Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.     

Molluskan Grazing Traces (Ichnogenus Radulichnus Voigt, 1977) on a Pleistocene Bivalve from Southern Brazil,With the Proposal of a New Ichnospecies

The ichnogenus Radulichnus Voigt, 1977 Voigt, E. 1977. On grazing traces produced by the radula of fossil and recent gastropods and chitons. In Trace fossils 2, eds. T. P. Crimes, and J. C. Harper, 335–346. Geological Journal, Special Issue 9. [Google Scholar] is recorded for the first time from a bivalve, Anomalocardia brasiliana (Gmelin, 1791 Gmelin, J. F. 1791. Caroli a Linné, Systema Naturae. Tom. I, Pars VI. Leipzig: G.E. Beer, pp. 3021–3910. [Google Scholar]), in a late Pleistocene molluskan assemblage from the southern Brazilian coastal plain. Grazing traces comprise short (<1?mm), parallel furrows, arranged in rows on the inner (concave) shell surface and mostly concentrated in its central area. Radulichnus accommodates scratches on hard substrates produced by the radula of grazing gastropod or polyplacophorans. Our literature survey on fossil and extant traces, as well as studies on the grazing mechanism in living mollusks, document at least two distinct morphotypes that are related to differences in the feeding modes of the producers. We propose to distinguish a second ichnospecies of Radulichnus, in addition to the type, R. inopinatus Voigt, 1977 Voigt, E. 1977. On grazing traces produced by the radula of fossil and recent gastropods and chitons. In Trace fossils 2, eds. T. P. Crimes, and J. C. Harper, 335–346. Geological Journal, Special Issue 9. [Google Scholar] (produced by gastropods), which is named R. transversus isp. nov., and attributed to polyplacophorans. Grazing traces on the shell of A. brasiliana match the morphotype produced by polyplacophorans mollusks, and are indicative of its complex taphonomic history in comparison with other shells in this assemblage.     

Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αβ) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αβ production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.     

The study of gene GJB2/DFNB1 causing deafness in humans by linkage analysis from district Peshawar

Families with at least 2 or more individuals having hereditary hearing loss were enrolled from different areas of Khyber Pakhtoonkhwa, mainly from district Peshawar. Detailed history was taken from each family to minimize the presence of other abnormalities and environmental causes for deafness. Families were questioned about skin pigmentation, hair pigmentation, and problems relating to balance, vision, night blindness, thyroid, kidneys, heart, and infectious diseases like meningitis, antibiotic usage, injury, and typhoid. The pedigree structures were based upon interviews with multiple family members, and pedigrees of the enrolled families were drawn using Cyrillic program (version 2.1). All families showed recessive mode of inheritance. I studied 8 families of these 10. For linkage analyses, studies for DFNB1 locus, 3 STR markers (D13S175, D13S292, and D13S787) were genotyped using polyacrylamide gel electrophoresis (PAGE) and haplotypes were constructed to determined, linkage with DFNB1 locus. From a total of 8 families, a single family-10 showed linkage to DFNB1 locus.     

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.     

In vitro cytogenetic studies of cypermethrin on human lymphocytes

Assessment of cytotoxicity and response to external factors like pesticides were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) or MTT assay, which measures mitochondrial metabolism in the entire cell culture and provides information about the percentage of cell survival. Utilizing the MTT assay, the cytotoxicity of cypermethrin was determined on lymphocyte cultures from human peripheral blood samples, the short-term lymphocyte cultures were incubated with various aliquots of the cypermethrin and the LC50 was found to be 33.6 microM. Lymphocytes treated with low-doses (1/10 of LC50) of cypermethrin showed an increase in the frequency of chromosomal aberrations and found to be significant. Karyotype analysis revealed more satellite associations and chromosomal breaks in cypermethrin treated samples. Low-doses of the pesticide also induced single-strand breaks in the DNA as assessed by comet assay. The pesticide caused increase in the comet tail length with increase in pesticide concentration, implicating genotoxicity in somatic cells. It is concluded that In vitro assays could give important information of the mechanism of toxicity at low dosages and impact on genetic material of human origin.     

Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.     

Carotenoid inhibitors reduce strigolactone production and Striga hermonthica infection in rice

The strigolactones are internal and rhizosphere signalling molecules in plants that are biosynthesised through carotenoid cleavage. They are secreted by host roots into the rhizosphere where they signal host-presence to the symbiotic arbuscular mycrorrhizal (AM) fungi and the parasitic plants of the Orobanche, Phelipanche and Striga genera. The seeds of these parasitic plants germinate after perceiving these signalling molecules. After attachment to the host root, the parasite negatively affects the host plant by withdrawing water, nutrients and assimilates through a direct connection with the host xylem. In many areas of the world these parasites are a threat to agriculture but so far very limited success has been achieved to minimize losses due to these parasitic weeds. Considering the carotenoid origin of the strigolactones, in the present study we investigated the possibilities to reduce strigolactone production in the roots of plants by blocking carotenoid biosynthesis using carotenoid inhibitors. Hereto the carotenoid inhibitors fluridone, norflurazon, clomazone and amitrole were applied to rice either through irrigation or through foliar spray. Irrigation application of all carotenoid inhibitors and spray application of amitrole significantly decreased strigolactone production, Striga hermonthica germination and Striga infection, also in concentrations too low to affect growth and development of the host plant. Hence, we demonstrate that the application of carotenoid inhibitors to plants can affect S. hermonthica germination and attachment indirectly by reducing the strigolactone concentration in the rhizosphere. This finding is useful for further studies on the relevance of the strigolactones in rhizosphere signalling. Since these inhibitors are available and accessible, they may represent an efficient technology for farmers, including poor subsistence farmers in the African continent, to control these harmful parasitic weeds.     

The Effects of Demographic Factors on the Environmental Awareness of Omani Citizens

In year 2007, a survey was conducted in the Sultanate of Oman in order to evaluate the current environmental awareness of the Omani general public and their willingness to protect the environment. The focus of the survey was to explore the role played by demographic factors (sex, age, and education level) in determining the environmental awareness of the Omani public. The survey was administered to 425 respondents among all areas of the entire Muscat governorate in Oman. The results of the survey revealed that the environmental awareness of the Omani public was related to gender, age, and education level. Males were found to have a higher level of knowledge about environmental issues than females. Males were also more environmentally concerned and tended to engage in more environmental behaviors than females. Younger and more educated respondents tended to be more knowledgeable and concerned about the environment than older and less educated respondents.