Replacement Of Aspartic Acid Research Articles

Case Report: Hemoglobin Hasharon with Concurrent Alpha Thalassemia in a Patient with Maple Syrup Urine Disease

Abstract Hemoglobin Hasharon (Hb Hasharon) is an unstable hemoglobin variant stemming from a mutation in the alpha globin gene. This mutation involves the replacement of aspartic acid with histidine at position 47 of the alpha globin chain. Initially identified in 1967 in Israel, Hb Hasharon has since been sporadically reported in various populations, including in Italy, Greece, Germany, and among Latin Americans of Mediterranean descent. Only three English articles have reported several cases of the combined alpha thalassemia deletion and Hasharon mutation, all between 1978-1980. Notably, there has been no prior report of Hb Hasharon with concurrent alpha thalassemia in patients with maple syrup urine disease (MSUD). We hereby present the first case of Hb Hasharon with alpha thalassemia in a patient diagnosed with MSUD. This patient, a 16-month-old female, received a prenatal diagnosis of MSUD through amniocentesis, confirming homozygosity for a known pathogenic variant in DBT (c.75_76delAT, p.C26wfsX2). Prompt initiation of formula feeding has effectively controlled her MSUD. The patient was referred for further evaluation due to hemoglobin newborn screen showing hemoglobin A, Hb F and “variant hemoglobin”. Her parents denied any family history of hemoglobinopathy. Her complete blood count was predominantly normal except for mild microcytosis, with a mean corpuscular volume of 69.1 fl (normal range 70.0-86.0 fl). The patient didn’t exhibit neonatal jaundice or any other signs or symptoms of hemolysis. The reticulocyte count was also within normal limits. The hemoglobin electrophoresis results revealed a complex pattern, with 28.8% of an unknown hemoglobin between zone 4 and 5 on capillary zone electrophoresis (CZE) as well as a possible A2 variant in zone 1. High-performance liquid chromatography (HPLC) indicated a 29.1% peak at a retention time of 4.72 minutes, along with several smaller peaks, that were indicative of the degradation of the unstable unknown hemoglobin. Although the zones and retention time aligned with Hb Hasharon, the percentage differed from the expected range of 19-20% in CZE and 18-21% in HPLC for this variant, prompting the team to order alpha and beta globin sequencing tests. Beta globin gene complete sequencing yielded negative results, while the sequencing of alpha globin gene revealed that the patient carries one copy of the -alpha3.7 alpha-globin deletion and one copy of the c.142G>C (p.Asp48His) variant in the alpha2-globin (HBA2) gene, resulting in an -alpha/alpha2alpha1 genotype with Hb Hasharon. While Hb Hasharon is generally not clinically significant, its co-occurrence with alpha-thalassemia can lead to hemolytic anemia. In our patient, clinic and chemical parameters were predominantly within normal ranges, except for mild microcytosis. The potential interaction between MSUD and Hb Hasharon with concurrent alpha thalassemia remains unknown. Continued patient monitoring will enable us to evaluate whether these conditions may influence each other’s clinical and biochemical profiles.

In-silico genomic landscape characterization and evolution of SARS-CoV-2 variants isolated in India shows significant drift with high frequency of mutations

In-silico studies on SARS-CoV-2 genome are considered important to identify the significant pattern of variations and its possible effects on the structural and functional characteristics of the virus. The current study determined such genetic variations and their possible impact among SARS-CoV-2 variants isolated in India. A total of 546 SARS-CoV-2 genomic sequences (India) were retrieved from the gene bank (NCBI) and subjected to alignment against the Wuhan variant (NC_045512.2), the corresponding amino acid changes were analyzed using NCBI Protein-BLAST. These 546 variants revealed 841 mutations; most of these were non-synonymous 464/841 (55.1%), there was no identical variant compared to the original strain. All genes; coding and non-coding showed nucleotide changes, most of the structural genes showed frequent nonsynonymous mutations. The most affected genes were ORF1a/b followed by the S gene which showed 515/841 (61.2%) and 120/841 (14.3%) mutations, respectively. The most frequent non-synonymous mutation 486/546 (89.01%) occurred in the S gene (structural gene) at position 23,403 where A changed to G leading to the replacement of aspartic acid by glycine in position (D614G). Interestingly, four variants also showed deletion. The variants MT800923 and MT800925 showed 12 consecutive nucleotide deletion in position 21982–21993 resulting in 4 consecutive amino acid deletions that were leucine, glycine, valine, and tyrosine in positions 141, 142, 143, and 144 respectively. The present study exhibited a higher mutations rate per variant compared to other studies carried out in India.

Structural basis of sterol binding and transport by a yeast StARkin domain

The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Saccharomyces cerevisiae. Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.

Molecular epidemiology of canine parvovirus in Morocco

Since it first emergence in the mid-1970's, canine parvovirus 2 (CPV-2) has evolved giving rise to new antigenic variants termed CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original strain and had been variously distributed worldwide. In Africa limited data are available on epidemiological prevalence of these new types. Hence, the aim of the present study was to determine circulating variants in Morocco. Through TaqMan-based real-time PCR assay, 91 samples, collected from symptomatic dogs originating from various cities between 2011 and 2015, were diagnosed. Positive specimens were characterised by means of minor groove binder (MGB) probe PCR. The results showed that all samples but one (98.9%) were CPV positive, of which 1 (1.1%) was characterised as CPV-2a, 43 (47.7%) as CPV-2b and 39 (43.3%) as CPV-2c. Interestingly, a co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples and 3 (3.3%) samples were not characterised. Sequencing of the full VP2 gene revealed these 3 uncharacterised strains as CPV-2c, displaying a change G4068A responsible for the replacement of aspartic acid with asparagine at residue 427, impacting the MGB probe binding. In this work we provide a better understanding of the current status of prevailing CPV strains in northern Africa.

Molecular and Functional Characterization of Two Novel Missense Mutations in Iron Transport Protein Causing Hypochromic Microcytic Anemia and Hemosiderosis

Abstract 3202Atransferrinemia is a rare autosomal recessive disorder characterized by hypochromic microcytic anemia and iron overload. It is extremely rare with 10 cases reported worldwide and only half of them have been characterized at the genetic level. In this report we describe two novel mutations in the transferrin gene found in two patients in two unrelated families from India.The proband-I was a 9 year old girl who had severe hypochromic microcytic anemia requiring intermittent transfusions since the age of four years. She presented at 7 years of age with symptomatic anemia requiring blood transfusion not responding to oral iron therapy. She had pallor and mild hepato splenomegaly and her hemoglobin was 6.0g/dL with a MCV of 67.1fL. Her hemoglobin F (HbF) and hemoglobin A2 (HbA2) was normal and screening of beta globin gene did not reveal any mutation. Bone marrow examination done to rule out CDA & MDS showed a cellular marrow with absent iron stores. She didn’t respond to oral iron supplements and her serum iron was 12ug/dL and TIBC was 110ug/dL with a ferritin of 3205ng/ml. Atransferrinemia was suspected because of a low TIBC and transferrin level of 5.7mg/dL confirmed the diagnosis. The proband-II was a 4 year old boy who had presented with hypochromic microcytic anemia and elevated ferritin. He was transfusion dependent from the age of two. His Hb was 6.4g/dl and MCV of 54.8fL with normal hemoglobin F (HbF) and hemoglobin A2 (HbA2) levels. His serum iron was 16ug/dL and TIBC 19 ug/dL with elevated ferritin (12910ng/ml). Low TIBC is correlated with very low transferrin level (TF-7.5mg/dl) found in this patient. DNA was extracted from the patients and parents by standard protocol. Both coding and adjacent regions of the exons in the transferrin gene (TF) were sequenced. Mutation analysis revealed the presence of a mutation c.1940 A>T in homozygous state resulting in a missense mutation p.Asp647Val in the C-lobe in proband-I. Both parents were heterozygous for this mutation and this change was absent in fifty healthy controls. Proband–II showed a homozygous c.1825 C>T variation in the transferrin gene resulting in p.Arg609Trp. Both parents were heterozygous for this mutation. These amino acids were highly conserved across species. When the protein sequence of TF was interrogated using SIFT and Polyphen algorithm for p.Asp647Val and p.Arg609Trp, both mutations were predicted to be pathological and deleterious. SPDBV modeling showed the p.Asp647Val will result in loss of interaction with a crucial amino acid Arg at 632 position which is essential for solvent accessibility and conformational change during iron release. There was a conformational change observed for the substitution of Arg by Trp at 609 position in TF protein by SPDBV modeling that may lead to instability or defective secretion and this has to be evaluated by invitro functional assays. To assess whether p.Asp647Val mutation leads to TF deficiency, in vitro TF production was assayed. T cells from the patient and controls were stimulated with phytohaemagglutinin and IL2 and the transferrin released was measured by ELISA. The amount of transferrin released from proband-I’s T cells at 72hrs was 4.75ng/ml which was much lower when compared to transferrin secreted (72.14ng/ml) from control T cells. In vitro experimental observations clearly suggest that p.Asp647Val mutation in transferrin gene results in protein deficiency. As predicted, replacement of Aspartic acid with valine in C-lobe of transferrin leads to ineffective protein secretion. Proband-I was started on monthly plasma infusions. Her hemoglobin levels improved and she became transfusion independent. Oral iron chelation was started to reduce tissue iron overload. Proband-II has just been infused with plasma and the outcome is awaited. This is the first report from India where prevalence of thalassemia is high and identification of two cases underlines the importance of screening of atransferrinemia in cases with hypochromic microcytic anemia and iron overload in order to start appropriate treatment to reduce iron overload related complications and anemia. Disclosures:Edison:Department Of Biotechnology, Government of India: Research Funding.

Structure–activity and immunochemical data provide evidence of developmental- and tissue-specific myosuppressin signaling

Myosuppressin peptides dramatically diminish contractions of the gut and heart. Thus, delineating mechanisms involved in myosuppressin signaling may provide insight into peptidergic control of muscle contractility. Drosophila myosuppressin (DMS, TDVDHVFLRFamide) structure–activity relationship (SAR) was investigated to identify an antagonist and explore signaling. Alanyl-substituted, N-terminal truncated, and modified amino acid analogs identified residues and peptide length required for activity. Immunochemistry independently provided insight into myosuppressin mechanisms. DMS decreased gut motility and cardiac contractility dose dependently; the different effective concentrations at half maximal-response were indicative of tissue-specific mechanisms. Replacement of aspartic acid 2 (D2) generated an analog with different developmental- and tissue-specific effects; [A2] DMS mimicked DMS in adult gut (100% inhibition), yet decreased larval gut contractions by only 32% with increased potency in pupal heart (126% inhibition). The DMS active core differed across development and in tissues; adult (DHVFLRFamide) and larval gut (TDVDHVFLRFamide), and adult (VFLRFamide) and pupal heart (VFLRFamide). Substitution of D2 and D4 with a modified amino acid, p-benzoyl-phenylalanine, produced developmental- and tissue-specific antagonists. In the presence of protease inhibitors, DMS and VFLRFamide were more effective in adult gut, but lower or unchanged in pupal heart compared to peptide or analog alone, respectively. DMS-specific antisera stained neurons that innervated the gut or heart. This study describes novel antagonists and data to identify developmental- and tissue-specific mechanisms underlying the pleotropic effects of myosuppressin in muscle physiology.

Hemoglobin Q-Iran detected in family members from Northern Iran: a case report

IntroductionHemoglobin Q-Iran (α75Asp→His) is an important member of the hemoglobin Q family, molecularly characterized by the replacement of aspartic acid by histidine. The first report of hemoglobin Q-Iran and the nomenclature of this hemoglobinopathy dates back to 1970. Iran is known as a country with a high prevalence of α- and β-thalassemia and different types of hemoglobinopathy. Many of these variants are yet to be identified as the practice of molecular laboratory techniques is limited in this part of the world. Applying such molecular methods, we report the first hemoglobin Q-Iran cases in Northern Iran.Case presentationAn unusual band was detected in an isoelectric focusing test and cellulose acetate electrophoresis of a sample from a 22-year-old Iranian man from Mazandaran Province. Capillary zone electrophoresis analysis identified this band as hemoglobin Q. A similar band was also detected in his mother's electrophoresis (38 years, Iranian ethnicity). The cases underwent molecular investigation and the presence of a hemoglobin Q-Iran mutation was confirmed by the amplification refractory mutation system polymerase chain reaction method. Direct conventional sequencing revealed a single guanine to cytosine missense mutation (c.226G > C; GAC >CAC) at codon 75 in the α-globin gene in both cases.ConclusionThe wide spectrum and high frequency of nondeletional α-globin mutations in Mazandaran Province is remarkable and seem to differ considerably from what has been found in Mediterranean populations. This short communication reports the first cases of patients with hemoglobin Q found in that region.

Expression of human melanocortin 4 receptor in Saccharomyces cerevisiae

Abstract The melanocortin 4 receptor (MC4R) is involved in the regulation of energy homeostasis and is known as one of the major hypothalamic regulators of food intake. Several studies have shown that replacement of aspartic acid at position 126 of the MC4R abolishes the ligand binding. We used the modified yeast Saccharomyces cerevisiae strain MMY28 to functionally express the MC4R and characterise the importance of this amino acid for ligand based activation of the receptor. The efficiency of the functional expression system was estimated by activation with αMSH, ACTH and THIQ and compared with cAMP response in mammalian cells. We generated the library of MC4R mutants randomised at the amino acid position 126. Recombinant MC4R clones were screened for the αMSH induced activity in yeast. From 9 different amino acids obtained only the natural aspartic acid displayed the ligand dependent activity of MC4R. The MC4R variants with glutamic acid and leucine at position 126, however, displayed higher background activity than other amino acid substitutions. The results suggest that the yeast expression system is suitable for screening of the MC4R receptor ligands and that the substitution of aspartic acid at position 126 of MC4R by different amino acids functionally inactivates the receptor.

Genetic linkage analyses and Cx50 mutation detection in a large multiplex Chinese family with hereditary nuclear cataract

Purpose: The aim of the study was to characterize the underlying mutation in a large multiplex Chinese family with hereditary nuclear cataract.Methods: A 6-generation Chinese family having hereditary nuclear cataract was recruited and clinically verified. Blood DNA samples were obtained from 53 available family members. Linkage analyses were performed on the known candidate regions for hereditary cataract with 36 polymorphic microsatellite markers. To identify mutations related to cataract, a direct sequencing approach was applied to a candidate gene residing in our linkage locus.Results: A linkage locus was identified with a maximum 2-point LOD score of 4.31 (recombination fraction = 0) at marker D1S498 and a maximum multipoint LOD score of 5.7 between markers D1S2344 and D1S498 on chromosome 1q21.1, where the candidate gene Cx50 is located. Direct sequencing of Cx50 showed a 139 G to A transition occurred in all affected family members. This transitional mutation resulted in a replacement of aspartic acid by asparagine at residue 47 (D47N) and led to a loss-of-function of the protein.Conclusions: The D47N mutation of Cx50 causes the hereditary nuclear cataract in this family in an autosomal dominant mode of inheritance with incomplete penetrance.

SCA1-like Disease in Mice Expressing Wild-Type Ataxin-1 with a Serine to Aspartic Acid Replacement at Residue 776

Glutamine tract expansion triggers nine neurodegenerative diseases by conferring toxic properties to the mutant protein. In SCA1, phosphorylation of ATXN1 at Ser776 is thought to be key for pathogenesis. Here, we show that replacing Ser776 with a phosphomimicking Asp converted ATXN1 with a wild-type glutamine tract into a pathogenic protein. ATXN1[30Q]-D776-induced disease in Purkinje cells shared most features with disease caused by ATXN1[82Q] having an expanded polyglutamine tract. However, in contrast to disease induced by ATXN1[82Q] that progresses to cell death, ATXN1[30Q]-D776 failed to induce cell death. These results support a model where pathogenesis involves changes in regions of the protein in addition to the polyglutamine tract. Moreover, disease initiation and progression to neuronal dysfunction are distinct from induction of cell death. Ser776 is critical for the pathway to neuronal dysfunction, while an expanded polyglutamine tract is essential for neuronal death.

Mutation at a Single Position in the V2 Domain of the HIV-1 Envelope Protein Confers Neutralization Sensitivity to a Highly Neutralization-Resistant Virus

Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralization-resistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described α4β7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to α4β7.

Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I

To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I. Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system. A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls. The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

Natural Coinfection with 2 Parvovirus Variants in Dog

To the Editor: Canine parvovirus (CPV) emerged in the late 1970s, presumably by mutations in feline panleukopenia virus, and became a major viral pathogen of dogs worldwide (1). Between 1979 and 1981, the original type 2 virus (CPV-2) was replaced by a new genetic and antigenic variant, type 2a (CPV-2a). Between 1983 and 1984, CPV-2a was replaced by type 2b (CPV-2b), which differs from type 2a by only 1 epitope located at residue 426 of the VP2 capsid protein (2). CPV-2 does not replicate in cats, but the new variants replicate in dogs and cats (3). Recently, an antigenic change has been observed in a new strain, CPV-2c, isolated from domestic dogs in Italy (4). This variant was also detected in Vietnam (5), other countries in Europe (6), and the United States (7). CPV-2c was recently detected in cats (8) and is characterized by a replacement of aspartic acid with glutamic acid at residue 426 of the VP2 capsid protein. To identify CPV types 2, 2a, and 2b, PCR methods were developed (9). However, these methods could not distinguish type 2c from type 2b (4). Consequently, we used a PCR–restriction fragment-length polymorphism (RFLP) assay with endonuclease MboII. This enzyme can distinguish type 2c from other CPVs (4). Recently, a real-time PCR assay based on minor groove binder (MGB) probe technology was developed for rapid identification and characterization of the antigenic variants. This assay is based on 1 nucleotide polymorphism in the VP2 gene (10). In June 2006, a 10-week-old female dog (PT-32/07) was brought to the veterinary clinic in Figueira da Foz, Portugal, with clinical signs of parvovirus infection, after an episode of gastrointestinal disease in her littermates. Three littermates, also brought to the clinic, showed no signs of infection. None of the dogs were vaccinated against CPV. Clinical signs in dog PT-32/07 were lethargy, anorexia, vomiting, diarrhea, and a temperature of 39.3°C. Identical signs were observed in 1 littermate 3 days later; the 2 other dogs did not show any signs other than lethargy and loose stools. Rectal swab samples from all dogs were screened for CPV by using an immunomigration rapid test (Synbiotics Corporation, Lyon, France). Two of the dogs showed negative results, and 2 showed positive results. Feces, serum, and lingual swab samples were positive for parvovirus DNA. DNA was quantified by using a real-time PCR with TaqMan technology performed in an i-Cycler iQ (Bio-Rad Laboratories, Milan, Italy). CPV variants were characterized by using MGB probe technology. This technology uses type-specific probes labeled with different fluorophores (FAM and VIC) that can detect single nucleotide polymorphisms between CPV types 2a/2b and 2b/2c (10). MGB probes specific for type 2b were labeled with FAM in both type 2a/2b and 2b/2c assays, and MGB probes specific for type 2a (type 2a/2b assay) and type 2c (type 2b/2c assay) were labeled with VIC. All specimens from 1 dog (PT-32/07) were positive for the 2 variants of CPV type 2 (CPV 2b and CPV 2c). Conversely, of the 3 littermates, 2 were positive for CPV type 2b and 1 was positive for CPV type 2c in all samples (Table). Table Detection by minor groove binder probe assay of CPV antigenic variants in different specimens from dogs from the same litter (10 weeks old), Portugal, 2006* A conventional PCR and RFLP analyses were performed by using the method of Buonavoglia et al. (4) with known positive CPV-2b and CPV-2c samples as controls to confirm our findings. The 583-bp PCR product obtained from the coinfected dog by using primer pair 555for/555rev was digested with MboII. Digestion generated 2 fragments (≈500 and 80 bp) in all dog samples. The CPV-2c control sample showed 2 fragments (≈500 and 80 bp), and CPV-2b control sample was not digested with MboII. We report CPV-2b and CPV-2c variants in samples from a dog with littermates that were positive for CPV-2b or CPV-2c during an episode of gastrointestinal disease. Coinfection with multiple CPV variants that showed high genetic diversity in the VP2 gene has recently been reported in a domestic cat (8). Continuous and rapid evolution of CPV may cause serious problems in diagnostic testing and vaccine efficacy. Antigenic variation may negatively affect vaccine efficacy if changes occur at major antigenic sites. Thus, continuous monitoring for novel genetic and antigenic virus types is needed. Additional studies are in progress to characterize nucleotide sequences of all CPV isolates from this case.

The role of OXA-1 β-lactamase Asp66 in the stabilization of the active-site carbamate group and in substrate turnover

The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.

A novel connexin50 mutation associated with congenital nuclear pulverulent cataracts

Purpose:To screen for mutations of connexin50 (Cx50)/GJA8 in a panel of patients with inherited cataract and to determine the cellular and functional consequences of the identified mutation.Methods:All patients in the...

Mutations in the SF1-U2AF59-U2AF23 Complex Cause Exon Skipping in Schizosaccharomyces pombe

To identify genes involved in the mechanism to ensure ordered 5' to 3' exon joining in constitutively spliced pre-mRNAs, we screened for mutants that cause exon skipping in the fission yeast Schizosaccharomyces pombe using a reporter plasmid, which contains the ura4+ gene with the nda3 intron 1-exon 2-intron 2 sequence. The reporter plasmid was designed to produce the functional ura4+ mRNA, when the central nda3 exon is skipped during the splicing reaction. We mutagenized cells harboring the plasmid by UV irradiation and isolated 34 ura+ mutants that grew on minimal medium. Of those, eight mutants were found to be temperature sensitive (ts) for growth. Complementation analyses revealed that the ts mutants belong to three distinct complementation groups named ods (ordered splicing) 1, 2, and 3. RT-PCR analyses showed that products of exon skipping were actually generated in the ods mutants. We cloned the genes responsible for the ods mutations, and found that ods1+, ods2+, and ods3+ encode splicing factors Prp2p/U2AF59, U2AF23, and SF1, respectively, which form a SF1-U2AF59-U2AF23 complex involved in recognition of the branch-point and 3' splice site sequences in a pre-mRNA. We also showed that mutations in the SF1-U2AF59-U2AF23 binding sequences in the reporter plasmid result in exon skipping in wild-type S. pombe cells. In addition, drugs that decrease the rate of transcription elongation were found to suppress the exon skipping in the ods mutants. These results suggest that co-transcriptional recognition of a nascent pre-mRNA by the SF1-U2AF59-U2AF23 complex is essential for ordered exon joining in constitutive splicing in S. pombe.

Species specificity of mammalian connexin‐26 to form open voltage‐gated hemichannels

Mutations of connexin-26 (Cx26) cause nonsyndromic hearing loss and other syndromes affecting ectoderm-derived tissues. While the exact mechanisms underlying these diseases remain elusive, Cx's are generally considered to mediate cell-to-cell communication by forming gap junction channels. We show here that unlike rat Cx26, human and sheep Cx26 form voltage-gated hemichannels when expressed in oocytes and Neuro2A cells. A single evolutionary amino acidic change at position 159 of the rodent protein, the replacement of aspartic acid with asparagine in the human and sheep proteins, accounts for this species specificity. At the resting potential and in normal millimolar extracellular calcium, open human Cx26 hemichannels can be detected both electrophysiologically and by dye uptake, although they did not affect cell viability. These hemichannels opened at approximately -50 mV and their activation increased by depolarization until they inactivate at positive membrane potentials. Single-channel analysis revealed that activation and inactivation involved two distinct voltage gating mechanisms and that the fully open hemichannel displays a conductance twice that of the intercellular channel. The existence of a hemichannel that opens under physiological control of the membrane potential may have important implications for the normal and pathological activity of Cx26 in humans, particularly with respect to hearing and the epidermis.

Purification and molecular cloning of a DNA ADP-ribosylating protein, CARP-1, from the edible clam Meretrix lamarckii

The cabbage butterflies Pieris rapae and Pieris brassicae have unique enzymes, named pierisin-1 and -2, respectively, that catalyze the ADP-ribosylation of guanine residues of DNA, which has been linked with induction of apoptosis and mutation in mammalian cell lines. In the present study, we identified ADP-ribosylation activity targeting DNA in six kinds of edible clam. Similar to our observations with pierisin-1 and -2, crude extracts from the clams Meretrix lamarckii, Ruditapes philippinarum, and Corbicula japonica incubated with calf thymus DNA and beta-NAD resulted in production of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine. The DNA ADP-ribosylating protein in the hard clam M. lamarckii, designated as CARP-1, was purified by column chromatography, and its cDNA was cloned. The cDNA encodes a 182-aa protein with a calculated molecular mass of 20,332. The protein synthesized in vitro from the cDNA in a reticulocyte lysate exhibited the same ADP-ribosylating activity as that of purified CARP-1. Neither the nucleotide nor the deduced amino acid sequence of CARP-1 showed homology with pierisin-1 or -2. However, a glutamic acid residue (E128) at the putative NAD-binding site, conserved in all ADP-ribosyltransferases, was found in CARP-1, and replacement of aspartic acid for this glutamic acid resulted in loss of almost all ADP-ribosylating activity. CARP-1 in the culture medium showed no cytotoxicity against HeLa and TMK-1 cells; however, introduction of this protein by electroporation induced apoptosis in these cells. The finding of clam ADP-ribosylating protein targeting guanine residues in DNA could offer new insights into the biological significance of ADP-ribosylation of DNA.

Retinal Vein Occlusion Associated With Antithrombin Deficiency Secondary to a Novel G9840C Missense Mutation

To describe a novel missense mutation in the antithrombin gene associated with antithrombin deficiency type I in a 40-year-old man with retinal vein occlusion. Investigational case report. Ophthalmoscopy of the right eye showed hemicentral retinal vein occlusion. The patient's medical history was negative for glaucoma or cardiovascular risk factors. Screening for thrombophilic disorders revealed antithrombin deficiency type I. Based on a genetic analysis, a novel missense mutation of a transition of guanosine to cytosine at nucleotide position 9840 was detected, predicting the replacement of aspartic acid by histidine encoded by codon 366 (D366H) in exon 5. Selective screening may be helpful in identifying patients with retinal vein occlusion with thrombophilic defects. When ordering laboratory tests in patients with retinal vein occlusion, antithrombin deficiency type I should be considered in the differential diagnosis. Our results contribute to a better understanding of the molecular bases of antithrombin deficiency, adding a novel entry for the molecular defects causing antithrombin deficiency type I. Moreover, the identification of this thrombophilic disorder in retinal vein occlusion may be relevant to the issue of the initiation and duration of oral anticoagulant therapy.

Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64

In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.