A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

Auditing protein therapeutics management by professional APCs: toward prevention of immune responses against therapeutic proteins

Alloimmunization is a crippling concern in the management of patients undergoing administration of protein therapeutics as evidenced in replacement therapy and other treatment procedures. Several issues in the genesis and modulation of such deleterious immune responses have been studied. While authors have focused on the downstream events of the specific immune response and suggested modification of protein therapeutics to eliminate epitopes that interact with B cell receptors, T cell receptors, or MHCII molecules, the mechanisms underlying Ag interaction with APCs, a step upstream of immune effectors, have been grossly neglected. We wish to emphasize that the recent knowledge in understanding the capacities of an APC to handle an Ag and the importance of the surrounding microenvironment in this process are crucial for designing novel protein therapeutics with reduced immunogenicity.     

Identification of novel endogenous cytochrome p450 arachidonate metabolites with high affinity for cannabinoid receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.     

Pseudoguaianolides from the flowers of Parthenium hysterophorus

Chemical investigation on the flowers of Parthenium hysterophorus has resulted in the isolation of four new pseudoguaianolides, hysterones A-D along with the known compounds, parthenin, coronopilin, 2beta-hydroxycoronopilin and tetraneurin-A. The structures of the new compounds were established by interpretation of their spectral (1D and 2D NMR) data. The X-ray crystallographic analysis of hysterones A and C was also carried out.     

1-(1-Naphthyl)piperazine as a novel template for 5-HT6 serotonin receptor ligands

4-Sulfonyl analogs of 1-(1-naphthyl)piperazine bind at human 5-HT6 receptors and represent a novel class of human 5-HT6 receptor ligands.     

Common variable immunodeficiency: the immune system in chaos

Common variable immunodeficiency (CVID) is a heterogeneous disorder that is associated with low serum-immunoglobulin concentrations, defective specific-antibody production and an increased susceptibility to bacterial infections of the respiratory and gastrointestinal tracts. In spite of the identification of genes that are associated with several known primary immunodeficiencies, the basic immunologic and molecular defects of the majority of patients with CVID have remained obscure. Most of the studies aimed at understanding the immunopathogenesis of CVID suggest that this condition is primarily a T-cell disorder, although renewed attention on the genetic linkage and haplotype analysis in families of patients with CVID and on the role of dendritic cells and B cells has revealed several interesting features. This new information should assist in understanding the pathogenesis of CVID and improving the therapeutic strategies.     

Protective Effects of 7‐Hydroxycoumarin on Dyslipidemia and Cardiac Hypertrophy in Isoproterenol‐Induced Myocardial Infarction in Rats

This study evaluates the protective effects of 7‐hydroxycoumarin (7‐HC) on dyslipidemia and cardiac hypertrophy in isoproterenol (ISO) induced myocardial infarction (MI) in rats. Rats were pre‐ and co treated with 7‐HC (16 mg/kg) daily for 8 days. ISO (100 mg/kg) was subcutaneously injected into rats on seventh and eighth days to induce MI. Increased activity/levels of serum creatine kinase‐MB (CK‐MB), troponin‐T, plasma lipid peroxidation products, and altered levels of lipids in the serum and heart and serum lipoproteins were noted in ISO‐induced rats. ISO‐induced myocardial infarcted rats revealed increased hypertrophy (cardiac and left ventricular) and hepatic 3‐hydroxyl 3‐methylglutaryl‐coenzyme‐A reductase (HMG‐CoA reductase) activity. Pre and cotreatment with 7‐HC revealed significant protective effects on all the biochemical parameters evaluated. The in vitro study demonstrated its free radical scavenging property. Thus, 7‐HC protects ISO‐induced MI in rats by its free radical scavenging and antihyperlipidaemic and antihypertrophic properties.     

Evolutionary rates for tuf genes in endosymbionts of aphids

The gene encoding elongation factor Tu (tuf) in aphid endosymbionts (genus Buchnera) evolves at rates of 1.3 x 10(-10) to 2.5 x 10(-10) nonsynonymous substitutions and 3.9 x 10(-9) to 8.0 x 10(-9) synonymous substitutions per position per year. These rates, which are at present among the most reliable substitution rates for protein-coding genes of bacteria, have been obtained by calibrating the nodes in the phylogenetic tree produced from the Buchnera EF-Tu sequences using divergence times for the corresponding ancestral aphid hosts. We also present data suggesting that the rates of nonsynonymous substitutions are significantly higher in the endosymbiont lineages than in the closely related free-living bacteria Escherichia coli and Salmonella typhimurium. Synonymous substitution rates for Buchnera approximate estimated mutation rates for E. coli and S. typhimurium, as expected if synonymous changes act as neutral mutations in Buchnera. We relate the observed differences in substitution frequencies to the absence of selective codon preferences in Buchnera and to the influence of Muller's ratchet on small asexual populations.      

Human placental calcium activated neutral proteinase: Separation and functional characterization of subunits

The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.     

Population analysis of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> by using endogenous repetitive DNA sequences and mating-type alleles in different districts of Karnataka,India

Rice is the staple food crop of more than 60% of the population of the world. This crop suffers from blast disease caused by Magnaporthe oryzae. Information on the mating-type allele distribution and diversity of the pathogen population for the state of Karnataka, India is scanty. With this background, a total of 72 isolates of M. oryzae from rice in different districts of Karnataka were examined for identifying sexual mating alleles MAT1, MAT2 and understanding the genetic diversity based on DNA fingerprint of pot2, an inverted repeat transposon. Among 72 isolates, 44 isolates belonged to MAT1 type (male fertile) and 28 isolates were of MAT2 (female fertile) and there were no hermaphrodite isolates. In a given geographical location, only one mating type was identified. Results revealed that the isolates obtained from these regions are not sexually fertile showing predominant asexual reproduction. Hence, genetic variation observed in the pathogen may be mainly because of high copy number of transposons. A high copy number transposon, namely Pot2, was selected in our study to detect genetic diversity of the pathogen. Pot2 rep-PCR DNA fingerprinting profile showed 27 polymorphic bands with bands ranging in size from 0.65 to 4.0 kb and an average of 10 to 14 bands per isolate. Five distinct clusters were formed with two major, two minor, and one outlier. Clusters 4 and 5 are further subdivided into three sub-clusters. Some of the isolates belonging to clusters 3, 4, and 5 are interlinked as these locations are close to one another sharing common geographical parameters and boundaries. This knowledge on the sexual behavior and genetic diversity of M. oryzae is important with respect to breeding for disease resistance.