Characterization of copper resistant ciliates: Potential candidates for consortia of organisms used in bioremediation of wastewater

Metals are environmental pollutants of major concern due to their ecological, sanitary and even economic consequences. Both prokaryotes and eukaryotes inhabiting such environments carry cellular systems that maintain the metal homeostasis. The ciliate protists tolerate elevated concentrations of metals, which are accumulated, bound to metallothioneins (MTs) peculiar to these organisms. Copper is one of such contaminant found in the wastewater of local industries. The concentrations of copper which caused 50% reduction (LC50) in the cell population of Tetrahymena sp RT1, and two Euplotes spp. RE-1 and RE-2, isolated from the industrial waste, were found to be 60, 48 and 49 ppm, respectively, compared to those of the cultures without copper in the media. RT-1 showed significantly high tolerance to copper ions and could uptake 52.66% of the copper ions from the medium. The axenic culture of RT-1 could uptake 61.2% of copper from the medium compared to 68.41 and 59.16% by the ATCC culture of Tetrahymena thermophila and T. pyriformis, respectively. RT-1 tolerated about 500 μM copper in the medium without affecting its movement. This ciliate showed promise as a member of the consortium used for bioremediation of copper contaminated wastewater.Key words: Copper toxicity, metallothionein, growth curve of ciliates, metal uptake, bioremediation

Gene study within the 5' flanking regions of growth hormone gene of first exon in Bos indicus

Growth hormone (GH) is a main regulatory protein secreted by pituitary gland and placenta, involved in most anabolic processes in mammals. Expression of more than one gene for GH has been reported, indicating polymorphism at gene and protein level; apart from this, silent mutations has also been reported, relating to the level of expression of GH gene. The aim of this study is to identify silent mutations within the 5' flanking regions of GH2 gene of the first exon in Bos indicus. DNA was isolatedfrom the blood of freshly slaughtered animal and a set of primer was used for gene amplification, binding at 5' flanking region of GH gene at chromosome 19. DNA was amplified and the resultant product of about 453 bp was sequenced. The results showed that there were 9 changes including 8 replacements and one addition, for GH 2.3 allele, as compared to Bos taurus Genome (Btau_4.0 ) and two earlier reported alleles 2.1 and 2.2 for GH2 gene in B indicus. As this region is related to higher milk production, growth regulation, carcass and immune response traits in livestock, these changes could be used as a genetic marker. The reported sequence has been deposited to the European Molecular Biology Laboratory (EMBL) with the Accession Number FN666263.Key words: Bos indicus, growth hormone gene, silent mutation, growth hormone (GH) gene, allele

Detection of FLT3/TKD and IDH1 Mutations in Pakistani Acute Myeloid Leukemia Patients by Denaturing HPLC

Acute myeloid leukemia (AML) is characterized by an increase in the number of myeloid cells in the marrow and an arrest in their maturation. Various genetic mutations are associated with AML. FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays an important role in stem cell survival, and the development of dendritic and natural killer cells. FLT3/TKD mutations are generally missense mutations or in-frame alterations of residues D835 and I836 within the activation loop of the FLT3 protein. D835 mutations have been reported to occur in ≈ 7% of AML patients. Mutations have also been reported in exon 4 of isocitrate dehydrogenase 1 (IDH1) in ≈9% of AML patients. Mutations in FLT3/TKD and IDH1 genes were studied in AML patients from Pakistan and correlated with the laboratory findings. FLT3/TKD mutations were found in 7%, while IDH1 mutations were found in 10% Pakistani AML patients. Neither of these mutations was significantly correlated with age and sex, although the incidence of these mutations was higher in female patients. These mutations were found to be positively associated with each other. IDH1 mutations were positively associated with FAB type M1 and negatively associated with FAB type M2. In conclusion, the overall incidence of all these mutations in Pakistani patients was within the globally reported ranges

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens

BACKGROUND: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). METHODS: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. RESULTS: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. CONCLUSION: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2 Œ-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production

A comparative study on the effects of a pesticide (cypermethrin) and two metals (copper, lead) to serum biochemistry of Nile tilapia, Oreochromis niloticus

The present study was designed to compare the responses in freshwater fish Oreochromis niloticus exposed to a synthetic pyrethroid, cypermethrin (CYP); an essential metal, copper (Cu); and a nonessential metal, lead (Pb). Fish were exposed to 0.05 μg/l CYP, 0.05 mg/l Cu, and 0.05 mg/l Pb for 4 and 21 days, and the alterations in serum enzyme activities, metabolite, and ion levels were determined. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased in response to CYP, Cu, and Pb exposures at both exposure periods. While elevations in alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities and in cholesterol level were observed in pesticide-exposed fish at 4 and 21 days, they increased in Cu- and Pb-exposed fish at 21 days. Although metal-exposed fish showed increases in cortisol and glucose levels at 4 days followed by a return to control levels at the end of the exposure period, their levels elevated in pesticide-exposed fish at both exposure periods. Total protein levels decreased in Pb- and pesticide-exposed fish at 21 days. Na+ and Cl− levels decreased in pesticide-exposed fish at both exposure periods and in Cu- and Pb-exposed fish at 21 days. The exposures of pesticide and metals caused an elevation in K+ level at the end of the exposure period. The present study showed that observed alterations in all serum biochemical parameters of fish-treated pesticide were higher than those in fish exposed to metals

Differential Proteome Analysis of Bone Marrow Mesenchymal Stem Cells from Adolescent Idiopathic Scoliosis Patients

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. The cause and pathogenesis of scoliosis and the accompanying generalized osteopenia remain unclear despite decades of extensive research. In this study, we utilized two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) to analyze the differential proteome of bone marrow mesenchymal stem cells (BM-MSCs) from AIS patients. In total, 41 significantly altered protein spots were detected, of which 34 spots were identified by MALDI-TOF/TOF analysis and found to represent 25 distinct gene products. Among these proteins, five related to bone growth and development, including pyruvate kinase M2, annexin A2, heat shock 27 kDa protein, γ-actin, and β-actin, were found to be dysregulated and therefore selected for further validation by Western blot analysis. At the protein level, our results supported the previous hypothesis that decreased osteogenic differentiation ability of MSCs is one of the mechanisms leading to osteopenia in AIS. In summary, we analyzed the differential BM-MSCs proteome of AIS patients for the first time, which may help to elucidate the underlying molecular mechanisms of bone loss in AIS and also increase understanding of the etiology and pathogenesis of AIS

Study on Ecological Growth Conditions of Cattle Hyalomma Ticks in Punjab, Pakistan

"nBackground: The survey for the prevalence of different species of cattle Hyalomma ticks was carried out in three districts (Rawalpindi, Multan and Lahore) of Punjab province in Pakistan. The bionomical conditions suitable for Hyalomma were also studied in laboratory."nMethods:  One hundred specimens of ticks of different genera were collected from each district. After identification, the Hyalomma ticks were reared in laboratory under the influence of varying temperature and humidity."nResults:  The   results showed highest prevalence (67%) of ticks in district Lahore. The highest prevalence (12%) of Hya­lomma ticks and lowest prevalence (3.1%) of Rhipicephalus in cattle was recorded. The bionomical study showed the high­est mean pre oviposition period was   during spring while it was lowest   in autumn .The  mean oviposition period was  also highest in spring  . The incubation period of the ova of Hyalomma varied in different seasons. No oviposition was   recorded at the temperature 100C and 85% humidity. The maximum number of eggs was laid at 340C and lowest egg production oc­curred at 150C. The maximum number of eggs hatched at 320C and 85% humidity."nConclusion: The variation in relative humidity had no appreciable effect on rate of development of ticks while the number of eggs laid increase with rise in temperature