Distinct Amino Acid Substitutions Research Articles

Erg11mutations associated with azole resistance in clinical isolates ofCandida albicans

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).

Ciprofloxacin-resistant Escherichia coli in Central Greece: mechanisms of resistance and molecular identification

BackgroundFluoroquinolone resistant E. coli isolates, that are also resistant to other classes of antibiotics, is a significant challenge to antibiotic treatment and infection control policies. In Central Greece a significant increase of ciprofloxacin-resistant Escherichia coli has occurred during 2011, indicating the need for further analysis.MethodsA total of 106 ciprofloxacin-resistant out of 505 E. coli isolates consecutively collected during an eight months period in a tertiary Greek hospital of Central Greece were studied. Antimicrobial susceptibility patterns and mechanisms of resistance to quinolones were assessed, whereas selected isolates were further characterized by multilocus sequence typing and β-lactamase content.ResultsSequence analysis of the quinolone-resistance determining region of the gyrA and parC genes has revealed that 63% of the ciprofloxacin-resistant E. coli harbored a distinct amino acid substitution pattern (GyrA:S83L + D87N; ParC:S80I + E84V), while 34% and 3% carried the patterns GyrA:S83L + D87N; ParC:S80I and GyrA:S83L + D87N; ParC:S80I + E84G respectively. The aac (6’)-1b-cr plasmid-mediated quinolone resistance determinant was also detected; none of the isolates was found to carry the qnrA, qnrB and qnrS.Genotyping of a subset of 35 selected ciprofloxacin-resistant E. coli by multilocus sequence typing has revealed the presence of nine sequence types; ST131 and ST410 were the most prevalent and were exclusively correlated with hospital and health care associated infections, while strains belonging to STs 393, 361 and 162 were associated with community acquired infections. The GyrA:S83L + D87N; ParC:S80I + E84V substitution pattern was found exclusively among ST131 ciprofloxacin-resistant E. coli. Extended-spectrum β-lactamase-positive ST131 ciprofloxacin-resistant isolates produced CTX-M-type enzymes; eight the CTX-M-15 and one the CTX-M-3 variant. CTX-M-1 like and KPC-2 enzymes were detected in five and four ST410 ciprofloxacin-resistant E. coli isolates, respectively.ConclusionsOur findings suggest that, ST131 and ST410 predominate in the ciprofloxacin resistant E. coli population.

Antibody Pressure by a Human Monoclonal Antibody Targeting the 2009 Pandemic H1N1 Virus Hemagglutinin Drives the Emergence of a Virus with Increased Virulence in Mice

ABSTRACTIn 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift.

Different degrees of loss of function between GEFS+ and SMEI Nav1.1 missense mutants at the same residue induced by rescuable folding defects

Generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) differ in their clinical severity and prognosis even though mutations of the Na(v) 1.1 sodium channel are responsible for both disorders. We compared the electrophysiologic properties of two mutant Na(v) 1.1 channels characterized by distinct amino acid substitutions at the same residue position: GEFS+ (A1685V) and SMEI (A1685D). Both the mutants showed complete loss of function when expressed alone. However, the function of A1685V can be partly rescued by the β(1) subunit, consistently with a folding defect, whereas that of A1685D was not rescued. These electrophysiologic differences are consistent with the divergence in clinical severity between GEFS+ and SMEI.

A novel method for protein–protein interaction site prediction using phylogenetic substitution models

Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. On the basis of this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and nonbinding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins.

Gene Polymorphism of Thymidine Kinase and Dna Polymerase in Clinical Strains of Herpes Simplex Virus

The thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are important targets for genotypic determination of HSV resistance to antiviral drugs. The knowledge of gene polymorphism is an absolute requirement for the correct interpretation of genotypic findings. In this study, the natural polymorphism of TK and DNA pol genes was examined by DNA sequencing in 56 HSV-1 and 12 HSV-2 strains sensitive to acyclovir. In 56 HSV-1 strains, 26 different non-synonymous polymorphism-associated mutations were detected in the TK gene. To our knowledge, 8 of them have never been reported in the literature. In the TK gene of 12 HSV-2 strains, 6 polymorphism-related non-synonymous mutations were observed, whereas there was 1 novel mutation in 1 strain. The DNA pol gene of 53 HSV-1 isolates contained 47 distinct polymorphism-associated amino acid substitutions and 11 substitutions were found in the DNA pol gene of 12 HSV-2 strains. Altogether, 31 novel substitutions could be identified in the DNA pol gene of HSV-1 and 3 in HSV-2 strains. In these strains, any resistance to foscarnet was excluded. The 43 novel non-synonymous mutations enrich the knowledge about the natural genetic polymorphism of TK and DNA pol in clinical HSV strains. The findings have to be considered for genotypic analysis of HSV in case of clinical resistance.

Geo-spatial hotspots of hemorrhagic fever with renal syndrome and genetic characterization of Seoul variants in Beijing, China.

BackgroundHemorrhagic fever with renal syndrome (HFRS) is highly endemic in mainland China, and has extended from rural areas to cities recently. Beijing metropolis is a novel affected region, where the HFRS incidence seems to be diverse from place to place.Methodology/Principal FindingsThe spatial scan analysis based on geographical information system (GIS) identified three geo-spatial “hotspots” of HFRS in Beijing when the passive surveillance data from 2004 to 2006 were used. The Relative Risk (RR) of the three “hotspots” was 5.45, 3.57 and 3.30, respectively. The Phylogenetic analysis based on entire coding region sequence of S segment and partial L segment sequence of Seoul virus (SEOV) revealed that the SEOV strains circulating in Beijing could be classified into at least three lineages regardless of their host origins. Two potential recombination events that happened in lineage #1 were detected and supported by comparative phylogenetic analysis. The SEOV strains in different lineages and strains with distinct special amino acid substitutions for N protein were partially associated with different spatial clustered areas of HFRS.Conclusion/SignificanceHotspots of HFRS were found in Beijing, a novel endemic region, where intervention should be enhanced. Our data suggested that the genetic variation and recombination of SEOV strains was related to the high risk areas of HFRS, which merited further investigation.

Aligning protein sequence and analysing substitution pattern using a class-specific matrix

Aligning protein sequences using a score matrix has became a routine but valuable method in modern biological research. However, alignment in the 'twilight zone' remains an open issue. It is feasible and necessary to construct a new score matrix as more protein structures are resolved. Three structural class-specific score matrices (all-alpha, all-beta and alpha/beta) were constructed based on the structure alignment of low identity proteins of the corresponding structural classes. The class-specific score matrices were significantly better than a structure-derived matrix (HSDM) and three other generalized matrices (BLOSUM30, BLOSUM60 and Gonnet250) in alignment performance tests. The optimized gap penalties presented here also promote alignment performance. The results indicate that different protein classes have distinct amino acid substitution patterns, and an amino acid score matrix should be constructed based on different structural classes. The class-specific score matrices could also be used in profile construction to improve homology detection.

Molecular characterization of moxifloxacin resistance from Canadian Clostridium difficile clinical isolates

Fluoroquinolone resistance in Clostridium difficile has been implicated in recent outbreaks of C. difficile infection. The purpose of this report was to characterize the molecular mechanism conferring resistance to moxifloxacin among C. difficile clinical isolates. Eighty-four C. difficile clinical isolates (collected as part of the Canadian Nosocomial Infection Surveillance Program) were evaluated in the current study. Pulsed-field gel electrophoresis was used to type the isolates. Susceptibility testing was performed using Clinical and Laboratory Standards Institute agar dilution methods. The quinolone resistance-determining region of both gyrA and gyrB was amplified using polymerase chain reaction and sequenced for each isolate. The proportion of isolates studied by the North American pulsed-field (NAP) type was as follows: NAP1 (47.6%), NAP2 (20.2%), NAP3 (5.9%), NAP4 (4.8%), NAP5 (2.4%), NAP6 (3.6%), and other patterns (15.5%). All isolates were resistant to ciprofloxacin. Among moxifloxacin-susceptible isolates (MIC ≤2 μg/mL), no amino acid substitutions were detected in either GyrA or GyrB. Three distinct amino acid substitutions were observed among the 3 isolates that had a moxifloxacin MIC of 8 μg/mL (GyrA Asp71 to Val, GyrB Asp426 to Asn, or Glu466 to Val). Isolates with a moxifloxacin MIC of 16 or 32 μg/mL (moderate-level resistance) all had a single identical amino acid substitution in GyrA (Thr82 to Ile). For isolates with a moxifloxacin MIC of ≥64 μg/mL (high-level resistance), this Thr82 to Ile substitution in GyrA was accompanied by at least 1 other amino acid substitution in either GyrA (Asp71 to Glu, Pro116 to Ala, or Ala118 to Ser) or GyrB (Ser366 to Ala, Asp426 to Asn, Asp426 to Val, or Leu444 to Phe) in all but 1 case. Moderate-level moxifloxacin resistance was associated with a single substitution in GyrA. High-level moxifloxacin resistance was associated with this GyrA substitution plus at least 1 other substitution in GyrA or GyrB.

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

Resistance to Targeted Therapy in Chronic Myelogenous Leukemia

The advent of the Bcr-Abl selective tyrosine kinase inhibitor imatinib mesylate (Glivec, Gleevec, Novartis, East Hanover, NJ) has substantially changed the treatment landscape for chronic myelogenous leukemia (CML). However, some patients, primarily those with advanced disease, are either initially refractory to imatinib or eventually develop imatinib resistance. Imatinib resistance or intolerance frequently depends on the re-emergence of Bcr-Abl kinase activity, but can also indicate Bcr-Abl-independent disease progression. Results from phase II/III trials suggest rates of resistance and relapse correlate with stage of disease and with the monitoring parameters: hematologic, cytogenetic, and molecular responses. To date, more than 40 different point mutations that code for distinct single amino acid substitutions in the Bcr-Abl kinase domain have been isolated from imatinib-resistant patients. These mutations affect amino acids involved in imatinib binding or in regulatory regions of the Bcr-Abl kinase domain, resulting in decreased sensitivity to imatinib while retaining aberrant kinase activity. Early mutation detection may aid in risk stratification and molecular-based treatment decisions. To overcome imatinib-resistant disease, novel tyrosine kinase inhibitors with activity against imatinib-resistant mutations and/or with inhibition of alternative pathways, such as Src activation, have recently been developed. Additional strategies include imatinib dose escalation, combination therapy, and treatment interruption to stop clonal selection of resistant cells.

Retinitis pigmentosa associated with rhodopsin mutations: Correlation between phenotypic variability and molecular effects

Similar retinitis pigmentosa (RP) phenotypes can result from mutations affecting different rhodopsin regions, and distinct amino acid substitutions can cause different RP severity and progression rates. Specifically, both the R135L and R135W mutations (cytoplasmic end of H3) result in diffuse, severe disease (class A), but R135W causes more severe and more rapidly progressive RP than R135L. The P180A and G188R mutations (second intradiscal loop) exhibit a mild phenotype with regional variability (class B1) and diffuse disease of moderate severity (class B2), respectively. Computational and in vitro studies of these mutants provide molecular insights into this phenotypic variability.

Functional restraints on the patterns of amino acid substitutions: Application to sequence–structure homology recognition

Amino acid residues that are involved in functional interactions in proteins have strong evolutionary pressure to remain unchanged and consequently their substitution patterns are different from those that are noninteracting. To characterize and quantify the differences between amino acid substitution patterns due to structural restraints and those under functional restraints, we have made a comparative analysis of families of homologous proteins. Residues classified as having the same amino acid type, secondary structure, accessibility, and side-chain hydrogen bonds are shown to be better conserved if they are close to the active site. We have focused on enzyme families for this analysis since they have functional sites that are easily defined by their catalytic residues. We have derived new sets of environment-specific substitution tables, which we term function-dependent environment-specific substitution tables, where amino acid residues are classified according to their distance from the functional sites. The residues that are within a distance of 9 A from the active site have distinct amino acid substitution patterns when compared to the other sites. The function-dependent environment-specific substitution tables have been tested using the sequence-structure homology recognition program FUGUE and the results compared with the recognition performance obtained using the standard environment-specific substitution tables. Significant improvements are obtained in both recognition performance and alignment accuracy using the function-dependent environment-specific substitution tables (P-value = 0.02, according to the Wilcoxon signed rank test for alignment accuracy). The alignments near the active site are greatly improved with pronounced improvements at lower percentage identities (less than 30%).

Tumor galectinology: insights into the complex network of a family of endogenous lectins.

Beta-Galactosides of cell surface glycoconjugates are docking sites for endogenous lectins of the galectin family. In cancer cells, primarily galectins-1 and -3 have been studied to date. With the emergence of insights into their role in growth control, resistance to or induction of apoptosis and invasive behavior the notion is supported that they can be considered as functional tumor markers. In principle, the same might hold true for the other members of the galectin family. But their expression in tumors has hitherto been a subject of attention only to a very limited extent. Pursuing our concept to define the complexity of the galectin network in cancer cells and the degree of functional overlap/divergence with diagnostic/therapeutic implications, we have introduced comprehensive RT-PCR monitoring to map their galectin gene expression. The data on so far less appreciated galectins in this context such as galectins-4 and -8 vindicate this approach. They, too, attach value to extend the immunohistochemical panel accordingly. Our initial histopathological and cell biological studies, for example on colon cancer progression, prove the merit of this procedure. Aside from the detection of gene expression profiles by RT-PCR, the detailed molecular biological monitoring yielded further important information. We describe different levels of regulation of galectin production in colon cancer cells in the cases of the tandem-repeat-type galectins-8 and -9. Isoforms for them are present with insertions into the peptide linker sequence attributed to alternative splicing. Furthermore, variants with distinct amino acid substitutions (galectin-8, Po66-CBP, PCTA-1, CocaI/II and galectin-9/ecalectin) and generation of multiple mRNA species, notably those coding for truncated galectin-8 and -9 versions with only one lectin site, justify to portray these two family members not as distinct individuals but as groups. In aggregate, the ongoing work to thoroughly chart the galectin network and to disentangle the individual functional contributions is expected to make its mark on our understanding of the malignant phenotype in certain tumor types.

Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C.

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of sigma54 holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated ntrC genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of glnA transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrCWT). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrCWT at low concentrations. Their elevated activation capacities remain to be explained.

Activating mutations of the thyrotropin receptor--unanswered questions

The cloning of the TSH receptor (TSHr) in 1989-1990 was a seminal event in thyroidology. Much has been learned regarding the pathogenesis of a variety of thyroid diseases since that milestone. In recent months, it has become apparent that specific mutations of TSHr involving the transmembrane domain result in the expression of an altered receptor protein that possesses constitutive activity, i.e. increased signal transduction in the absence of its hormone ligand (l-3). These anomalous receptors are the apparent cause of some autonomously functioning thyroid nodules, where they appear as somatic mutations. More recently, germ line transmission of these mutations was found in families of patients with congenital, nonimmune hyperthyroidism (toxic thyroid hyperplasia, TTH) (4-6). In the current issue of JCEM (see page 0000), de Roux et al. describe an infant suffering from congenital, nonimmune hyperthyroidism (7). Following polymerase chain reaction (PCR) amplification of genomic DNA followed by direct sequence analysis, the authors showed that the infant expressed a TSH receptor gene containing a mutation within the second membrane-spanning segment of the transmembrane domain. Neither parent possessed the abnormal codon, indicating that the event was, in this case, a spontaneous mutation in the infant. When expressed in vitro, this mutant receptor demonstrated constitutive activity, similar to that described previously in other cases of congenital, nonimmune hyperthyroidism and autonomously functioning thyroid nodules (AFTN). The importance of the finding in this patient, other than the description of yet another mutation resulting in the phenotype, is that it is the first reported involving the second transmembrane segment, thereby expanding regions of the receptor harboring activating mutations. To date, all of the mutations found in patients with AFTN or TTH have been confined to exon 10, which codes for the entire transmembrane domain (TMD) of the receptor as well as the carboxyl terminal portion of the extracellular domain. As of the writing of this review, 13 distinct amino acid substitutions within the TMD that result in expression of a constitutively active TSH receptor and the phenotype of hyperthyroidism have been described. The locations of these mutations are well scattered across the TMD and involve the second, third, sixth, and seventh membrane-spanning segments as well as the first and second extracellular and the third intracellular loops (6, 7). Mutations within the large extracellular domain encoded by exons l-9 have also been described (8,9). However, none of these later alterations have

Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita

The periodic paralyses are a group of autosomal dominant muscle diseases sharing a common feature of episodic paralysis. In one form, paramyotonia congenita (PC), the paralysis usually occurs with muscle cooling. Electrophysiologic studies of muscle from PC patients have revealed temperature-dependent alterations in sodium channel (NaCh) function. This observation led to demonstration of genetic linkage of a skeletal muscle NaCh gene to a PC disease allele. We now report the use of the single-strand conformation polymorphism technique to define alleles specific to PC patients from three families. Sequencing of these alleles defined base pair changes within the same codon, which resulted in two distinct amino acid substitutions for a highly conserved arginine residue in the S4 helix of domain 4 in the adult skeletal muscle NaCh. These data establish the chromosome 17q NaCh locus as the PC gene and represent two mutations causing the distinctive, temperature-sensitive PC phenotype.

Thyroid hormone resistance syndrome. Inhibition of normal receptor function by mutant thyroid hormone receptors.

Thyroid hormone (T3) resistance is inherited in most cases in an autosomal dominant manner. The disorder is characterized by elevated free thyroid hormone levels and partial resistance to thyroid hormone at the cellular level. Distinct single amino acid substitutions in the ligand binding domain of the beta form of the thyroid hormone receptor have been described in two kindreds with this disorder. We used transient expression assays to characterize the functional properties of these receptor mutants, one containing a Gly to Arg change at amino acid 340 (G340R) and the other a Pro to His change at amino acid 448 (P448H). A nine amino acid carboxy terminal deletion (delta 448-456), analogous to an alteration that occurs in v-erbA, was also studied for comparison with the mutations that occur in the T3 resistance syndrome. None of the receptor mutants were able to mediate thyroid hormone dependent activation (TreTKCAT) or repression (TSH alpha CAT) of reporter genes when compared with the wild type receptor. In addition, the mutants inhibited the activity of normal alpha and beta receptor isoforms when examined in coexpression assays. This activity, referred to as dominant negative inhibition, was manifest with respect to both the positively and negatively regulated reporter genes. Although mutant receptor binding to DNA was unaffected, ligand binding studies showed that the G340R and delta 448-456 mutants failed to bind T3, whereas the P448H mutant bound hormone with reduced affinity (approximately 10% of normal) compared to the wild type receptor. Consistent with this finding, the P448H mutant receptor was partially active at higher T3 concentrations. Furthermore, the dominant negative inhibition elicited by the P448H receptor mutant at higher T3 concentrations was reversed in the presence of high doses of T3. These findings indicate that mutant beta receptors in patients with thyroid hormone resistance have reduced affinity for T3 and are functionally deficient, but impair the activity of normal receptors, thereby providing a mechanism for the dominant mode of inheritance in this disorder.

The Antigenic Structure of a Human Influenza A (H1N1) Virus Isolate Grown Exclusively in MDCK Cells

A panel of monoclonal antibodies has been produced against a 1983 human influenza A (H1N1) virus that has been isolated and grown exclusively in MDCK cells. Several of these antibodies were used to select variants from MDCK-derived virus in cells and their epitopes were then located by determining the HA1 amino acid sequence. The operational antigenic map of the haemagglutinin indicates the presence of two distinct immunodominant antigenic regions which correspond but are not identical to antigenic sites Sa and Sb of the A (H1N1) virus A/PR/8/34. Also during this study, we characterized a unique group of antibodies for which variants require two distinct and specific HA1 amino acid substitutions to escape neutralization.