In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.
We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.
Septins belong to GTPases that are involved in vital cellular activities, including cytokinesis. Although present in many organisms, they are yet to be isolated from Aedes albopictus. This study reports for the first time on a serendipitous isolation of a partial septin sequence from Ae. albopictus and its developmental expression profile. The Ae. albopictus partial septin sequence contains 591 nucleotides encoding 197 amino acids. It shares homology with several insect septin genes and has a close phylogenetic relationship with Aedes aegypti and Culex quinquefasciatus septins. The Ae. albopictus septin fragment was differentially expressed in the mosquito's developmental stages, with an increased expression in the adults.
Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding.
Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.
The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.
Nine Newcastle disease virus (NDV) isolates from Newcastle disease (ND) outbreaks in different regions of Iran were characterized at molecular level. Sequence analysis revealed that the isolates shared two pairs of arginine and a phenylalanine at the N-terminus of the fusion (F) protein cleavage site similarly to other velogenic isolates of NDV characterized earlier. Eight of the nine isolates had the same amino acid sequence as VOL95, a Russian NDV isolate from 1995. However, one isolate, MK13 showed 5 amino acid substitutions, of which 3 have been reported for other velogenic NDV isolates. These results suggest that the origin of the outbreaks of ND in different parts of Iran in 1995-1998 is VOL95.
Partial nucleotide sequences of 1D gene of 38 isolates of foot-and-mouth disease virus (FMDV) of serotypes O, A and Asia 1 originating from various parts of India were determined. Field materials were subjected straight to RNA extraction, reverse transcription - PCR (RT-PCR) and sequencing. Also 3 FMDV vaccine strains, IND R2/75 (serotype O), IND 63/72 (serotype Asia 1) and IND 17/77 (serotype A) were included in the analysis. The seqences were compared mutually as well as with available corresponding sequences of other FMDV isolates, and their phylogenetic relationships were calculated. The deduced amino acid sequences showed that the serotype O isolates were relatively conserved as compared to serotype Asia 1 or A isolates from India. In phylogenetic analysis, the serotype O viruses clustered in two genotypes, one including the European vaccine strain (O1/K) and the other represented by the isolates from Bangladesh, India, Nepal and Turkey. The serotype Asia 1 viruses clustered in two groups of single genotype where the prototype strain from Pakistan (PAK 1/54) formed one group and the other was formed by the isolates from Bangladesh, Bhutan, India, Israel and Nepal. In serotype A viruses three well-differentiated genotypes were observed. The isolates from Azerbaijan, Bangladesh, Malaysia and India formed the first genotype. The second genotype was formed by isolates from Iran, Saudi Arabia and Turkey, while two recent Iranian isolates represented the third genotype. In India, the prevalence of at least one genotype could be identified in each serotype. This evolutionary clustering of isolates from the neighbor countries is not surprising, since these countries share border with India. The genetic relatedness between sequences of isolates from India and those from distant places is indicative of spread of the virus between the countries. Of importance is the fact that clinical materials proved useful for rapid generation of sequences and subsequent studying of molecular epidemiology of the disease.
Ebola virus (EBOV) is one of the lethal viruses, causing more than 24 epidemic outbreaks to date. Despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of EBOV infections in humans. Disclosing this, the present study described an epitope-based peptide vaccine against EBOV, using a combination of B-cell and T-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. Here, protein sequences of all glycoproteins of EBOV were collected and examined via in silico methods to determine the most immunogenic protein. From the identified antigenic protein, the peptide region ranging from 186 to 220 and the sequence HKEGAFFLY from the positions of 154-162 were considered the most potential B-cell and T-cell epitopes, correspondingly. Moreover, this peptide (HKEGAFFLY) interacted with HLA-A*32:15 with the highest binding energy and stability, and also a good conservancy of 83.85% with maximum population coverage. The results imply that the designed epitopes could manifest vigorous enduring defensive immunity against EBOV.
Separate studies have reported spina bifida patients to be especially allergic to proteins of 27 and 23 kDa found in the serum of centrifuged natural rubber latex. An insoluble latex protein located on the surface of small rubber particles, Hev b 3, has similarly been found to be allergenic to spina bifida patients. In this study, internal amino acid sequences of Hev b 3 showed similarity to the published sequences for the 27- and 23-kDa latex proteins. The latter allergens are hence identified as Hev b 3. Determination of the molecular weight of Hev b 3 revealed various species of 22-23 kDa. The consistent gaps of about 266 Da observed between various forms of the intact protein suggest that the protein undergoes post-translational modification. To determine whether Hev b 3 also occurs in a soluble form in the latex serum, its presence in molecular-filtered serum was checked by ELISA and Western blot. The results showed Hev b 3 to be largely absent in the C-serum from fresh latex. The protein is therefore insoluble in its native state. However, a small amount of the solubilized protein was detected in ammonia-stabilized latex (commonly used in the manufacture of latex products).
Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis.
We describe four families with affected siblings showing unique clinical features: early-onset (before 1 year of age) progressive diffuse brain atrophy with regression, postnatal microcephaly, postnatal growth retardation, muscle weakness/atrophy, and respiratory failure. By whole-exome sequencing, we identified biallelic TBCD mutations in eight affected individuals from the four families. TBCD encodes TBCD (tubulin folding co-factor D), which is one of five tubulin-specific chaperones playing a pivotal role in microtubule assembly in all cells. A total of seven mutations were found: five missense mutations, one nonsense, and one splice site mutation resulting in a frameshift. In vitro cell experiments revealed the impaired binding between most mutant TBCD proteins and ARL2, TBCE, and β-tubulin. The in vivo experiments using olfactory projection neurons in Drosophila melanogaster indicated that the TBCD mutations caused loss of function. The wide range of clinical severity seen in this neurodegenerative encephalopathy may result from the residual function of mutant TBCD proteins. Furthermore, the autopsied brain from one deceased individual showed characteristic neurodegenerative findings: cactus and somatic sprout formations in the residual Purkinje cells in the cerebellum, which are also seen in some diseases associated with mitochondrial impairment. Defects of microtubule formation caused by TBCD mutations may underlie the pathomechanism of this neurodegenerative encephalopathy.
RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration.
Bioassay and immunohistochemical studies have detected the presence of prosaposin in the central nervous system (CNS) of mammals. Here, first time, we have determined the partial cDNA sequence of pigeon prosaposin and mapped the distribution of its mRNA in the pigeon CNS. The predicted amino acid sequence of pigeon prosaposin showed 93 and 60% identity to chicken and human prosaposin, respectively. In situ hybridization, autoradiograms showed that the prosaposin mRNA expression was found in the olfactory bulb, prepiriform cortex, Wulst, mesopallium, nidopallium, hippocampal formation, thalamus, tuberis nucleus, pre-tectal nucleus, nucleus mesencephalicus lateralis, pars dorsalis, nucleus isthmi, pars parvocellularis and magnocellularis, Edinger-Westphal nucleus, optic tectum, cerebellar cortex and nuclei, vestibular nuclei and gray matter of the spinal cord. These results suggest that the cDNA sequence of pigeon prosaposin is comparable to other vertebrates, and the general distribution pattern of prosaposin mRNA resembles those are found in mammals.