PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “mutational profiling”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 1,045 records · Page 58Linked to original sources
PubMed2005

Pneumococcal HtrA protease mediates inhibition of competence by the CiaRH two-component signaling system.

Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.

Amino Acid Sequence↗
PubMed2026

Identification and characterization of non-canonical azole antifungal resistance pathways in Aspergillus fumigatus.

UNLABELLED: Human fungal infections, especially those caused by Aspergillus fumigatus, pose a significant global health threat, particularly in immunocompromised individuals. Azole antifungals are the primary treatment for this pathogen; however, the prevalence of azole-resistant A. fumigatus strains is steadily increasing. Mutations in cyp51A, which encodes an enzyme involved in ergosterol biosynthesis and the molecular target of the azoles, are well established to confer resistance in this fungal species. However, additional mechanisms governing resistance to this antifungal class remain understudied and poorly characterized, despite growing recognition of their importance in clinical resistance. In this study, we investigated the genetic basis of azole resistance in A. fumigatus isolates from clinical settings worldwide, with a particular focus on mechanisms independent of cyp51A (non-canonical). Using a combination of genomic and functional approaches, including whole-genome sequencing and transcriptomic analysis, we identified novel genetic variants and characterized population structure, advancing our understanding of the genetic diversity and evolutionary dynamics of resistance in A. fumigatus. By expanding our understanding of the complex genetic and molecular factors underlying azole resistance in this important human fungal pathogen, this research is poised to inform the development of novel antifungal strategies and contribute to global efforts to combat fungal infections. IMPORTANCE: Azole antifungals are the frontline therapy for infections caused by the opportunistic mold Aspergillus fumigatus, yet resistance to these drugs is rapidly increasing worldwide. Most studies have focused on mutations in cyp51A, the canonical target of azoles; however, a growing proportion of resistant clinical isolates lack these mutations, indicating that alternative resistance mechanisms are emerging. Here, we integrate population genomics, transcriptomics, and functional analyses across a global collection of isolates to define the architecture of cyp51-independent (non-canonical) azole resistance. We show that this resistance phenotype is strongly associated with a distinct population lineage and is driven by a highly polygenic network of metabolic, mitochondrial, and regulatory adaptations rather than single target site mutations. These isolates exhibit extensive transcriptional rewiring and metabolic remodeling under azole stress, suggesting distinct survival strategies beyond canonical resistance. Our findings reveal that azole resistance in A. fumigatus can evolve through diverse evolutionary routes and emphasize the need to monitor and therapeutically target non-canonical pathways that may increasingly contribute to antifungal treatment failure.

Aspergillus fumigatus↗
PubMed2005

Flavonoid structure-activity studies identify 6-prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2.

Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 micromol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 micromol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 micromol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology.

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3/SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17/gp160-CVT. Phenotypic analysis of the NL4.3/SPL2923 p17- and p17/gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3/SPL2923 towards SPL2923.

Anti-HIV Agents↗
PubMed2005

Expression analysis of candidate breast tumour suppressor genes on chromosome 16q.

INTRODUCTION: Chromosome arm 16q is the second most frequent target of loss of heterozygosity in breast cancer and is, therefore, a candidate to contain one or more classic tumour suppressor genes (TSGs). E-cadherin at 16q22 was identified as a TSG in lobular breast cancer, but TSGs in ductal breast cancer remain elusive. Several genes have been suggested as potential candidates (e.g. CBFA2T3, CTCF and WWOX) but no inactivating mutations could be identified in these genes and they thus fail to fit the classic two-hit model for a TSG. With the completion of the human transcriptome, new candidate genes can be distinguished. Besides mutational inactivation, a TSG could, at least in a subset of the tumours, be transcriptionally suppressed or even inactivated. Studying candidate genes for expression and somatic mutations could thus identify the TSGs. METHODS: Possible candidates CBFA2T3, TERF2 and TERF2IP, FBXL8 and LRRC29 and FANCA were studied for insertion and deletion mutations and for expression differences using quantitative RT-PCR in a panel of tumour cell lines and primary tumours with and without loss of 16q. RESULTS: None of the genes showed mutations or obvious expression differences. FANCA expression increased with tumour grade. CONCLUSION: Apparently, the underlying genetics at chromosome 16q are complex or the TSGs remain to be identified. Multiple mechanisms, such as mutations, promoter hypermethylation or haploinsufficiency, might lead to the inactivation of a TSG.

Breast Neoplasms↗
PubMed2002

Analysis of antibody gene rearrangement, usage, and specificity in chronic focal encephalitis.

BACKGROUND: Autoantibodies have been implicated in the development of chronic focal encephalitis (CFE) or Rasmussen's disease, a progressive and intractable form of epilepsy characterized by uncontrollable unilateral focal seizures, brain atrophy, and inflammation. OBJECTIVE: To investigate the origin and characteristics of the B cell population that trigger or sustain brain inflammation in patients with CFE. METHODS: The authors used immunoglobulin (Ig) complementary determining region 3 (CDR3)-size spectratyping and DNA sequencing to examine the rearranged IgG heavy chain (IgGH) transcript repertoire in resected brain samples from four patients with CFE. They also performed Western blotting on human and rat brain homogenates and immunostaining on a human neuronal cell line to test the reactivity of sera from patients with CFE. RESULTS: The authors observed substantial perturbations from the normal, unstimulated repertoire of immunoglobulin genes. Sequencing of randomly selected clones confirmed the restricted profile and provided evidence for somatic mutation patterns characteristic of antigen-specific stimulation. They also observed IgGVH-CDR3 sequence diversity among patients. When sera were assayed from patients with CFE for specificity against rat and human brain homogenates, heterogeneous reactivity patterns were detected among patients. Immunostaining of postmitotic human neuronal cells demonstrated reactivity of some patients' sera against neural antigens. CONCLUSIONS: These findings support an important role for clonally expanded B lymphocytes in some forms of epilepsy, but also indicate a wide spectrum of reactivity characteristic of antigenic heterogeneity.

Animals↗
PubMed1999

Isolation of two novel alternative splicing variants of allograft inflammatory factor-1.

Allograft inflammatory factor-1 (AIF-1) was initially cloned from rat cardiac allografts undergoing chronic rejection. AIF-1, possessing an EF-hand-like motif, is thought to be a Ca2+ binding protein. In this study, we identified two novel alternatively spliced variants of AIF-1 by reverse-transcription polymerase chain reaction. One variant encodes an AIF-1 protein that lacks 14 amino acids corresponding to one exon. The other variant encodes a truncated AIF-1 protein due to a frameshift introduced by an 85-bp insertion, and its C-terminal region differs from that of AIF-1. Interestingly, these variants have incomplete and no EF-handlike motifs, respectively. The expression level of the latter variant is high in peripheral blood leukocytes, and it is selectively expressed in macrophage-like cell lines.

Alternative Splicing↗
PubMed2005

Dynamic force spectroscopy of glycoprotein Ib-IX and von Willebrand factor.

The first stage in hemostasis is the binding of the platelet membrane receptor glycoprotein (GP) Ib-IX complex to the A1 domain of von Willebrand factor in the subendothelium. A bleeding disorder associated with this interaction is platelet-type von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Ibalpha subunit of GP Ib-IX. Using optical tweezers and a quadrant photodetector, we investigated the binding of A1 to GOF and loss-of-function mutants of GP Ibalpha with mutations in the region containing the two known naturally occurring mutations. By dynamically measuring unbinding force profiles at loading rates ranging from 200-20,000 pN/s, we found that the bond strengths between A1 and GP Ibalpha GOF mutants (233, 235, 237, and 239) were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined (p < 0.05). In addition, mutants 231 and 232 exhibited significantly lower bond strengths with A1 than the wild-type receptors (p < 0.05). We computed unloaded dissociation rate constant (k(off)(0)) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k(off)(0) value of 5.47 +/- 0.25 s(-1) to be significantly greater than the GOF k(off)(0) values and significantly less than the loss-of-function k(off)(0) values. Our data illustrate the importance of the bond kinetics associated with the VWF/GP Ib-IX interaction in hemostasis and also demonstrate the drastic changes in binding that can occur when only a single amino acid of GP Ibalpha is altered.

Amino Acid Sequence↗
PubMed2005

Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa.

The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.

Base Sequence↗
PubMed2001

Candidate-gene cloning and targeted marker enrichment of wheat chromosomal regions using RNA fingerprinting-differential display.

The usefulness of the RNA fingerprinting-differential display technique in gene cloning and targeted marker enrichment in wheat is demonstrated. A small region of chromosome 5BL was targeted that contains Ph1, a chromosome-pairing regulator gene. The cultivar Chinese Spring (CS) and mutant ph1b are almost identical except for chromosome 5BL, which, in the mutant line, carries an interstitial deletion encompassing the Ph1 gene. Poly(A)+ RNA of the two lines from anthers at developmental stages ranging from pre-meiotic mitosis to anaphase II was PCR-amplified using 38 pairwise combinations of 19 primers. The 35S-labeled amplified products were size-separated on denaturing polyacrylamide-urea gels. A total of 3154 fragment bands were observed, of which 43 were present in CS but absent in the ph1b mutant. These 43 fragment bands were eluted, re-amplified, and used as probes in gel-blot DNA analyses of wheat group 5 nullisomic-tetrasomic lines and the ph1b mutant. Twenty-four of these 43 probes were single- or few-copy sequences. Eight of the 24 probes mapped to wheat group 5 and five mapped to the deletion of the ph1b mutant. Three of these five probes were further localized to the submicroscopic region containing the Ph1 gene, by using two deletion lines flanking the region. Northern-blot analysis revealed that the gene corresponding to one of these three probes expresses mainly during meiosis and is from the B genome.

Blotting, Northern↗
PubMed2000

A gene network inference method from continuous-value gene expression data of wild-type and mutants.

In this paper we introduce a new inference method of a gene regulatory network from steady-state gene expression data. Our method determines a regulatory structure consistent with an observed set of steady-state expression profiles, each generated from wild-type and single deletion mutant of the target network. Our method derives the regulatory relationships in the network using a graph theoretic approach. The advantage of our method is to be able to deal with continuous values of steady-state data, while most of the methods proposed in past use a Boolean network model with binary data. Performance of our method is evaluated on simulated networks with varying the size of networks, indegree of each gene, and the data characteristics (continuous-value/binary), and is compared with that of predictor method proposed by Ideker et al. As a result, we show the superiority of using continuous values to binary values, and the performance of our method is much better than that of the predictor method.

Animals↗
PubMed1997

beta-Lactamases: quality and resistance.

beta-Lactamase-mediated resistance to beta-lactam antibiotics is a feature of great clinical significance. beta-Lactamases are a diverse group of bacterial enzymes that vary in their abilities to hydrolyze beta-lactam antibiotics. beta-Lactamases possess an active site containing either a serine moiety or a zinc atom; serine beta-lactamases are currently of greater clinical prevalence. This review considers the molecular classification of beta-lactamases, the structure of the serine beta-lactamase active site and the mechanisms by which beta-lactamase production may become derepressed. The spread of beta-lactamases in the clinical setting and some important structural mutations that have extended the hydrolysis profiles of serine beta-lactamases are also discussed.

Journal Article↗
PubMed1991

Mutant profiles of selectable genetic elements.

A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.

Amino Acid Sequence↗
PubMed2004

Gene expression profiling of fibroblasts from a human progeroid disease (mandibuloacral dysplasia, MAD #248370) through cDNA microarrays.

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder caused basically by a missense mutation within the LMNA gene, which encodes for lamin A/C. We have used gene expression profiling to characterize the specificity of molecular changes induced by the prevalent MAD mutation (R527H). A total of 5531 transcripts expressed in human dermis were investigated in two MAD patients, both carrying the R527H mutation, and three control subjects (age and sex matched). Transcription profiles revealed a differential expression in MAD vs. control fibroblasts in at least 1992 genes. Sixty-seven of these genes showed a common altered pattern in both patients with a threshold expression level >+/-2. Nevertheless, a large number of these genes (43.3%) are ESTs or encode for protein with unknown function; the other genes are involved in biological processes or pathways such as cell adhesion, cell cycle, cellular metabolism, and transcription. Quantitative RT-PCR was applied to validate the microarray results (R2= 0.76). Analysis of the effect of the prevalent MAD mutation (R527H) over the transcriptional pattern of genes expressed in the human dermis showed that this LMNA gene mutation has pleiotropic effects on a limited number of genes. Further characterization of these effects might contribute to understanding the molecular pathogenesis of this disorder.

Abnormalities, Multiple↗
PubMedDate not supplied

Resistance profiles of (+)2'-deoxy-3'-oxa-4'-thiocytidine and (-)2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine.

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.

Anti-HIV Agents↗
PubMed2005

Detection of alpha-galactosidase a mutations causing Fabry disease by denaturing high performance liquid chromatography.

Mutations in the alpha-galactosidase A (alpha-Gal A, GLA) gene cause Fabry disease, an X-linked recessive lysosomal storage disease. The majority of mutations are private, and confirmation of carrier status in females requires the definitive identification of a DNA mutation. In addition, knowledge of a family's mutation enables rapid and precise preimplantation and prenatal genetic testing. Here we report the development and use of DHPLC to rapidly and cost-effectively screen for alpha-Gal A mutations. Optimal DHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to the seven alpha-Gal A exons and their adjacent intronic/flanking sequences. At least five known mutations in each exon (45 in total) were screened by DHPLC to validate the method. Mutation detection was then performed in 14 affected males diagnosed by enzyme assay and 39 at-risk females, and the amplicons with abnormal DHPLC profiles were sequenced. In all affected males, and in 32 of the 39 at-risk females, four and 16 previously reported and 10 and 15 new mutations were identified, respectively. Sequencing all seven alpha-Gal A gene amplicons in the seven at-risk females who had normal DHPLC profiles excluded them as mutation carriers. Only one mutation (p.P362L) was not initially identified by its DHPLC profile, but in retrospect the profile was abnormal, emphasizing the need for experience in inspecting the profiles. In addition, this technique detected two new intronic polymorphisms, c.640-16A>G and c.1000-22C>T, with frequencies of 0.14 and 0.25 in both normal individuals and Fabry patients, respectively. This DHPLC method should improve the rapidity and cost-effectiveness of alpha-Gal A mutation identification in affected males and carrier females for Fabry disease.

Amino Acid Substitution↗
PubMed2006

Metastasis predictive signature profiles pre-exist in normal tissues.

Previous studies from our laboratory have demonstrated that metastatic propensity is significantly influenced by the genetic background upon which tumors arise. We have also established that human gene expression profiles predictive of metastasis are not only present in mouse tumors with both high and low metastatic capacity, but also correlate with genetic background. These results suggest that human metastasis-predictive gene expression signatures may be significantly driven by genetic background, rather than acquired somatic mutations. To test this hypothesis, gene expression profiling was performed on inbred mouse strains with significantly different metastatic efficiencies. Analysis of previously described human metastasis signature gene expression patterns in normal tissues permitted accurate categorization of high or low metastatic mouse genotypes. Furthermore, prospective identification of animals at high risk of metastasis was achieved by using mass spectrometry to characterize salivary peptide polymorphisms in a genetically heterogeneous population. These results strongly support the role of constitutional genetic variation in modulation of metastatic efficiency and suggest that predictive signature profiles could be developed from normal tissues in humans. The ability to identify those individuals at high risk of disseminated disease at the time of clinical manifestation of a primary cancer could have a significant impact on cancer management.

Animals↗