Asp Substitution Research Articles

Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module.

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Structure-Guided Rational Design of a Mono- and Diacylglycerol Lipase from Aspergillus oryzae: A Single Residue Mutant Increases the Hydrolysis Ability.

Engineering of enzymes on the basis of protein structures are rational and efficient approaches to acquire biocatalysts of desired performances. In this study, we focused on a special mono- and diacylglycerol lipase (MDGL) isolated from the lipolytic enzyme-enriched fungus Aspergillus oryzae and discovered improved variants based on its crystal structure. We first solved the crystal structure of Aspergillus oryzae lipase (AOL) at 1.7 Å resolution. Structure analysis and sequence alignment of AOL and other MDGLs revealed that the residue V269 is of vital importance for catalysis. Replacement of the V269 in AOL with the corresponding residues in other MDGLs has led to noticeable changes in hydrolysis without sacrificing the thermostability and substrate specificity. Among the investigated variants, V269D exhibited about a six-fold higher hydrolysis activity compared to the wild type. Molecular dynamics simulations and protein-ligand interaction frequency analyses revealed that the Asp substitution enhanced the substrate affinity of AOL. Our work sheds light on understanding the catalytic process of AOL and helps tailoring MDGLs with desired catalytic performance to fulfill the demand for biotechnological applications.

Asp-376-Glu substitution endows target-site resistance to AHAS inhibitors in Limnocharis flava, an invasive weed in tropical rice fields.

Limnocharis flava (L.) Buchenau is a problematic weed in rice fields and water canals of Southeast Asia, and in Malaysia this invasive aquatic weed species has evolved multiple resistance to synthetic auxin herbicide and acetohydroxyacid synthase (AHAS) inhibitors. In this study, it was revealed that, a single nucleotide polymorphism (SNP) at amino acid position 376, where C was substituted to G at the third base of the same codon (GAC to GAG), resulting in Aspartate (Asp) substitution by Glutamate (Glu) was the contributing resistance mechanism in the L. flava population to AHAS inhibitors. In vitro assay further proved that, all the L. flava individuals carrying AHAS resistance mutation exhibited decreased-sensitivity to AHAS inhibitors at the enzyme level. In the bensulfuron-methyl whole-plant bioassay, high resistance indices (RI) of 328- and 437-fold were recorded in the absence and presence of malathion (the P450 inhibitor), respectively. Similarly, translocation and absorption of bensulfuron-methyl in both resistant and susceptible L. flava populations showed no remarkable differences, hence eliminated the possible co-existence of non-target-site resistance mechanism in the resistant L. flava. This study has confirmed another new case of a target-site resistant weed species to AHAS-inhibitors.

SUN-043 ENDOTHELIAL NITRIC OXIDE SYNTHASE (ENOS) GENE POLYMORPHISM IN MALARIA ASSOCIATED ACUTE KIDNEY INJURY

In endemic areas like odisha state in India, the incidence of AKI (acute kidney injury) may be >4% of malaria cases. The ENOS(Endothelial Nitric Oxide Synthase) derived NO (nitric oxide) mediates vasodilation, inhibits platelet aggregation and and modulates expression of cell adhesion molecules. We hypothesize that functionally important variants of ENOS could influence individual susceptibility to malaria associated AKI by altering the amount of NO generated by the endothelium. Plasma levels of nitric oxide will help in predicting malarial AKI Study was conducted between November 2018 to september 2019. Consecutive patients with acute kidney injury were included in the study. Clinically suspected malaria patients were screened for plasmodium falciparum infection using rapid diagnostic test, thick and thin film method and confirmed by species-specific PCR diagnosis. Venous blood were collected in EDTA-containing vials after informed consent was obtained from all enrolled patients (for analysis of ENOS gene polymorphism and plama nitric oxide level) and immediately centrifuged for 3 min. AKI were staged into 3 stages based on severity according to KDIGO criteria. ENOS gene polymorphism and plasma nitric oxide level measured in malaria associated AKI patients and were compared with other groups of AKI. Patients were followed for monthly basis to determine the course of the disease and progression of renal failure 128 patients with febrile illness associated with AKI were enrolled in the study. Around 38 had malarial AKI. 19 of them were stage 3 AKI. 16 of them underwent hemodialysis. 8 underwent intermittent hemodialysis more than 2 weeks. One patient expired. Of 30 patients who were followed up to 3 months, none developed CKD. We genotyped three commonly defined polymorphic loci of eNOS, Glu298->Asp, intron 4 variable number of tandem repeat region, and T-7863C, in these patients. The median plasma nitric oxide was found to be increased in individuals with the Glu298->Asp substitution and was significantly raised in mild malarial AKI (P < 0.0001), but the increase was not significant in stage 3 malarial AKI. When the genotype and allele frequencies for the three polymorphic sites ofthe eNOS gene (Glu298->Asp, intron 4 VNTR, and T-786->C) were compared for the groups of patients with mild and severe malarial AKI, a significant difference was observed only for Glu2983Asp polymorphism. In patients with mild malarial AKI, the Glu2983Asp substitution is associated with a significant difference in the median plasma NO levels. In patients with severe malaria with the Glu2983Asp substitution, the median plasma NO level was higher for the genotype with the T allele, but the difference was not statistically significant. Our findings suggest that the eNOS Glu298->Asp substitution enhance ENOS expression and NO production and thus have protective effects against malaria associated AKI. Median plasma nitric oxide level were significantly not increased in patients with stage 3 malarial AKI compared to other stages of AKI. These findings will increase our understanding of the pathogenesis of malaria associated AKI,

Characterization of the pleiotropic LysR-type transcription regulator LeuO of Escherichia coli.

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO. This yielded nine mutants carrying amino acid substitutions in the dimerization interface of the regulatory EBD, as shown by solving the EBD’s crystal structure. Superimposing of the crystal structures of LeuO-EBD and LeuO-S120D-EBD suggests that the Ser120 to Asp substitution triggers a structural change that is related to effector-induced structural changes of LTTRs. Corresponding functional analyses demonstrated that LeuO-S120D has a higher DNA-binding affinity than wild-type LeuO. Further, a palindromic DNA-binding core-site and a consensus sequence were identified by DNase I footprinting with LeuO-S120D as well as with the dimeric DBD. The data suggest that LeuO-S120D mimics an effector-induced form of LeuO regulating a distinct set of target loci. In general, constitutive mutants and determining the DNA-binding specificity of the DBD-dimer are feasible approaches to characterize LTTRs of unknown function.

A novel COL11A1 missense mutation in siblings with non-ocular Stickler syndrome.

Stickler syndrome (STL) is an autosomal, dominantly inherited, clinically variable and genetically heterogeneous connective tissue disorder characterized by ocular, auditory, orofacial and skeletal abnormalities. We conducted targeted resequencing using a next-generation sequencer for molecular diagnosis of a 2-year-old girl who was clinically suspected of having STL with Pierre Robin sequence. We detected a novel heterozygous missense mutation, NM_001854.3:n.4838G>A [NM_001854.3 (COL11A1_v001):c.4520G>A], in COL11A1, resulting in a Gly to Asp substitution at position 1507 [NM_001854.3(COL11A1_i001)] within one of the collagen-like domains of the triple helical region. The same mutation was detected in her 4-year-old brother with cleft palate and high-frequency sensorineural hearing loss.

Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death.

Holins are phage-encoded small transmembrane proteins that perforate the bacterial cytoplasmic membrane. In most cases, this process allows the phage-encoded peptidoglycan hydrolases to act on the cell wall, resulting in host cell lysis and phage release. We report a detailed functional characterization of Mycobacterium phage D29 gp11 coding for a putative holin that, upon expression, rapidly kills both Escherichia coli and Mycobacterium smegmatis. We dissected Gp11 by making several deletions and expressing them in E. coli. The shortening of Gp11 from its C-terminus results in diminished cytotoxicity and smaller holes. Evidently, the two transmembrane domains (TMDs) present at the N-terminus of Gp11 are incapable of integrating into the cytoplasmic membrane and do not show toxicity. Interestingly, the fusion of two TMDs and a small C-terminal region that bears the coiled-coil motif resulted in restoration of the cell killing ability of the protein. We further show that the second TMD is dispensable in protein toxicity because its deletion does not abolish Gp11-mediated cell death. We conclude that Gp11 C-terminal region is necessary but not sufficient for toxicity. These results shed light on a yet undiscovered role of Gp11 C-terminal region that will help clarify the mechanism of holin-mediated membrane perforation. Finally, we abolish the toxicity of Gp11 using a specific Gly to Asp substitution in the putative loop region of the protein; the mutant protein may help to clarify how holin functions in mycobacteriophage D29.

The Thr205 Phosphorylation Site within Respiratory Syncytial Virus Matrix (M) Protein Modulates M Oligomerization and Virus Production

Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

A transitional hydrolase to glycosynthase mutant by Glu to Asp substitution at the catalytic nucleophile in a retaining glycosidase

Glycosynthases from more than 16 glycosidase families have been developed for the efficient synthesis of oligosaccharides and glycoconjugates. β-1,3-1,4-Glucan oligo- and polysaccharides with defined sequences can be quantitatively achieved with the glycosynthases derived from Bacillus licheniformis β-1,3-1,4-glucanase. The screening of a nucleophile saturation library of this enzyme yielded the unexpected E134D mutant which has high glycosynthase efficiency (25% higher kcat than the best glycosynthase to date, E134S) but also retains some hydrolase activity (2% relative to the wild-type enzyme). Here, we report the biochemical and structural analyses of this mutant compared to E134S and wild-type enzymes. E134D shows a pH profile of general base catalysis for the glycosynthase activity, with a kinetic pKa (on kcat/KM) assigned to Glu138 of 5.8, whereas the same residue acts as a general acid in the hydrolase activity with the same pKa value. The pKa of Glu138 in the wt enzyme was 7.0, a high value due to the presence of the catalytic nucleophile Glu134 which destabilizes the conjugate base of Glu138. Thus, the pKa of Glu138 drops 1.1 pH units in the mutant relative to the wild-type enzyme meaning that the larger distance between carboxylates in positions 138 and 134 (5.6Å for wt, 7.0Å for E134D) and/or a new hydrogen bonding interaction with a third Asp residue (Asp136) in the mutant reduces the effect of the negatively charged Asp134. In consequence, the pKa of Glu138 has a similar pKa value in the E134D mutant than in the other glycosynthase mutants having a neutral residue in position 134. The behavior of the E134D mutant shows that shortening the side chain of the nucleophile, despite maintaining a carboxylate group, confers glycosynthase activity. Therefore E134D is a transitional hydrolase to glycosynthase mutation.

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

Human Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding proteins (Ggas) bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide-sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p Vps27, Hrs, Stam (VHS) domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently autoinhibitory sequence in the Gga2p hinge. Ser780in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS–GGA and TOM1 (GAT) and glutathione S-transferase–Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities Asp780&gt; Ser780&gt; Ala780. Affinity-purified antibody against a peptide containing phospho-Ser­780recognizes wild-type Kex2p but not S780A Kex2p, showing that Ser780is phosphorylated in vivo; phosphorylation of Ser780is up-regulated by cell wall–damaging drugs. Finally, mutation of Ser780alters trafficking of Kex2p both in vivo and in cell-free trans-Golgi network (TGN)–prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN–PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules.

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

Human Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins (Ggas) bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide-sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p Vps27, Hrs, Stam (VHS) domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently autoinhibitory sequence in the Gga2p hinge. Ser(780) in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS-GGA and TOM1 (GAT) and glutathione S-transferase-Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities Asp(780) > Ser(780) > Ala(780). Affinity-purified antibody against a peptide containing phospho-Ser-(780) recognizes wild-type Kex2p but not S(780)A Kex2p, showing that Ser(780) is phosphorylated in vivo; phosphorylation of Ser(780) is up-regulated by cell wall-damaging drugs. Finally, mutation of Ser(780) alters trafficking of Kex2p both in vivo and in cell-free trans-Golgi network (TGN)-prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN-PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules.

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

Human Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding proteins (Ggas) bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide-sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p Vps27, Hrs, Stam (VHS) domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently autoinhibitory sequence in the Gga2p hinge. Ser780 in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS–GGA and TOM1 (GAT) and glutathione S-transferase–Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities Asp780 > Ser780 > Ala780. Affinity-purified antibody against a peptide containing phospho-Ser­780 recognizes wild-type Kex2p but not S780A Kex2p, showing that Ser780 is phosphorylated in vivo; phosphorylation of Ser780 is up-regulated by cell wall–damaging drugs. Finally, mutation of Ser780 alters trafficking of Kex2p both in vivo and in cell-free trans-Golgi network (TGN)–prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN–PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules.

Characterization of a novel missense mutation on murine Pax3 through ENU mutagenesis

N-ethyl-N-nitrosourea (ENU) mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant, including exencephaly, spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes. This novel mutant was named as Sp(xG). Through genome-wide linkage analysis in backcross progenies with microsatellite markers, the Sp(xG) was confined to a region between D1MIT415 and D1MIT7 on chromosome 1, where notable Pax3 gene was located. Direct sequencing revealed that Sp(xG) carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain (HD) DNA recognition module, which was the first point mutation found in this domain in mice. This N269D mutation impaired the transactivation capacity of Pax3 protein, but exerted no effect on Pax3 protein translation. The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.

Casein Kinase 1-dependent Phosphorylation of Familial Advanced Sleep Phase Syndrome-associated Residues Controls PERIOD 2 Stability

The mammalian circadian clock component PERIOD2 (PER2) plays a critical role in circadian rhythm entrainment. Recently, a missense mutation at a putative phosphorylation site in hPER2, Ser-662, was identified in patients that suffer from familial advanced sleep phase syndrome (FASPS). Patients with FASPS display abnormal sleep-wake patterns characterized by a lifelong pattern of sleep onset in the early evening and offset in the early morning. Although the phosphorylation of PER2 is strongly implied from functional studies, it has not been possible to study the site-specific phosphorylation of PER2 on Ser-662, and the biochemical functions of this residue are unclear. Here, we used phospho-specific antibodies to show that PER2 is phosphorylated on Ser-662 and flanking casein kinase (CK) sites in vivo. The phosphorylation of PER2 was carried out by the combined activities of casein kinase 1δ (CK1 δ) and casein kinase 1ε (CK1ε) and was antagonized by protein phosphatase 1. PER2 phosphorylation was rapidly induced in response to circadian entrainment of mammalian cell lines and occurred in both cytosolic and nuclear compartments. Importantly, we found that the pool of Ser-662-phosphorylated PER2 proteins was more stable than the pool of total PER2 molecules, implying that the FASPS phosphorylation cluster antagonizes PER2 degradation. Consistent with this idea, a Ser-662→Ala mutation that abrogated PER2 phosphorylation significantly reduced its half-life, whereas a phosphomimetic Ser-662→Asp substitution led to an elevation in half-life. Our combined findings provide new insights into PER2 regulation and the biochemical basis of FASPS.

Highest prevalence of the Mycobacterium tuberculosis Beijing genotype isolates in patients newly diagnosed with tuberculosis in the Novosibirsk oblast, Russian Federation

In order to assess the genetic diversity of Mycobacterium tuberculosis in the Novosibirsk Region and determine profiles of resistance, 106 M. tuberculosis isolates were analysed. Fifty (47 %) isolates were identified using variable number tandem repeat typing as being in the Beijing family, of which eight (16 %) were type M2 isolates with the genetic profile 233325153533424 and eight (16 %) were type M11 isolates with the genetic profile 233325173533424, both of which are widespread in Russia. Mutations associated with resistance to isoniazid and rifampicin were identified. Of 48 isolates with resistance to isoniazid, 42 (87.5 %) contained a Ser(315)→Thr substitution in the katG gene and one contained a T→A substitution at position -34 of the promoter region of the ahpC gene. Of 31 isolates with resistance to rifampicin, 19 (61 %) each contained a mutation (TCG→TTG) at codon 531 of the rpoB gene. Two isolates each contained a mutation (GAC→GTC) at codon 516 of the rpoB gene and two others each contained a substitution at codon 526 of the rpoB gene, leading to a His→Asn substitution in one case and a His→Asp substitution in another case. One isolate contained a mutation (CTG→CCG) at codon 533 of the rpoB gene. An association between the Beijing genotype and multidrug resistance was demonstrated (R = 0.2, P = 0.032). However, it was interesting to note that a significant proportion (46 %) of isolates were sensitive to all drugs tested.

Amantadin resistant variants of influenza A virus from Flu-like infected suspects in a Children Infectious Research Center in Tehran, Iran

The matrix protein 2 (M2) blocker, Amantadin, is approved by FDA to treat and control of influenza A virus infections in children and adults. Recently, some substitutions at amino acid positions are causing the emergence of anti viral drug-resistant strains of influenza virus. To investigate the frequency of Amantadin resistance among influenza A viruses isolated in children (up to 10 years old) referring to Children Infectious Research Center in Tehran – Iran during the 2008 – 2009 Flu season, 124 samples were collected. Forty cases from them were detected as influenza A virus by Reverse transcriptase polymerase chain reaction (RT-PCR). The Large subunit of hemagglutinin (HA1) and M2 genes were amplified by RT-PCR followed by sequencing. 62.5% of positive cases were 0-3 years, 10% of them were 4-6 years, and 27.5% were 7- 10 years. The result shows 70% were male. 50% of positive cases affected sporadic. Clinical symptoms in these positive cases were as follows: Sudden onset 51%, fever 69.4%, headache 21.8%, coryza 82.5%, weakness 54.8%, pharingitis 41.1%, bronchodyspenia 4%, cough 11.3%, agitation 58%, sputum 59.7%, and muscle pain 8%, gastrointestinal problem, 23.4%, otitis 13%. 97.5% of these forty cases did not received influenza vaccine and 40% received antibiotic. Our data show that Amantadin resistant A/H3 N2 is caused by a single amino acid mutation that makes a Ser 31 Asp substitution in M2 protein. Since these children might not used Amantadin, the drug resistance determined in this project is not related to direct usage of Amantadin by the patient. Continued surveillance is required to elucidate full Amantadin-resistant pattern of influenza virus in Iranian children, especially in the recent pandemics.

Runx2Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316-421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of Runx2 AD3, and nuclear HIPK3 colocalized with MINT. HIPK3 antisense oligodeoxynucleotide selectively reduced Runx2 protein accumulation and OC gene expression in C3H10T1/2 cells. Thus, HIPK3 participates in MINT+FGF2 regulation of Runx2 AD3 activity and controls Runx2-dependent OC expression.

Contribution of Asparagine Residues to the Stabilization of a Proteinaceous Antigen-Antibody Complex, HyHEL-10-Hen Egg White Lysozyme

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

Gene polymorphisms in angiotensin I converting enzyme (ACE I/D) and angiotensin II converting enzyme (ACE2 C→T) protect against cerebral malaria in Indian adults

To explore the hypothesis that angiotensin II may play a role in the susceptibility to cerebral malaria (CM), we performed a genetic association study of malaria patients in Orissa, India analyzing three SNPs (ACE2 C-->T, iNOS C-->T, eNOS Glu-->Asp) and two I/D polymorphisms (ACE I/D and IL-4 B1/B2). Our results showed that the 'D' allele of ACE I/D polymorphism, responsible for increased Ang II production had a significant association with mild malaria and the ACE2 C-->T substitution had gender specific effect of possibly reduced expression of ACE2 in presence of 'T' allele in women leading to increased level of Ang II and hence protection against CM. Combined genotype analysis of eNOS Glu-->Asp substitution responsible for increased NO production in Plasmodium falciparum infected individuals and ACE I/D polymorphism also showed stronger association of (Glu-Asp+Asp-Asp/ID+DD) genotypes with mild malaria (P<0.0001). Whether by its antiplasmodial activity and/or by some unknown mechanisms, Ang II protects from susceptibility to cerebral malaria remains to be investigated. These genetic findings may contribute to the understanding of malaria pathogenesis.