Mutations in TAP genes are common in cervical cancinomas

Objective. To determine whether squamous cervical cancers exhibit mutations or deletions in MHC class I genes or transport-associated protein (TAP) genes. Methods. Polymerase chain reaction based protocols were used to examine HLA class I and TAP genes in a panel of cervical tumours, using DNA from corresponding blood samples as controls. SSP-PCR protocols were similarly used for examination of all TAP alleles in tumour and blood samples. Results. In a series of cervical carcinomas, 7 of 27 (26%) exhibited mutations in HLA-A genes, while 12 of 23 (52%) exhibited mutations in TAP genes. HLA gene mutations were detected in 2 of 14 CIN2-3 lesions, and TAP gene mutations in none of 14, a frequency significantly less than observed in the cervical carcinoma samples (P < 0.01). The TAP 2A/2B heterozygous genotype was observed with increased frequency in patients with cervical cancer compared to population controls (P < 0.02). Conclusion. These data suggest that TAP genes may be relevant to evolution of cervical cancer from precursor lesions. (C) 2004 Elsevier Inc. All rights reserved.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site. © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

DHPLC analysis of patients with nevoid basal cell carcinoma syndrome reveals novel PTCH missense mutations in the serol-sensing domain

Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one-third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray-based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SECOG. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.

Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers

Although MYB overexpression in colorectal cancer (CRC) is known to be a prognostic indicator for poor survival, the basis for this overexpression is unclear. Among multiple levels of MYB regulation, the most dynamic is the control of transcriptional elongation by sequences within intron I. The authors have proposed that this regulatory sequence is transcribed into an RNA stem-loop and 19-residue polyuridine tract, and is subject to mutation in CRC. When this region was examined in colorectal and breast carcinoma cell lines and tissues, the authors found frequent mutations only in CRC. It was determined that these mutations allowed increased transcription compared with the wild type sequence. These data suggest that this MYB regulatory region within intron I is subject to mutations in CRC but not breast cancer, perhaps consistent with the mutagenic insult that occurs within the colon and not mammary tissue. In CRC, these mutations may contribute to MYB overexpression, highlighting the importance of noncoding sequences in the regulation of key cancer genes. (c) 2006 Wiley-Liss, Inc.

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

Background and purpose: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. Experimental approach: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. Key results: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N + 1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N + 1) sites but not at the NR1(N0) site significantly reduced Mg2+ block. Conclusion and implications: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.

Effect of site-specific mutations in different phosphotransfer domains of the chemosensory protein ChpA on Pseudomonas aeruginosa motility

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa

Prevalence of drug-resistance mutations and non-subtype B strains among HIV-infected infants from New York State

Summary: Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001-2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998-1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998-1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non-subtype B strain of HIV. It seems that drug-resistant and non-subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies.