Colicinogenic plasmids and inhibitor sensitivity in Escherichia coli

The effects of Col plasmids on sensitivities of E.coli K-12 derivatives, especially 1829 and P678-54" to hydrophobic, hydrophilic and aminoglycoside antibiotics and to other inhibitors such as copper ions and Tris-EDTA have been examined. It was found that the colicinogenic plasmid ColVIK-94 sensitised the strains to hydrophobic antibiotics as well as to some of the hydrophilic and aminoglycoside antibiotics whilst Col BK-98 sensitised the strains to rifampicin and some of the aminoglycosides and hydrophilic antibiotics but not to most of the hydrophobic agents. The various effects of the above mentioned antibiotics and inhibitors were also tested on strains carrying mutant derivatives of the Col VIK-94. This was to investigate which plasmid components were responsible for sensitivities particularly to rifampicin and novobiocin. It was shown that both mutant plasmids sensitise 1829 to these two agents. It seems that both transfer and colicin components were Important for increased rifampicin sensitivity whereas transfer components were needed for novobiocin sensitivity. The effects of divalent cations, namely magnesium and calcium ions were investigated and it was found that they reversed the Inhibitory effects of the hydrophobic and aminoglycoside antibiotics on strain 1829 bearing the Col VIK-94 but there was no effect on those strains bearing Col BK-98. This probably indicates that the divalent cations in some way stabilise the outer membrane preventing the entry of some antibiotics into the cell. A prior growth temperature of 25° C seemed to reduce the sensitisation effects to hydrophobic antibiotics of the ColV plasmids without affecting the sensitivity of the parent strain 1829 whereas the effects of the hydrophilic and aminoglycoside e antibiotics were unaffected by a 25° C prior growth temperature. A Synthesis of colicin and transfer components are reduced at 25° C, hence the results are In accordance with the conclusion concerning the components involved in sensitivity especially with regards to rifampicin and novobiocin. The Col V and Col B plasmids sensitised the strains 1829 and P678-54 to copper sulphate and to the effects of the Tris-EDTA. The sensitivity to copper ions is due to the uptake of the ions by the Omp F porin. The plasmid may affect porin function or open up another entry pathway. The effects of the plasmids on the Tris-EDTA sensitivity was probably due to the weakening of the LPS-LPS bonds by this combination of agents

Multidrug resistant yeasts in synanthropic wild birds

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to investigate the presence of multidrug resistant yeasts in the faeces of synanthropic wild birds from the Bangsar suburb of Kuala Lumpur.</p> <p>Methods</p> <p>Species characterisations of yeast isolates and determinations of antimycotic susceptibility profiles were undertaken using the commercial characterization kit, Integral System Yeasts Plus (Liofilchem, Italy).</p> <p>Results</p> <p>Fourteen species of yeasts were detected in the bird faecal samples.<it>Candida albicans </it>was present in 28.89% of bird faecal samples, <it>Candida krusei </it>(13.33%), <it>Candida tropicalis </it>(4.44%), <it>Candida glabrata </it>(4.44%), <it>Candida parapsilosis </it>(2.22%), <it>Candida lambica </it>(2.22%), <it>Candida stellatoidea </it>(2.22%), <it>Candida rugosa </it>(2.22%) and <it>Candida lusitaniae </it>(2.22%). Amongst the non-candidal yeast isolates, <it>Cryptococcus laurentii </it>was present in 6.67% of bird faecal samples, <it>Cryptococcus uniguttulatus </it>(4.44%), <it>Saccharomyces cerevisiae </it>(4.44%), <it>Trichosporon pullulans </it>(2.22%), <it>Trichosporon pullulans/Cryptococcus albidus </it>(8.89%) and <it>Rhodotorula rubra/Rhodotorula glutinis </it>(4.44%). Of the isolated yeasts, 18.1% (or 26/144) were found to be resistant to all 11 antimycotic agents they were tested against i.e. Nystatin, Amphotericin B, Flucytosine, Econazole, Ketoconazole, Clotrimazole, Miconazole, Itraconazole, Voriconazole, Fluconazole 16 and Fluconazole 64. 45.8% (or 66/144) of the bird faecal yeast isolates were resistant to four or more of the 11 antimycotic agents they were tested against.</p> <p>Conclusions</p> <p>This finding is of public health significance as these synanthropic wild birds may be reservoirs for transmission of drug resistant yeast infections to humans.</p

Unravelling the neuroprotective mechanisms of carotenes in differentiated human neural cells: biochemical and proteomic approaches

Carotenoids, fat-soluble pigments found ubiquitously in plants and fruits, have been reported to exert significant neuroprotective effects against free radicals. However, the neuroprotective effects of total mixed carotenes complex (TMC) derived from virgin crude palm oil have not been studied extensively. Therefore, the present study was designed to establish the neuroprotective role of TMC on differentiated human neural cells against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. The human neural cells were differentiated using retinoic acid for six days. Then, the differentiated neural cells were pre-treated for 24 hr with TMC before exposure to 6-OHDA. TMC pre-treated neurons showed significant alleviation of 6-OHDA-induced cytotoxicity as evidenced by enhanced activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes. Furthermore, TMC elevated the levels of intra-neuronal dopamine and tyrosine hydroxylase (TH) in differentiated neural cells. The 6-OHDA induced overexpression of α-synuclein was significantly hindered in neural cells pre-treated with TMC. In proteomic analysis, TMC altered the expression of ribosomal proteins, α/β isotypes of tubulins, protein disulphide isomerases (PDI) and heat shock proteins (HSP) in differentiated human neural cells. The natural palm phytonutrient TMC is a potent antioxidant with significant neuroprotective effects against free radical-induced oxidative stress

A Glimpse into the Genome-wide DNA Methylation Changes in 6-hydroxydopamine-induced In Vitro Model of Parkinson’s Disease

A cell-based model of Parkinson’s disease (PD) is a well-established in vitro experimental prototype to investigate the disease mechanism and therapeutic approach for a potential anti-PD drug. The SH-SY5Y human neuroblastoma cells and 6-OHDA combo is one of the many neurotoxin-induced neuronal cell models employed in numerous neuroscience-related research for discovering neuroprotective drug compounds. Emerging studies have reported a significant correlation between PD and epigenetic alterations, particularly DNA methylation. However, the DNA methylation changes of PD-related CpG sites on the 6-OHDA-induced toxicity on human neuronal cells have not yet been reported. We performed a genome-wide association study (GWAS) using Infinium Epic beadchip array surveying 850000 CpG sites in differentiated human neuroblastoma cells exposed to 6-OHDA. We identified 236 differentially methylated probes (DMPs) or 163 differentially methylated regions (DMRs) in 6-OHDA treated differentiated neuroblastoma cells than the untreated reference group with p&lt;0.01, Δbeta cut-off of 0.1. Among 236 DMPs, hypermethylated DMPs are 110 (47%), whereas 126 (53%) are hypomethylated. Our bioinformatic analysis revealed 3 DMRs that are significantly hypermethylated and associated with neurological disorders, namely AKT1, ITPR1 and GNG7. This preliminary study demonstrates the methylation status of PD-related CpGs in the 6-OHDA-induced toxicity in the differentiated neuroblastoma cells model.</p

Influence of serum concentration in retinoic acid and phorbol ester induced differentiation of SH-SY5Y human neuroblastoma cell line

Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum used. In this paper, we compared the effects of high (10%), low (3%) and descending (2.5%/1%) serum concentration in differentiation medium containing different proportion of retinoic acid (RA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) or RA-only on the undifferentiated SH-SY5Y cells with regards to cell morphology, biochemical and gene expression alterations. Cells differentiated in culture medium containing low and descending serum concentrations showed increased number of neurite projections and reduced proliferation rates when compared to undifferentiated cells. The SH-SY5Y cells differentiated in culture medium containing 3% RA and low serum or descending (2.5%/1% RA/TPA) were found to be more susceptible to 6-hydroxydopamine (6-OHDA) induced cytotoxicity. Cells differentiated with RA/TPA or RA differentiated showed increased production of the α-synuclein (SNCA) neuroprotein and dopamine neurotransmitter compared to undifferentiated cells, regardless serum concentrations used. There was no significant difference in the expression of tyrosine hydroxylase (TH) gene between undifferentiated and differentiated SH-SY5Y cells. However, the expression of dopamine receptor D2 (DRD2) gene was markedly increased (p<0.05) in differentiated cells with 3% serum and RA only when compared to undifferentiated cells. In conclusion, to terminally differentiate SH-SY5Y cells to be used as a cell-based model to study Parkinson's disease (PD) to investigate molecular mechanisms and drug discovery, the optimal differentiation medium should contain 3% serum in RA-onl

6-Hydroxydopamine Induces Neurodegeneration in Terminally Differentiated SH-SY5Y Neuroblastoma Cells via Enrichment of the Nucleosomal Degradation Pathway:a Global Proteomics Approach

The SH-SY5Y human neuroblastoma cells have been used for decades as a cell-based model of dopaminergic neurons to explore the underlying science of cellular and molecular mechanisms of neurodegeneration in Parkinson’s disease (PD). However, data revealing the protein expression changes in 6-OHDA induced cytotoxicity in differentiated SH-SY5Y cells remain void. Therefore, we investigated the differentially regulated proteins expressed in terminally differentiated SH-SY5Y cells (differ-SH-SY5Y neural cells) exposed to 6-hydroxydopamine (6-OHDA) using the LC–MS/MS technology and construed the data using the online bioinformatics databases such as PANTHER, STRING, and KEGG. Our studies demonstrated that the neuronal development in differ-SH-SY5Y neural cells was indicated by the overexpression of proteins responsible for neurite formations such as calnexin (CANX) and calreticulin (CALR) besides significant downregulation of ribosomal proteins. The enrichment of the KEGG ribosome pathway was detected with significant downregulation (p < 0.05) of all the 21 ribosomal proteins in differ-SH-SY5Y neural cells compared with undifferentiated cells. Whereas in the PD model, the pathological changes induced by 6-OHDA were indicated by the presence of unfolded and misfolded proteins, which triggered the response of 10 kDa heat shock proteins (HSP), namely HSPE1 and HSPA9. Moreover, the 6-OHDA-induced neurodegeneration in differ-SH-SY5Y neural cells also upregulated the voltage-dependent anion-selective channel protein 1 (VDAC1) protein and enriched the KEGG systemic lupus erythematosus (SLE) pathway that was regulated by 17 histone proteins (p < 0.05) in differ-SH-SY5Y neural cells. These results suggest that the nucleosomal degradation pathway may have regulated the 6-OHDA induced neurodegeneration in PD cell-based model, which is reflected by increased apoptosis and histone release in differ-SH-SY5Y neural cells

Tocotrienol-rich fraction and levodopa regulate proteins Involved in Parkinson’s disease-associated pathways in differentiated neuroblastoma cells:Insights from quantitative proteomic analysis

Tocotrienol-rich fraction (TRF), a palm oil-derived vitamin E fraction, is reported to possess potent neuroprotective effects. However, the modulation of proteomes in differentiated human neuroblastoma SH-SY5Y cells (diff-neural cells) by TRF has not yet been reported. This study aims to investigate the proteomic changes implicated by TRF in human neural cells using a label-free liquid-chromatography-double mass spectrometry (LC-MS/MS) approach. Levodopa, a drug used in the treatment of Parkinson’s disease (PD), was used as a drug control. The human SH-SY5Y neuroblastoma cells were differentiated for six days and treated with TRF or levodopa for 24 h prior to quantitative proteomic analysis. A total of 81 and 57 proteins were differentially expressed in diff-neural cells following treatment with TRF or levodopa, respectively. Among these proteins, 32 similar proteins were detected in both TRF and levodopa-treated neural cells, with 30 of these proteins showing similar expression pattern. The pathway enrichment analysis revealed that most of the proteins regulated by TRF and levodopa are key players in the ubiquitin-proteasome, calcium signalling, protein processing in the endoplasmic reticulum, mitochondrial pathway and axonal transport system. In conclusion, TRF is an essential functional food that affects differential protein expression in human neuronal cells at the cellular and molecular levels

Tocotrienols protect differentiated SH-SY5Y human neuroblastoma cells against 6-hydroxydopamine-induced cytotoxicity by ameliorating dopamine biosynthesis and dopamine receptor D2 gene expression

Oxidative stress is a critical factor that triggers a "domino" cascade of events leading to the degeneration of dopaminergic neurons in Parkinson disease. Tocotrienols (T3) have antioxidant effects and can protect neuronal cells against oxidative damage. In the present study, we investigated the neuroprotective effects of different forms of T3 (alpha, delta, gamma) or tocotrienol-rich fraction (TRF) against 6-hydroxydopamine (6-OHDA)-induced oxidative damage in differentiated SH-SY5Y human neural cells. Differentiating the SH-SY5Y cells with retinoic acid and a low-serum culture medium for 6 days allowed development of human dopamine-like neural cells. Subsequently, the differentiated SH-SY5Y neural cells were pretreated with different forms of T3 for 24 hours before these cells were exposed to 6-OHDA. The T3 analogues and TRF displayed neuroprotective effects (P < .05) via restoration of cell viability and activation of antioxidant enzymes (e.g., superoxide dismutase, catalase). Notably, TRF was highly efficient in scavenging reactive oxygen species and upregulating dopamine and tyrosine hydroxylase levels in the differentiated SH-SY5Y cells. Gamma-T3 exhibited the most potent effects in attenuating apoptosis, whereas alpha-T3 was most effective in preventing 6-OHDA-induced leakage of α-Synuclein. Delta-T3 displayed a noticeable effect in upregulating the dopamine receptor D2 gene expression compared with controls. These findings suggest T3 isoforms and TRF demonstrate significant neuroprotective effects in protecting differentiated neural cells against 6-OHDA-mediated oxidative stress

Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA

Financial support was provided by The Wellcome Trust (grant ref. 055487), the European Commission, and the University of London Central Research FundLondon School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.London School of Hygiene and Tropical Medicine. Department of Infectious and Tropical Diseases. Keppel St, London, UK.University of Ibadan. College of Medicine. Ibadan, Nigeria.Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale. Malaria Department. Yaounde, Cameroon.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Universiti Malaysia Sarawak. Kota Samarahan. Sarawak, Malaysia.Biomedical Primate Research Centre. Rijswijk, The Netherlands.The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately