Kinetic characterization and ligand binding studies of His351 mutants of Bacillus anthracis adenylate cyclase

Edema factor is a calmodulin dependent adenylyl cyclase secreted as one of the primary exotoxins by Bacillus anthracis. A histidine residue at position 351 located in its active site has been implicated in catalysis but direct evidence of its functional role is still lacking. In the present study, we introduced mutations in full-length edema factor (EF) to generate alanine (H351A), asparagine (H351N), and phenylalanine (H351F) variants. Spectral analysis of these variants displayed no gross structural deformities. Kinetic characterization showed that the adenylyl cyclase activity of H351N and H351F mutants decreased 34- and 40-fold, respectively, whereas H351A mutant completely lost activity. K(m) and K(i) values for ATP, pH activity profiles, and calmodulin activation curves of asparagine and phenylalanine mutants were not altered markedly. This kinetic data corroborated our ligand binding studies. Apparent K(d) values for calmodulin and ATP binding were found to be similar for wild-type EF and these active site variants. The effective substitution of H351 by asparagine and phenylalanine, albeit at a greatly reduced K(cat), without perturbing the ATP binding highlights the importance of this residue in transition-state stabilization. This was also evident from the positive free energy difference calculated for these mutants. However, equilibrium dialysis experiments revealed noticeable increase in ATP binding constant of H351A mutant, suggesting an additional role of H351 in precise substrate binding in the catalytic pocket. This is the first comprehensive study that describes the kinetic and ligand binding properties of H351 mutants and validates the importance of this residue in EF catalysis.     

Melanoma-induced suppression of tumor antigen-specific T cell expansion is comparable to suppression of global T cell expansion

We have observed that in vivo interaction between melanoma and resting T cells promotes suppression of antigen-driven proliferative T cell expansion. We hypothesized that this suppression would affect tumor antigen-specific T cell populations more potently than tumor-unrelated T cell populations. A B16F10 cell line was stably transfected to express low levels of the lymphocytic choriomeningitis virus (LCMV) glycoprotein GP33 (B16GP33). Mice bearing B16F10 or B16GP33 tumors were infected with LCMV, and proliferative expansion of LCMV epitope-specific T cell populations was quantified. In vitro and in vivo assays confirmed low levels of antigenic GP33 expression by B16GP33 tumors. Suppressed expansion of GP33-specific T cells was equivalent between mice bearing B16F10 and B16GP33 tumors. These observations suggest that the ability of growing melanoma tumors to impair antigen-driven proliferative expansion of activated T cells is global and not antigen-specific, and provide further insight into the influence of cancer on activated T cell homeostasis.     

Identification of Tsetse (Glossina spp.) Using Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies'' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the future.     

Microwave-assisted preparation of bagasse and rice straw for the removal of emulsified oil from wastewater

The present study was carried out to examine modified bagasse and rice straw as absorbents for the removal of emulsified oil by a microwave-assisted technique. The raw adsorbents were chemically modified with sodium hydroxide and surfactant solution for better adsorption of oil. The surface modification was confirmed through Fourier transform infrared (FTIR) spectroscopy and scanning electron microscope (SEM) analysis. Batch studies were performed to identify the influence of contact time, pH, dosage, speed, and temperature on the percentage of removal of emulsified oil from the wastewater. A maximum oil removal of 98.07% and 98.72% was achieved in microwave-assisted bagasse and rice straw at 313 K. The obtained data were compared with isotherm and kinetic models to understand the mechanism behind the adsorption. From the Langmuir isotherm model, a maximum adsorption capacity of 192.58 and 276.82 mg/g was observed for modified bagasse and rice straw. The oil-loaded raw and microwave-assisted adsorbents can act as a potential energy source for renewable energy.     

Effect of Different Light Regimes on Pre-Adult Fitness in Drosophila melanogaster Populations Reared in Constant Light for over Six Hundred Generations

Egg to eclosion development time and survivorship were assayed on four laboratory populations of Drosophila melanogaster that had been reared for over 600 generations in continuous light (LL) and constant temperature. The assays were performed in three environments: continuous light (LL), periodically varying light/dark cycles (LD 12:12 hr), and continuous darkness (DD). Development time in LL was significantly less than that in LD, which, in turn, was significantly less than that in DD, whereas survivorship did not differ significantly among the three treatments. The results indicate that individuals from Drosophila populations routinely maintained in LL do not suffer any deleterious effects of LL treatment on pre-adult fitness. Other studies on these populations have shown that free-running period (t) of the eclosion rhythm in DD is greater than that in LD. Our results are, thus, also consistent with the notion that development time may be a function of the free-running period.     

Neuronal synchrony during anesthesia: a thalamocortical model

There is growing evidence in favor of the temporal-coding hypothesis that temporal correlation of neuronal discharges may serve to bind distributed neuronal activity into unique representations and, in particular, that θ (3.5-7.5 Hz) and δ (0.5 < 3.5 Hz) oscillations facilitate information coding. The θ- and δ-rhythms are shown to be involved in various sleep stages, and during anesthesia, they undergo changes with the depth of anesthesia. We introduce a thalamocortical model of interacting neuronal ensembles to describe phase relationships between θ- and δ-oscillations, especially during deep and light anesthesia. Asymmetric and long-range interactions among the thalamocortical neuronal oscillators are taken into account. The model results are compared with experimental observations. The δ- and θ-activities are found to be separately generated and are governed by the thalamus and cortex, respectively. Changes in the degree of intraensemble and interensemble synchrony imply that the neuronal ensembles inhibit information coding during deep anesthesia and facilitate it during light anesthesia.     

Developmental plasticity of the locomotor activity rhythm of Drosophila melanogaster

We used four replicate outbred populations of Drosophila melanogaster to investigate whether the light regimes experienced during the pre-adult (larval and pupal) and early adult stages influence the free-running period (τ_DD) of the circadian locomotor activity rhythm of adult flies. In a series of two experiments four different populations of flies were raised from egg to eclosion in constant light (LL), in light/dark (LD) 12:12 h cycle, and in constant darkness (DD). In the first experiment the adult male and female flies were directly transferred into DD and their locomotor activity was monitored, while in the second experiment the locomotor activity of the emerging adult flies was first assayed in LD 12:12 h for 15 days and then in DD for another 15 days. The τ_DD of the locomotor activity rhythm of flies that were raised in all the three light regimes, LL, LD 12:12 h and in DD was significantly different from each other. The τ_DD of the locomotor activity rhythm of the flies, which were raised in DD during their pre-adult stages, was significantly shorter than that of flies that were raised as pre-adults in LL regime, which in turn was significantly shorter than that of flies raised in LD 12:12 h regime. This pattern was consistent across both the experiments. The results of our experiments serve to emphasise the fact that in order to draw meaningful inferences about circadian rhythm parameters in insects, adequate attention should be paid to control and specify the environment in which pre-adult rearing takes place. The pattern of pre-adult and early adult light regime effects that we see differs from that previously observed in studies of mutant strains of D. melanogaster, and therefore, also points to the potential importance of inter-strain differences in the response of circadian organisation to external influences.     

Effect of pH,temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain fromAnanas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.