Three actinomycetes isolated from the surfaces of rocks in a medieval slate mine were examined in a polyphasic taxonomic study. Chemotaxonomic and morphological characteristics of the isolates were typical of strains of the genus Amycolatopsis. The isolates had identical 16S rRNA gene sequences and formed a distinct phyletic line towards the periphery of the Amycolatopsis mediterranei clade, being most closely related to Amycolatopsis rifamycinica. The organisms shared a wide range of genotypic and phenotypic markers that distinguished them from their closest phylogenetic neighbours. On the basis of these results, a novel species, Amycolatopsis saalfeldensis sp. nov., is proposed. The type strain is HKI 0457(T) (=DSM 44993(T)=NRRL B-24474(T)).
Global surveillance of HIV-1 subtypes for genetic characterization is hampered by the biohazard of processing and the difficulties of shipping whole blood or cells from many developing country regions. We developed a technique for the direct automated sequencing of viral DNA from dried blood spot (DBS) specimens collected on absorbent paper, which can be mailed unrefrigerated in sturdy paper envelopes with low biohazard risk. DBS were collected nonrandomly from HIV-1-infected, mostly asymptomatic, patients in five Asian countries in 1991, and shipped via airmail or hand carried without refrigeration to Bangkok, and then transshipped to North America for processing. After more than 2 years of storage, including 6 months at ambient temperatures, proviral DNA in the DBS was amplified by nested PCR, and a 389-nucleotide segment of the C2-V3 env gene region was sequenced, from which 287 base pairs were aligned and subtyped by phylogenetic analysis with neighbor-joining and other methods. From southern India, there were 25 infections with subtype C and 2 with subtype A. From Myanmar (Burma), we identified the first subtype E infection, as well as six subtype BB, a distinct cluster within subtype B that was first discovered in Thailand and that has now appeared in China, Malaysia, and Japan. From southwest China, one BB was identified, while a "classical" B typical of North American and European strains was found in Indonesia. From Thailand, five DBS of ambiguous serotype were identified as three B, one BB, and one E. A blinded control serotype E specimen was correctly identified, but a serotype BB control was not tested. Most HIV-1 in southern India appears to be env subtype C, with rare A, as others have reported in western and northern India. The subtypes BB and E in Myanmar, and the BB in China, suggest epidemiological linkage with these subtypes in neighboring Thailand. DBS are a practical, economical technique for conducting large-scale molecular epidemiological surveillance to track the global distribution and spread of HIV-1 variants.
The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.
Eleusine indica (goosegrass) populations resistant to fluazifop, an acetyl-CoA carboxylase (ACCase: EC6.4.1.2)-inhibiting herbicide, were found in several states in Malaysia. Dose-response assay indicated a resistance factor of 87.5, 62.5 and 150 for biotypes P2, P3 and P4, respectively. DNA sequencing and allele-specific PCR revealed that both biotypes P2 and P3 exhibit a single non-synonymous point mutation from TGG to TGC that leads to a well known Trp-2027-Cys mutation. Interestingly, the highly resistant biotype, P4, did not contain any of the known mutation except the newly discovered target point Asn-2097-Asp, which resulted from a nucleotide change in the codon AAT to GAT. ACCase gene expression was found differentially regulated in the susceptible biotype (P1) and highly resistant biotype P4 from 24 to 72h after treatment (HAT) when being treated with the recommended field rate (198gha(-1)) of fluazifop. However, the small and erratic differences of ACCase gene expression between biotype P1 and P4 does not support the 150-fold resistance in biotype P4. Therefore, the involvement of the target point Asn-2097-Asp and other non-target-site-based resistance mechanisms in the biotype P4 could not be ruled out.
During January and February 2001, an outbreak of febrile illness associated with altered sensorium was observed in Siliguri, West Bengal, India. Laboratory investigations at the time of the outbreak did not identify an infectious agent. Because Siliguri is in close proximity to Bangladesh, where outbreaks of Nipah virus (NiV) infection were recently described, clinical material obtained during the Siliguri outbreak was retrospectively analyzed for evidence of NiV infection. NiV-specific immunoglobulin M (IgM) and IgG antibodies were detected in 9 of 18 patients. Reverse transcription-polymerase chain reaction (RT-PCR) assays detected RNA from NiV in urine samples from 5 patients. Sequence analysis confirmed that the PCR products were derived from NiV RNA and suggested that the NiV from Siliguri was more closely related to NiV isolates from Bangladesh than to NiV isolates from Malaysia. NiV infection has not been previously detected in India.
Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.
Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation.
Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.
A draft genome sequence of Pichia kudriavzevii M12 is presented here. The genome reveals the presence of genes encoding enzymes involved in xylose utilization and the pentose phosphate pathway for bioethanol production. Strain M12 is also a potential producer of phytases, enzymes useful in food processing and agriculture.
Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.
Enterococcus sp. strain C1 is a facultative anaerobe which was coisolated with Citrobacter sp. strain A1 from a sewage oxidation pond. Strain C1 could degrade azo dyes very efficiently via azo reduction and desulfonation in a microaerophilic environment. Here the draft genome sequence of Enterococcus sp. C1 is reported.
Citrobacter sp. strain A1, isolated from a sewage oxidation pond, is a facultative aerobe and mesophilic dye-degrading bacterium. This organism degrades azo dyes efficiently via azo reduction and desulfonation, followed by the successive biotransformation of dye intermediates under an aerobic environment. Here we report the draft genome sequence of Citrobacter sp. A1.
Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.
Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C(6). Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.
A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 microg AHL h(-1) per 10(9) CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif (106)HXDH-59 amino acids-H(169)-21 amino acids-D(191) for N-acylhomoserine lactone lactonases.
A new species of Ansonia is described based on genetic and morphological differentiation. Ansonia lumut sp. nov. is most closely related to three other Peninsular Malaysian species, A. penangensis, A. malayana, and A. jeetsukumarani but differs from these and other congeners by at least 6.9% sequence divergence at the 12S, 16S rRNA and t-RNA-val genes and the following combination of morphological characters: (1) SVL 21.0-23.6 mm in males, 27.7-31.6 mm in females; (2) first finger shorter than second; (3) interorbital and tarsal ridges absent; (4) light interscapular spot absent; (5) presence of large, yellow rictal tubercle; (6) dorsum black with greenish-yellow reticulations; (7) flanks with small yellow spots; (8) fore and hind limbs with yellow cross-bars; and (9) venter light gray with fine, white spotting.
During a study on the quality of the indoor environment, Acanthamoeba spp. were detected in 20 out of 87 dust samples collected from air-conditioners installed in a four-story campus building located in Kuala Lumpur, Malaysia. Twenty-one cloned Acanthamoeba isolates designated as IMU1 to IMU21 were established from the positive primary cultures. Five species were identified from the 16 isolates according to the morphological criteria of Pussard and Pons; i.e. A. castellanii, A. culbertsoni, A. griffini, A. hatchetti and A. polyphaga. Species identities for the remaining five isolates (IMU4, IMU5, IMU15, IMU20 and IMU21), however, could not be determined morphologically. At genotypic characterization, these isolates were placed into T3 (IMU14); T5 (IMU16 and IMU17) and T4 (all the remaining isolates). To predict the potential pathogenicity of these Acanthamoeba isolates, thermo- and osmotolerance tests were employed; many isolates were predicted as potential human pathogens based on the outcome of these tests. This is the first time potentially pathogenic Acanthamoeba have been isolated from air-conditioners in Malaysia.
The relationship between a putative metallothionein gene (MT) and exposure to cadmium (Cd) in blood cockles (Anadara granosa) is reported. In a 96-h dose-response experiment, mortality of cockles was found to proportionately increase in the range of 0.2-5.0 mg/l Cd with a calculated LC(50) of 2.94 mg/l. Exposure to 0.25 mg/l Cd for 16 days caused significant increases (P<0.05) in Cd concentrations in whole tissues, gills and hepatopancreas, and the accumulation of Cd in these tissues increased with the duration of exposure. Two cDNA libraries constructed using the hepatopancreas from control and Cd-treated cockles gave titres of 5.62 x 10(5) and 1.94 x 10(5) pfu/microg vector, respectively. A putative MT gene, AnaMT, of 510 nucleotides in length, was isolated from the treated cDNA library using a heterologous probe MT20 from the blue mussel, Mytilus edulis. Northern analyses using AnaMT as a probe indicated low expression of the MT mRNA in control animals. In cockles treated with 0.25 mg/l Cd for 4 days, MT mRNA level increased to approximately 168%, but declined to 108% at day 8. After 12 and 16 days of Cd treatment, expression of the MT gene was 138% and 187%, respectively, compared to the controls. These observations suggest that induction of the MT gene by a sublethal dose of Cd is rapid, occurring within 4 days of treatment.
A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.
Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5'/3' rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis.