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Results for “mutational profiling”

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PubMed1994

Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation).

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.

Journal Article↗

PubMed2005

The kinetochore and cancer: what's the connection?

The molecular mechanisms ensuring accurate chromosome segregation during meiosis and mitosis are critical to the conservation of euploidy (normal chromosome number) in eukaryotic cells. A dysfunctional kinetochore represents one possible source for chromosome instability (CIN) and the generation of aneuploidy. The kinetochore is a large complex of proteins and associated centromeric DNA that is responsible for mediating the segregation of sister chromatids to daughter cells via its interactions with the mitotic spindle. Continued identification of conserved kinetochore components in model systems such as yeast has provided a rich resource of candidate genes that may be mutated or misregulated in human cancers. Systematic mutational testing and transcriptional profiling of CIN candidate kinetochore genes should shed light on the kinetochore's role in tumorigenesis, and on the general role CIN plays in cancer development.

Animals↗

PubMed1991

Discrimination of mutagenic intermediates derived from alkylating agents by mutational patterns generated in Escherichia coli.

Reactive intermediates (ultimate mutagens/carcinogens) generated by alkylating agents are unstable and difficult to characterize by chemical means. We have used a genetic system to distinguish the in vivo interactions of eight carcinogenic methylating agents and five ethylating agents by the patterns of induced mutations at different target sites in Escherichia coli WU3610. For this multiple locus assay, target sites were an amber (TAG) and an ochre (TAA) triplet, DNA encoding five suppressor tRNA anticodons, and one unidentified locus. Most of the mutations could be classified as specific sequence changes at the target loci by suppressor analysis using T4 bacteriophage. Ratios of the slopes of dose-response curves for induced mutations were used to generate a profile of preferred sites for mutagenesis independent of mutagen potency. 'Mutational fingerprints' derived from different methylating and ethylating agents were compared, as evidence for the existence of common intermediates responsible for their biological effects. Six methylating agents thought to act via SN1 mechanisms were found to generate similar mutational patterns, indicative of a common mechanism, while two methylating agents reacting via SN2 mechanisms gave different patterns. The mutational fingerprints of SN1- and SN-type ethylating agents were also distinct. Mutational fingerprints may be useful in distinguishing the interactions of different ultimate mutagens.

Alkylating Agents↗

PubMed2003

Mutation of the SBF2 gene, encoding a novel member of the myotubularin family, in Charcot-Marie-Tooth neuropathy type 4B2/11p15.

Autosomal recessive hereditary motor and sensory neuropathy or Charcot-Marie-Tooth disease (CMT) is a severe childhood-onset neuromuscular disorder. Autosomal recessive CMT is genetically heterogeneous with one locus mapped to chromosome 11p15 (CMT4B2). The histopathological hallmarks of CMT4B2 are focal outfoldings of myelin in nerve biopsies. Homozygosity mapping, in a Turkish inbred family with four children affected by CMT characterized by focally folded myelin, provided linkage to the CMT4B2 locus. We identified a large, novel gene, named SET binding factor 2 (SBF2), that lies within this interval and is expressed in various tissues, including spinal cord and peripheral nerve. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and was an obvious candidate for CMT4B2 by virtue of its striking homology to myotubularin-related protein 2 (MTMR2), causing another form of autosomal recessive CMT with outfoldings of the myelin sheaths. Molecular study of the SBF2 gene in the CMT4B family demonstrated the presence of a homozygous inframe deletion of SBF2 exons 11 and 12 in all four affected individuals. On the protein level, this mutation is predicted to disrupt an N-terminal domain that is conserved in SBF2 and its orthologues across species. Myotubularin-related proteins have been suggested to work in phosphoinositide-mediated signalling events that may also convey control of myelination. Localization of SBF2 within the candidate interval, cosegregation with the disease, expression in the peripheral nervous system, and resemblance of the histopathological phenotype to that related to mutations in its paralogue MTMR2 indicate that this gene is the CMT4B2 gene.

Charcot-Marie-Tooth Disease↗

PubMed2006

Genetic and histopathologic evaluation of BRCA1 and BRCA2 DNA sequence variants of unknown clinical significance.

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Alleles↗

PubMed1997

Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent inherited disorders. The majority of cases are due to mutation of the PKD1 gene, on 16p13.3, while in most of the remainder the disease maps to the PKD2 locus, at chromosome 4q21-q23. Recently, the PKD2 gene has been positionally cloned and three nonsense mutations within the coding sequence of the gene identified. Here we report a systematic mutation screening of all 15 exons of the PKD2 gene in chromosome 4-linked ADPKD families, using heteroduplex and SSCP analyses. We have identified and characterized seven novel mutations, with a detection rate of approximately 90% in the population studied. All of the mutations result in the premature stop of translation: four nonsense changes and three deletions. The deletions are all frameshifting, of four T nucleotides in one case and one G nucleotide in the other two. All mutations are unique and are distributed throughout the gene without evidence of clustering. Comparison of specific mutations with the clinical profile in ADPKD2 families shows no clear correlation.

Chromosomes, Human, Pair 4↗

PubMed2004

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

PURPOSE: Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. MATERIALS AND METHODS: One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. RESULTS: Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. CONCLUSION: The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Adult↗

PubMed2002

Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase.

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the hydrolysis of beta-(1, 4)-glycosidic bonds in chitin, the major structural polysaccharide present in the cuticle and gut peritrophic matrix of insects. Two conserved regions have been identified from amino acid sequence comparisons of family 18 glycosyl hydrolases, which includes Manduca sexta (tobacco hornworm) chitinase as a member. The second of these regions in M. sexta chitinase contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. In this study the functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins, D142E, D142N, D144E, D144N, E146D and E146Q, as well as the wild-type enzyme, were produced using a baculovirus-insect cell line expression system. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic and substrate binding properties were determined. Circular dichroism spectra of the mutant proteins were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was required for enzymatic activity because mutants E146Q and E146D were devoid of activity. D144E retained most of the enzymatic activity, but D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity relative to the wild-type enzyme. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicate that E146 probably functions as an acid/base catalyst in the hydrolytic mechanism, as do homologous residues in other glycosyl hydrolases. D144 apparently functions as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.

Amino Acid Sequence↗

PubMed2005

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil.

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasilia, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).

Anti-HIV Agents↗

PubMed2002

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain.

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.

Alkylating Agents↗

PubMed2003

Familial haemolytic uraemic syndrome and an MCP mutation.

BACKGROUND: Mutations in factor H (HF1) have been reported in a consistent number of diarrhoea-negative, non-Shiga toxin-associated cases of haemolytic uraemic syndrome (D-HUS). However, most patients with D-HUS have no HF1 mutations, despite decreased serum concentrations of C3. Our aim, therefore, was to assess whether genetic abnormalities in other complement regulatory proteins are involved. METHODS: We screened genes that encode the complement regulatory proteins-ie, factor H related 5, complement receptor 1, and membrane cofactor protein (MCP)-by PCR-single-strand conformation polymorphism (PCR-SSCP) and by direct sequencing, in 25 consecutive patients with D-HUS, an abnormal complement profile, and no HF1 mutation, from our International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura). FINDINGS: We identified a heterozygous mutation in MCP, a surface-bound complement regulator, in two patients with a familial history of HUS. The mutation causes a change in three aminoacids at position 233-35 and insertion of a premature stop-codon, which results in loss of the transmembrane domain of the protein and severely reduced cell-surface expression of MCP. INTERPRETATION: Results of previous studies on HF1 indicate an association between HF1 deficiency and D-HUS. Our findings of an MCP mutation in two related patients suggest that impaired regulation of complement activation might be a factor in the pathogenesis of genetic forms of HUS. MCP could be a second putative candidate gene for D-HUS. The protein is highly expressed in the kidney and plays a major part in regulation of glomerular C3 activation. We propose, therefore, that reduced expression of MCP in response to complement-activating stimuli could prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury.

Adolescent↗

PubMed2002

Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH.

We studied the effects of increase in the number of surface arginines on the enzyme activity and stability of Trichoderma reesei endo-1,4-beta-xylanase II. The number of arginines was increased in two mutant series. The first set contained six arginines on different sides of the protein surface. These arginines had no significant effect on the thermostability. However, the optimal pH region became narrower. Another series of five arginines was engineered into the 'Ser/Thr surface', formed of part of the double-layered beta-sheet located on one side of the 'right-hand-like' xylanase. These mutations shifted the activity profile to the alkaline region by approximately 0.5-1.0 pH units. In addition, the arginines on the Ser/Thr surface increased the enzyme activity at high temperature, although the enzyme stability in the absence of substrate decreased significantly at 50-55 degrees C. In the presence of the substrate, the thermostability increased 4-5-fold at 60-65 degrees C. Thus, the substrate neutralized the destabilizing effect of Ser/Thr surface arginines and revealed a stabilizing effect of the same mutations. The stabilizing effect of arginines at high temperatures was seen clearly only when five arginines were introduced into the Ser/Thr surface.

Arginine↗

PubMed1992

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.

Amino Acid Sequence↗

PubMed2026

Integrating germline and tumor sequencing to improve hereditary cancer diagnosis and care.

A subset of cancers arises due to inherited germline pathogenic variants in specific genes, known as hereditary cancers. These genes typically include tumor suppressors, DNA repair and replication fidelity genes, and occasionally oncogenes. In most hereditary cancer syndromes, Knudson's two-hit hypothesis applies, where a second somatic event inactivates the remaining allele of a tumor suppressor or DNA repair gene, leading to tumorigenesis. Advancements in genome-wide sequencing have significantly enhanced our understanding of the mutational processes involved in hereditary cancers. In particular, the assessment of microsatellite instability (MSI), tumor mutational burden (TMB), and mutational signatures has emerged as a powerful tool for the identification of hereditary tumors. Tumors with high or ultra-high TMB often reflect underlying DNA repair deficiencies, while specific mutational signatures can pinpoint the defective pathway. These tumor mutational features are especially informative in syndromes involving mismatch repair (MMR), homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and polymerase proofreading. Moreover, tumor sequencing aids in the interpretation of germline variants, identifies somatic mosaicism, and helps differentiate hereditary from sporadic cancers. Additionally, tumor molecular features associated with DNA repair deficiencies offer insights into personalized therapies, such as the use of PARP inhibitors for BRCA1/2-deficient tumors and immune checkpoint inhibitors for MMR- and polymerase proofreading-deficient cancers. Tumor profiling also uncovers actionable mutations in oncogenes like RET and VHL, which can be targeted with specific therapies. This review explores the integration of tumor molecular features with germline genetic data to refine diagnosis, risk assessment, and therapeutic strategies in hereditary cancer.

Humans↗

PubMed2007

K-ras mutation and p16 and preproenkephalin promoter hypermethylation in plasma DNA of pancreatic cancer patients: in relation to cigarette smoking.

OBJECTIVES: To examine the profiles of K-ras mutations and p16 and preproenkephalin (ppENK) promoter hypermethylation and their associations with cigarette smoking in pancreatic cancer patients. METHODS: In plasma DNA of 83 patients with untreated primary pancreatic ductal adenocarcinoma, DNA hypermethylation was determined by methylation-specific polymerase chain reaction and K-ras codon 12 mutations by enriched-nested polymerase chain reaction followed by direct sequencing. Information on smoking exposure was collected by in-person interview. Pearson chi test and Fisher exact test were used in statistical analysis. RESULTS: K-ras mutations, ppENK, and p16 promoter hypermethylation were detected in 32.5%, 29.3%, and 24.6% of the patients, respectively. Sixty-three percent (52/83) of patients exhibited at least one of the alterations. Smoking was associated with the presence of K-ras mutations (P = 0.003). A codon 12 G-to-A mutation was predominantly observed in regular smokers and in heavy smokers (pack-year of smoking > or =36). Smoking was not associated with p16 or ppENK hypermethylation. CONCLUSIONS: These preliminary observations suggest that plasma DNA might be a useful surrogate in detecting genetic and epigenetic alterations of pancreatic cancer. The findings on the association between K-ras mutation and smoking were in consistency with previous studies. Further studies on environmental modulators of epigenetic changes in pancreatic cancer are warranted.

Adenocarcinoma↗

PubMed2003

The Iranian Human Mutation Gene Bank: a data and sample resource for worldwide collaborative genetics research.

As Human Genome Project exploration continues, the necessity of having a broader spectrum of genomic DNA material from different nationalities to study various aspects of hereditary disease becomes more obvious. The existence of high genetic polymorphism within and between different communities in the world makes it necessary for the gene hunters to investigate many different populations. Iran, a large country with close to 66 million people, is a land of different nationalities, tribes, and religions that offers a highly heterogeneous gene pool to the genetics researcher. The purity of many different races in this country has been highly conserved by geographical borders and by an ancient culture that has always encouraged intrafamilial marriages. All these have created a population that is remarkably heterogeneous yet high in consanguinity rate. During the last five years of investigation we have established a DNA bank, the Iranian Human Mutation Gene Bank (www.IHMGB.com), which contains all genetic diseases studied in Iran that have the Mendelian mode of inheritance. Some of the samples are assigned to common or novel mutations and others belong to patients with clinical profiles associated with particular genetic diseases but undefined mutation. This bank stores samples of DNA from the patient and his/her first-degree relatives together with a comprehensive pedigree and clinical profile for each sample. To facilitate collaboration with other scientists around the world with the same interests, we decided to present our experimental projects online. This DNA bank provides opportunities for us to collaborate with scientists outside Iran. It offers a sample resource to research scientists around the world, at no charge, for the purpose of investigating the various aspects of genetic disorders from prenatal diagnosis to gene structure and function. It is strongly stressed that no commercial benefit is involved in the establishment of this DNA bank and the DNA samples are free of charge. However, to meet our goals and to respect ethical values, DNA samples can only be used under certain conditions stated in the User Consent Form.

Consanguinity↗

PubMed1998

Alteration of HERG current profile during the cardiac ventricular action potential, following a pore mutation.

HERG is believed to encode the major sub-unit of the cardiac 'rapid' delayed rectifier K channel (I(Kr)). Both I(Kr) and HERG exhibit marked inward rectification at positive membrane potentials due to rapid inactivation and this is thought to influence significantly the contribution of the current to cardiac action potential (AP) repolarisation. We investigated directly the role played by rapid inactivation, by measuring current activated by a ventricular AP waveform, from Chinese Hamster Ovary cells transfected with HERG cDNA with a point-mutation (S631A) in the pore region. Square command pulses elicited HERG-S631A current which increased progressively in magnitude with test potential up to +30/+40 mV (n=6). During test pulses to +40mV, HERG-S631A exhibited little inactivation compared to wildtype HERG. During an action potential command, WT-HERG current developed progressively during the AP plateau and slow repolarisation phase, showing maximal current between -30mV and -40 mV (n=10). In contrast, HERG-S631A current increased earlier during the AP plateau, with a maximal amplitude near +30mV (n=7). Current then declined as the AP proceeded, giving rise to a 'bow'- or 'inverted-U-' shaped current profile. A mathematical model with inactivation removed from the HERG current reproduced the I-V profile of HERG-S631A. These data provide a direct demonstration that rapid inactivation normally plays a critical role in determining both time-course and voltage dependence of HERG/I(Kr) -current during the cardiac ventricular AP.

Action Potentials↗

PubMed1994

Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.

Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.

Bacillus↗