Ecological studies of Euglenineae in certain polluted and unpolluted environments

Summary The ecology of Euglenineae was investigated for two years in certain polluted and unpolluted environments of Hyderabad, India. The paper includes discussions regarding the distribution and periodicity of euglenoid population in relation to the physicochemical complexes which existed in the three ponds during the period of investigation. Sewage and Garden Ponds were found to be considerably polluted, whereas, the Typha Pond was comparatively pure and less polluted. Sewage Pond exhibited the highest percentage of Euglenineae harbouring 13 species and Garden Pond harboured 2 species in large numbers. Typha Pond harboured typically Trachelomonas hispida and Euglena Sp. which were totally absent in the other two ponds. It seems that high average concentrations of free CO₂ were favourable for euglenoid growth in Sewage Pond and the oxygen deficiency was helpful to trigger the oxygen-iron-phosphate complex, releasing larger quantities of phosphorus and iron. Higher average values of percent Cl + NO₃ ratio were found to be responsible for the luxuriant growth of these flagellates and it was suggested that inorganic sources of nitrogen might be more important in their ecology as fluctuations of nitrate coincide closely with those of Euglenineae in all ponds. These flagellates were found to be abundant during the periods when water was not rich in total sulphides and concentrations higher than 2.0 ppm adversely effected the euglenoid development. These flagellates were found to develop usually after rains or during intermittent showers experiencing comparatively higher temperatures. A temperature range of 27° – 39°C was observed to be favourable for their growth. Higher concentrations of oxidizable organic matter showed a direct relationship to euglenoid population in Sewage Pond. It is suggested that certain species of Euglenineae, which very often developed in the polluted waters of Sewage Pond, could be used as biological indicators of pollution in the present investigation.Part of the thesis approved by Osmania University, Hyderabad, India for the award of the degree of Ph. D.     

A method for evaluating the impact of navigationally induced suspended sediments from the Upper Great Lakes Connecting Channels on the primary productivity

The impact of navigationally induced suspended sediments from the Upper Great Lakes Connecting Channels on the size-fractionated primary productivity was evaluated by the Carbon-14 technique. The method applied was on-site, rapid, sensitive, inexpensive, and provided dynamic-toxicological information essential for hazard assessment. Both enhancement and inhibition of the primary productivity was observed in various parts of the Upper Great Lakes Connecting Channels. These responses appear to depend on the type of natural plankton and their exposure to various contaminant/nutrient complexes generated by the disturbance of the bottom sediments during the passage of ships. Traditionally, only the inhibition of primary productivity has been monitored from a toxicity point of view, but it is important also to evaluate the implications of enhancement since it may result in increased eutrophication, propagation of nuisance blooms, and change of intricate food-web interactions. The procedure adopted in this study for the first time appears to possess considerable potential for a simple and rapid screening of environmental perturbations resulting from navigational activities.     

Flow‐injection determination of manganese (II) using surfactant enhanced diperiodatonickelate (IV)‐rhodamine 6G chemiluminescence

Chemiluminescence (CL) of the rhodamine 6‐G‐diperiodatonickelate (IV) (Rh6‐G‐Ni(IV) complex) in the presence of Brij‐35 was examined in an alkaline medium and implemented using flow‐injection analysis to analyze Mn(II) in natural waters. Brij‐35 was identified as the surfactant of choice that enhanced CL intensity by about 62% of the reaction. The calibration curves were linear in the range 1.7 × 10^?3 – 0.2 (0.9990, n = 7) and 8.0 × 10^?4 – 0.1 μg ml^?1 (0.9990, n = 7) with limits of detection (LODs) (S:N = 3) of 5.0 × 10^?4 and 2.4 × 10^?4 μg ml^?1 without and with using an in‐line 8‐hydroxyquinoline (8‐HQ) resin mini‐column, respectively. The sample throughput and relative standard deviation were 200 h^?1 and 1.7–2.2% in the range studied respectively. Mn(II) concentrations in certified reference materials and natural water samples was successfully determined. A brief discussion about the possible CL reaction mechanism is also given. In addition, analysis of V(III), Cr(III) and Fe(II) was also performed without and with using an in‐line 8–HQ column and selective elution of each metal ion was achieved by adjusting the pH of the sample carrier stream with aqueous HCl solution.     

An efficient protocol for transient transformation of intact fruit and transgene expression inCitrus

In this study, conditions were optimized for transient gene expression in Rough Lemon (Citrus jambhiri Lush.), a major rootstock used in the citrus growing regions of Pakistan.Agrobacterium tumefaciens carrying the binary vector p35GUSINT, containingNPT II andGUS genes, was used in the study. The transformation method was based on injection ofAgrobacterium intoCitrus fruits followed by histochemical assay ofGUS activity in different tissues. Different tissues of mature fruits exhibited significantly different percentages of transientGUS expression: in rind (76%), spongy tissue (92%), juice vesicles (0%) and seeds (83%) (P<0.01)., The incubation period after injecting theAgrobacterium culture also showed a significant (P<0.01) effect on the transient expression ofGUS in these tissues. An incubation period of 48 h was found to be the best (72%) for transformation of whole fruit, followed by 72 h (67%) and 96 h (49%). TransientGUS expression also varied significantly (P<0.01) in juice vesicles and seeds as fruit matured. Juice vesicles from mature fruits showed no transientGUS expression, while those from immature fruits showed 50% expression. Furthermore, transformation of seeds had no effect on their germination capability. Germinating seeds from mature fruits injected withAgrobacterium culture showed tolerance to kanamycin (100 mg/L), which varied with the incubation period (55% at 48 h, 25% at 72 h and 23% at 96 h). This report offers an easy protocol for transient expression studies of transgenes and has the potential to be used for stable transformation ofCitrus.     

Fam60a defines a variant Sin3a‐Hdac complex in embryonic stem cells required for self‐renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.     

Serological cross-reactivity of environmental isolates of Enterobacter, Escherichia, Stenotrophomonas, and Aerococcus with Shigella spp.-specific antisera

Using protocols designed for the isolation of Shigella from environmental freshwater samples from different regions of Bangladesh, 11 bacterial strains giving rise to Shigella-like colonies on selective agar plates and showing serological cross-reaction with Shigella-specific antisera were isolated. Phylogenetic analyses revealed that three of the isolates were most closely related to Escherichia coli, four to Enterobacter sp., two to Stenotrophomonas, and two isolates belonged to the Gram-positive genus Aerococcus. The isolates cross-reacted with six different serotypes of Shigella and were, in each case, highly type-specific. Two of the isolates belonging to the Enterobacter and Escherichia genera gave extremely strong cross-reactivity with Shigella dysenteriae and Shigella boydii antisera, respectively. The Aerococcus isolates gave relatively weak but significant cross-reactions with S. dysenteriae. Western blot analysis revealed that a number of antigens from the isolates cross-react with Shigella spp. The results indicate that important Shigella spp. surface antigens are shared by a number of environmental bacteria, which have implications for the use of serological methods in attempts for the detection and recovery of Shigella from aquatic environments.     

Native gel analysis of macromolecular protein complexes in cultured mammalian cells

Native gel electrophoresis enables separation of cellular proteins in their non‐denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co‐migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes.     

Iron Cycling Potentials of Arsenic Contaminated Groundwater in Bangladesh as Revealed by Enrichment Cultivation

The activities of iron-oxidizing and reducing microorganisms impact the fate of arsenic in groundwater. Phylogenetic information cannot exclusively be used to infer the potential for iron oxidation or reduction in aquifers. Therefore, we complemented a previous cultivation-independent microbial community survey covering 22 arsenic contaminated drinking water wells in Bangladesh, with the characterization of enrichments of microaerophilic iron oxidizers and anaerobic iron reducers, conducted on the same water samples. All investigated samples revealed a potential for microbial iron oxidation and reduction. Microbial communities were phylogenetically diverse within and between enrichments as was also observed in the previous cultivation-independent analysis of the water samples from which these enrichments were derived. Enrichment uncovered a larger diversity in iron-cycling microorganisms than previously indicated. The iron-reducing enrichments revealed the presence of several 16S ribosomal RNA (16S rRNA) gene sequences most closely related to Acetobacterium, Clostridium, Bacillus, Rhizobiales, Desulfovibrio, Bacteroides, and Spirochaetes, in addition to well-known dissimilatory iron-reducing Geobacter and Geothrix species. Although a large diversity of Geobacteraceae was observed, they comprised only a small part of the iron-reducing consortia. Iron-oxidizing gradient tube enrichments were dominated by Comamonadaceae and Rhodocyclaceae instead of Gallionellaceae. Forty-five percent of these enrichments also revealed the presence of the gene encoding arsenite oxidase, which converts arsenite to less toxic and less mobile arsenate. Their potential for ferric (oxyhydr)oxides precipitation and arsenic immobilization makes these iron-oxidizing enrichments of interest for rational bioaugmentation of arsenite contaminated groundwater.     

A simple and sensitive assay for evaluation of sediment toxicity using Lumbriculus variegatus (Müller)

The burrowing oligochaete Lumbriculus variegatus (Muller) was evaluated as a test organism for sediment bioassessment. As part of a battery of assays, the survival, growth, reproduction and burrowing behaviour of Lumbriculus in sediments from several areas of the North American Great Lakes were examined. Although not abundant, this species can be found in oligotrophic and mesotrophic environments. They are readily cultured and can be commercially purchased. Chronic tests indicated that Lumbriculus was as sensitive to contaminated sediments as Hyalella azteca, but less sensitive than the Holarctic amphipod Pontoporeia (Diporeia) hoyi. The growth of individuals or their reproduction by cloning can be measured after a two-week exposure at 20 °C.