 3' sides of stem segments, had little or no effect on selenocysteine incorporation. Effects of single- and double-nucleotide substitutions in three short, highly conserved elements in the GPX1 STE depended in large part on their computer-predicted perturbation of the stem-loop and its midstem bulge. Only in the conserved "AAA" apical loop sequence did mutations show major effects on function without predicted changes in secondary structure. Our results demonstrate the critical role of the three short, highly conserved sequences. However, outside of these elements, the function of the human GPX1 STE appears to depend strongly on the stem-loop secondary structure.

Animals↗
PubMed1994

Scanning the structure and antigenicity of HPV-16 E6 and E7 oncoproteins using antipeptide antibodies.

The structure and antigenicity of the HPV-16 E6 and E7 oncoproteins was studied using a set of antisera against overlapping synthetic peptides. We report that antigenic, mobile regions of the native proteins, as defined by reactivity with antipeptide antisera, occur at the N-termini of both E6 and E7 proteins, corresponding to regions of known or suspected protein-protein interactions. The putative zinc finger domains were consistently non-reactive, despite computer predictions of relatively high antigenicity, suggesting that the proposed zinc finger regions are held in stable secondary structures that the peptides were not able to mimic. In E6, the linker region between the two zinc fingers was antigenic, indicating that the two zinc finger structures might be able to articulate relative to one another by a flexible linker region. The highly antigenic N-terminal region of HPV-16 E7 was also found to be antigenic in E7 of both HPV-11 and HPV-18, indicating that the E7 proteins of different HPV types have similar antigenic structures. The identification of antigenic regions of the E6 and E7 proteins should be therefore be useful in the design of site-directed antibodies against E6 and E7 for numerous HPV types.

Amino Acid Sequence↗
PubMed1994

[Detection of antigenic determinants of alcohol dehydrogenase for certain biological species using antipeptide antibodies].

The structural and functional peculiarites of alcohol dehydrogenases (ADH) of some biological species have been studied using antipeptide antibodies (AB). The synthetic peptides were used which corresponded to the functionally important sites (1-14, 93-115, 265-276) of equine liver ADH. Conjugates of peptides with protein carriers were obtained and used for immunization of laboratory animals. It was shown that the antipeptide antibodies formed therefrom could interact with the corresponding synthetic peptides, with conjugates derived from them as well as with the native enzyme. The results of cross-reactivity of antipeptide AB with ADH from other biological sources suggest that amino acid sequences (1-14), (93-115) and (265-276) contain common antigenic determinants for the corresponding ADH from animal sources. According to computer prediction data for linear epitopes of equine liver ADH, sequence (93-115) comprises amino acid residues of the antigenic determinants with enhanced conformational rigidity which is consistent with the literary data on the functional significance of this site (the loop ligating the second Zn atom). Sequences (1-14) and (265-276) contain amino acid residues capable of interacting with AB and pertaining, in all probability, to antigenic determinants formed by mobile structures.

Alcohol Dehydrogenase↗