Insulin adsorption onto zinc oxide nanoparticle mediates conformational rearrangement into amyloid-prone structure with enhanced cytotoxic propensity

Background

Injection localized amyloidosis is one of the most prevalent disorders in type II diabetes mellitus (TIIDM) patients relying on insulin injections. Previous studies have reported that nanoparticles can play a role in the amyloidogenic process of proteins. Hence, the present study deals with the effect of zinc oxide nanoparticles (ZnONP) on the amyloidogenicity and cytotoxicity of insulin.

In vivo effects of cadmium on calmodulin and calmodulin regulated enzymes in rat brain.

Effect of chronic cadmium (Cd) exposure and the influence of diethyldithiocarbamate (DDC) on Cd absorption was studied on the brain of young male Wistar rats. A significant amount of Cd accumulated in cerebral cortices of rats after 4 weeks of Cd (6 mg/kg body wt) exposure (through gastric intubation). The biological activity of calmodulin (CaM) decreased significantly (p less than 0.001) in the cerebral cortices of these animals in comparison to the control group. 3'-5' Phosphodiesterase and synaptic membrane Ca(2+)-Mg(2+) ATPase were also significantly affected (p less than 0.01 and p less than 0.001 respectively). However, Cd treatment did not alter synaptic membrane adenylate cyclase activity and DDC (9.2 mg/kg body wt, intraperitoneal) treatment along with Cd (6 mg/kg body wt) enhanced Cd accumulation in cerebral cortices of treated animals resulting in an increased inhibition of CaM and CaM dependent enzymes. These data suggest that Cd may be acting via binding to CaM and uncoupling it from its normal cellular control of calcium.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

Conformational coupling,bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP–NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop–trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β′ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.     

Cadmium stress-induced oxidative stress and role of nitric oxide in rice (Oryza sativa L.)

Cadmium (Cd) is a potential environmental phytotoxicant. The generation of reactive oxygen species (ROS) due to Cd stress is responsible for the induction of oxidative stress in plants. On the other hand, SNP, a NO donor is known to have effect on Cd-induced oxidative stress in plants. We evaluated the effect of NO on the regulation of Cd stress in the rice (Oryza sativa L.) variety MSE-9. Cd treatment was given in the form of 50, 100 and 200 ??M, whereas for interaction study, 100 ??M of Cd and 100 ??M of SNP were used. The result showed that Cd-induced oxidative stress in MSE-9 by generating ROS. However, when SNP was given with Cd stress, it was seen that SNP treatment regulated the stress metabolism in rice seedlings under Cd toxicity by generating NO. It can be said that the SNP in combination with Cd treatment might possess the way to protect rice seedlings under Cd stress.     

Antioxidant properties and total phenolic contents of some tropical seaweeds of the Brazilian coast

Many types of macroalgae contain a wide range of bioactive compounds that have antioxidant potential. However, in contrast to terrestrial plants, only a few studies have reported the antioxidant activity of seaweeds. Therefore, extracts from 26 marine macroalgae species from the south and southeast coasts of Brazil were evaluated for their antioxidant activity, using the 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) method and β-carotene/linoleic acid assay, and their total phenolic contents, through Folin–Ciocalteu method. Padina gymnospora, Sargassum vulgare, and Osmundaria obtusiloba presented the highest values of total phenolic content. Using β-carotene bleaching assay, Colpomenia sinuosa, Dictyota sp., Dichotomaria marginata, Ganonema farinosum, and Spyridia clavata presented up to 65 % of antioxidant activity. Some of the extracts showed more than 60 % of inhibition of DPPH in the lowest concentration (0.01 mg/mL), including Amansia sp., Bostrychia tenella, Cryptonemia seminervis, Hypnea musciformis, Plocamium brasiliense (1), and S. clavata. Both Amansia sp., and C. seminervis presented the most relevant antioxidant potential, with percentage of inhibition greater than 70 % in the three tested concentrations. These two species were then analyzed by nuclear magnetic resonance spectroscopy (NMR) and were selected for guided fractionation bioassay. They both presented lipid compounds, fatty acids, esters of fatty acids, triglycerides, and sterols as major components. The fractionation of extracts revealed that the organic fractions were responsible for the antioxidant activity. The results obtained through this work indicate that the analyzed seaweeds are a promising source of compounds with high antioxidant potential.     

Quantification of protein transcytosis in the human colon carcinoma cell line CaCo-2

The transepithelial absorption of food-type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo-2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical-basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4 degrees C (7.41 +/- 1.26 pmoles/h.cm2 vs. 27.40 +/- 8.91 at 37 degrees C). The amount conveyed via the direct pathway was very small (0.89 +/- 0.35 pmoles/h.cm2) and did not diminish at 4 degrees C (1.43 +/- 0.59 pmoles/h.cm2). In the basal-apical direction, HRP transport along the degradative pathway at 37 degrees C was similar to the apical-basal value and was inhibited at 4 degrees C (16.40 +/- 4.05 vs. 2.72 +/- 2.52 pmoles/h.cm2), but along the direct pathway, it was eight times the apical-basal value (8.36 +/- 3.11 pmoles/h.cm2) and was inhibited at 4 degrees C (2.43 +/- 0.78 pmoles/h.cm2). Intact HRP fluxes were not correlated with the electrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10(-5) M did not affect direct or degradative transport in the apical-to-basal direction. These results suggest that in CaCo-2 cells HRP undergoes bidirectional transcytosis by a fluid-phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.     

A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min⁻¹ mg⁻¹) as compared to unmodified pH tuned lipase (128 nmol min⁻¹ mg⁻¹). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min⁻¹ mg⁻¹, respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content.