Aspects of the demetalation and remetalation of ceruloplasmin

This study dealt with the demetalation and remetalation chemistry of the copper-containing protein ceruloplasmin. For the enzyme from human plasma, dialysis against cyanide at 4°C readily removed copper. Although the apoprotein took up copper(I) at the same temperature, the characteristic blue color of the native protein did not return to any significant extent. However, excellent reconstitution occurred when we added copper(I) at room temperature. With porcine ceruloplasmin, it was more practical to carry out the copper removal step at room temperature, but the reconstitution went smoothly at 4°C. With either source of ceruloplasmin, the binding of the six essential copper ions was generally a highly cooperative process, but the results were different when we combined the apoprotein with Hg(II). After the protein took up 2 mercury ions, it would take up only 1–2 more metal ions even after exposure to a large excess of copper(I). In order to accomodate the various experimental results, we have proposed that a reversible conformational change must occur during the demetalation and remetalation processes. During the remetalation process, it is therefore important that metal uptake occurs in the proper sequence.     

Facile Synthesis of Benzimidazoles via Oxidative Cyclization of Acyclic Monoterpene Aldehyde with Diamines: Studies on Antimicrobial and in Vivo Evaluation of Zebrafish

Citral ( 1a ), a bioactive component of Cymbopogon citratus (lemongrass) could be isolated and semi-synthetic analogs synthesized with improved therapeutic properties. Herein we first report describes citral ( 1a ) as a primary material for the synthesis of benzimidazole derivatives between various o-phenylenediamines ( 2a – l ) in the presence of Diisopropylethylamine (DIPEA) as a commercially available environmentally benign base, ethanol as a green solvent and the yield of all benzimidazole derivatives ( 3a – l ) was between 68–76 %; The semi-synthetically prepared benzimidazole derivatives ( 3a – l ) were assessed for their anti-bacterial and anti-fungal properties. The benzimidazole compounds ( 3a – b , and 3g – j ) exhibit good anti-microbial activity. In addition, in silico study was carried out to determine the specific binding affinity of the diamine halogen substituted benzimidazole derivatives to the specific target proteins. In silico analysis revealed a high correlation between docking results and experimental results. Finally, benzimidazole demonstrated significant antibacterial and antifungal activity. Zebrafish embryos were subjected to In vivo toxicological test found that all of the benzimidazole compounds ( 3a – l ) were non-toxic and had low embryotoxicity after 96 h, with an LC₅₀ of 36.425 μg, which could facilitate the design of novel antimicrobial agents using a cost-effective method.     

Linoleic acid-induced endothelial cell injury: Role of membrane-bound enzyme activities and lipid oxidation

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca²⁺-ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [₃H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

Bionics and Structural Biology： A Novel Approach for Bio-energy Production

Cellular metabolism is a very complex process. The biochemical pathways are fundamental structures of biology. These pathways possess a number of regeneration steps which facilitate energy shuttling on a massive scale. This facilitates the biochemical pathways to sustain the energy currency of the cells. This concept has been mimicked using electronic circuit components and it has been used to increase the efficiency of bio-energy generation. Six of the carbohydrate biochemical pathways have been chosen in which glycolysis is the principle pathway. All the six pathways are interrelated and coordinated in a complex manner. Mimic circuits have been designed for all the six biochemical pathways. The components of the metabolic pathways such as enzymes, cofactors etc., are substituted by appropriate electronic circuit components. Enzymes are related to the gain of transistors by the bond dissociation energies of enzyme-substrate molecules under consideration. Cofactors and coenzymes are represented by switches and capacitors respectively. Resistors are used for proper orientation of the circuits. The energy obtained from the current methods employed for the decomposition of organic matter is used to trigger the mimic circuits. A similar energy shuttle is observed in the mimic circuits and the percentage rise for each cycle of circuit functioning is found to be 78.90. The theoretical calculations have been made using a sample of domestic waste weighing 1.182 kg. The calculations arrived at finally speak of the efficiency of the novel methodology employed.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Creating designed zinc-finger nucleases with minimal cytotoxicity

Zinc-finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination with investigator-provided donor DNA or induce gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining both in plant cells and in mammalian cells, including human cells. ZFNs are formed by fusing zinc-finger proteins to the nonspecific cleavage domain of the FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (> 10%) in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. The mechanism of DSB by ZFNs requires (1) two ZFN monomers to bind to their adjacent cognate sites on DNA and (2) the FokI nuclease domains to dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form; this could limit the efficacy and safety of ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of the FokI cleavage domain for the creation of custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the reengineered obligate heterodimer variants of the FokI cleavage domain were tested using the green fluorescent protein gene targeting reporter system. The three-finger and four-finger zinc-finger protein fusions to the REL_DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.     

Electroactive biofilms: how microbial electron transfer enables bioelectrochemical applications

Microbial biofilms are ubiquitous. In marine and freshwater ecosystems, microbe–mineral interactions sustain biogeochemical cycles, while biofilms found on plants and animals can range from pathogens to commensals. Moreover, biofouling and biocorrosion represent significant challenges to industry. Bioprocessing is an opportunity to take advantage of biofilms and harness their utility as a chassis for biocommodity production. Electrochemical bioreactors have numerous potential applications, including wastewater treatment and commodity production. The literature examining these applications has demonstrated that the cell–surface interface is vital to facilitating these processes. Therefore, it is necessary to understand the state of knowledge regarding biofilms’ role in bioprocessing. This mini-review discusses bacterial biofilm formation, cell–surface redox interactions, and the role of microbial electron transfer in bioprocesses. It also highlights some current goals and challenges with respect to microbe-mediated bioprocessing and future perspectives.     

Hypocone reduction and Carabelli's traits in contemporary Jordanians and the association between Carabelli's trait and the dimensions of the maxillary first permanent molar

The objective of this study is to determine the prevalence of expression and bilateralism of two dental morphological traits in contemporary Jordanians: The hypocone reduction trait on the maxillary second permanent molar and Carabelli's trait on maxillary permanent first and second molars. Furthermore, inter-trait correlation and the relationship of Carabelli's traits with upper first molar dimensions were investigated. Three hundred subjects of school children at their 10th grade and of an average age of 15.5 +/- 0.4 years were involved. Alginate impressions for the maxillary arch were taken, dental casts were reproduced. The selected accurate casts were of 132 male- and 155 female-students. The frequencies of hypocone reduction trait on the maxillary second molar and Carabelli's trait on the maxillary molars were examined. Buccolingual and mesiodistal diameters of the maxillary first molar were measured and recorded. Paired Sample t test and Nonparametric Correlation analysis were used for data analysis. Hypocone reduction trait on the maxillary second molar was found in 29.8% of the examined students. Positive forms of Carabelli's trait on first and second molars were observed in 65.0% and 3.8%, respectively. Nonparametric correlation analysis revealed positive association between Carabelli's trait on first molar and hypocone reduction trait on the maxillary second molar. The presence of Carabelli's trait on first molar was strongly associated with the increase of buccolingual, but not the mesiodistal, diameter. Bilateralism was found highly significant in the tested traits and both genders (p < 0.001). This finding might be a sign of relatively low environmental stresses in the living Jordanian population and/or great ability of its individuals to buffer the adverse effects of such stresses.