An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.     

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Abstract

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model

Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADU_DD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).     

The Action of Peptidases from Aspergillus sojae on Soybean Proteins

To elucidate the mechanism of light-activation of pyruvate P_L dikinase in maize leaf, the inactive form was purified to homogeneity from dark-treated leaves using an activation system to locate it. The purification procedure included ammonium sulfate-fractionation followed by conventional chromatography.

The homogeneous enzyme after maximal activation had a specific activity comparable to that of the active enzyme obtained from non-dark-treated plants. The enzyme was indistinguishable from the active one in its molecular size and charge and in the amino acid composition of its acid-hydrolysate.     

Biofumigation and Enhanced Biodegradation: Opportunity and Challenge in Soilborne Pest and Disease Management

Management of soilborne pests and diseases in cropping systems is often highly challenging—in implementation of acceptable methodologies and in dealing with secondary problems. The phase-out of methyl bromide brings this into particularly sharp focus. There is a need for diversified options and alternatives to fill different roles across the soilborne pest and disease management spectrum, but flexibility is limited, as practicalities demand that they fit into a prophylactic methodology. It is against such a backdrop that expectations and promotion of alternatives must be set. There is also a need to recognize potentially serious problems that may have been masked under historical management regimes, but for which the nature of the system offers little scope to avoid or manage.

Biofumigation is the beneficial use of Brassica green manures that release isothiocyanates chemically similar to methyl isothiocyanate, the active agent from the synthetic fumigant metam sodium, which is used as a substitute for methyl bromide in some systems. A systematic approach to research into biofumigation, specifically aimed at overcoming a long history of empiricism, has seen significant recent advances in both basic and applied knowledge. A key development has been achievement of maximal biofumigation potential through greatly enhanced release of appropriate isothiocyanates into soil. These advances have led to commercial adoption, demonstrating that biofumigation, when applied to appropriate production systems, can have efficacy and offer cost savings. Crucially, these systematically derived research and development findings and their adoption now provide the impetus for self-sustaining further development and market penetration of the concept. Despite this success, biofumigation is not seen as being sufficiently powerful or practical in implementation to be an alternative to methyl bromide on a broad scale and misdirection in that regard could be counterproductive to more appropriately targeted further development.

Enhanced microbial biodegradation is a cryptic phenomenon that can diminish the efficacy of soil-applied pesticides, including isothiocyanates and most other currently available methyl bromide substitutes. Because methyl bromide is not susceptible, the phenomenon has potentially serious implications in intensive production systems switching from methyl bromide to reliance on other compounds that are. It is an intractable problem once induced. Avoidance of its onset is the only feasible management strategy. This has been aided for some particularly vulnerable environments by recent clarification of key risk factors associated with soil type, soil pH, and calcium content.     

Effective cancer therapy based on selective drug delivery into cells across their membrane using receptor-mediated endocytosis

Cancer is one of the major causes of death globally. The current treatment options are insufficient, leading to unmet medical needs in cancer treatment. Off-target side effects, multidrug resistance, selective distribution to cancerous tissues, and cell membrane permeation of anti-cancer agents are critical problems to overcome. There is a method to solve these problems by using receptor-mediated endocytosis (RME). It is well known that proteins such as integrin, HER2, EGFR, or other cancer biomarkers are specifically overexpressed on the surface of target cancer cells. By taking advantage of such specific receptors, payloads can be transported into cells through endocytosis using a conjugate composed of the corresponding ligands connected to the payloads by an appropriate linker. After RME, the payloads released by endosomal escape into the cytoplasm can exhibit the cytotoxic activity against cancer cells. Cell-penetrating peptides (CPPs), tumor-homing peptides (THPs), and monoclonal antibodies (mAbs) are utilized as ligands in this system. Antibody drug conjugates (ADCs) based on RME have already been used to cure cancer. In addition to the canonical conjugate method, nanocarriers for spontaneous accumulation in cancer tissue due to enhanced permeability and retention (EPR) effect are extensively used. In this review, I introduce the possibilities and advantages of drug design and development based on RME for the treatment of cancer.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

The facile HPLC enantioresolution of amino acids,peptides on naphthylethylcarbamate-β-cyclodextrin bonded phases using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate: factors that affect the resolution

Summary. A variety of -amino acids are enantioresolved for the first time on naphthylethylcarbamate--cyclodextrin bonded phases (i.e., SN- and RN--CD) using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate in alkaline medium. The resolution is better obtained on RN--CD phase and fails to reproduce if the amino acid is N-benzoylated or N-carbobenzyloxylated under the same chromatographic conditions. The enhanced resolution is believed to be due to the re-location of the hydrogen receptor site from sulfur to nitrogen on the isothiocyanyl fragment of derivatizing reagent, which in turn changes the enantioselectivity. Also, the sulfur atom is larger in size and subject to steric hindrance more significantly in comparison with oxygen. The carboxyl group of amino acid is essential toward a satisfactory resolution. The position of the amino group on the backbone affects the resolution as well. Finally, the resolution is either not observed or unsatisfactory in the reversed- or normal phase mode for most of the amino acids examined in this study.