Inositol- and Galactose-Binding Lectins of Chicken Brain Fractions Enriched with Glial Cells

Galactose- and inositol-binding proteins with lectin activity (GL-GAL and GL-I, respectively) were isolated from membranes enriched with cells of chicken brain fractions. Both lectins are glycoproteins of molecular mass 13.5 and 11.5 kDa, respectively; they show a high affinity to EDTA (GL-I) and EGTA (GL-GAL, GL-I), which indicates an important role of Ca⁺² in molecular organization of these lectins. In brain glial cells of chick embryos, unlike adult chickens, a soluble form of lectins has been revealed; it is easily extracted with 2 mM EDTA and shows sensitivity to L-lactose, D-galactose, and N-acetyl-D-galactosamine. It is suggested that in the course of embryonal and postembryonal development of the chicken brain, a transformation and qualitative changes of the lectin spectrum occur due to a change of function of glial cells.     

Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.     

A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans

A polyclonal antibody to ubiquitin has been prepared and shown to react with both ubiquitin and ubiquitinated histone 2A (uH2A). Applying this antibody in Western blotting experiments, we have observed that the salivary glands of Chironomus tentans contain an unusually low amount of uH2A (1% of histone 2A), while the amount of free ubiquitin is as abundant as in other animal cells, e.g. HeLa cells. The same low content of uH2A was also found in diploid epidermal cells of Chironomus origin suggesting that the low amount is not a characteristic of the polytene state of chromatin in salivary gland cells but rather a property of C. tentans as a species. The significance of the low degree of ubiquitination is discussed in relation to the information available on the organization of Chironomus chromatin into unusually large chromomeric entities.     

Determination of dialkyl phosphates in human hair for the biomonitoring of exposure to organophosphate pesticides

A new, simple, fast and sensitive method that enables the measurement of four dialkyl phosphates (DAPs) in human head hair is presented in the current study. The dialkyl phosphates, dimethyl phosphate (DMP), diethyl phosphate (DEP), diethyl thiophosphate (DETP) and diethyl dithiophosphate (DEDTP) are non-selective metabolites of the organophosphate pesticides (OPs). The extraction of DAPs from hair matrix was achieved by one step methanolic extraction. Head hair samples from general population and population occupationally exposed to OPs were analysed using gas chromatography–mass spectrometry (GC–MS) after derivatization with pentafluorobenzylbromide. The recovery of the target compounds was estimated at 84.3% for DMP, 116.1% for DEP, 109.0% for DETP and 91.5% for DEDTP. The limit of quantitation (LOQ) and detection (LOD) was 20 and 6 pg/mg for DMP, 10 and 5 pg/mg for DEP and DETP and 5 and 3 pg/mg for DEDTP, respectively. With-run and between-run precision as well as accuracy was estimated. The percentage of positive hair samples for DMP, DEP, DETP and DEDTP for the group of general population was 63.0%, 96.3%, 66.7%, and 70.4% respectively. The samples from the group with occupational exposure were positive for all dialkyl phosphates analysed. The median concentrations for DMP were 165.0 and 181.7 pg/mg, for DEP were 51.2 and 812.9 pg/mg, for DETP were 54.0 and 660.1 pg/mg, and for DEDTP were 40.0 and 60.6 pg/mg for the general population group and the group with occupational exposure respectively. Significant differences in the levels of the total dialkyl phosphates amongst exposed and not exposed groups were observed (p < 0.001). More specifically, the total ethyl phosphate (DEPs) and DAPs median concentrations were 119.5 and 301.5 pg/mg for the general population group and 1498.8 and 1694.4 pg/mg for the group with occupational exposure.     

Intragroup serotyping of meningococci

The method for obtaining antisera to meningococci of different serotypes are described and the scheme for the preparation of serotyping is presented, as well as the method for the preparation of the determinate fraction of serotype 2. Antisera to typing antigens 1, 2, 2-7, 2-10, 4, 5, 6, 8 (1) have been obtained, their specificity tested in parallel experiments with American and French typing sera. When typing meningococci, the use of antisera to purified protein antigen 2 is recommended.     

The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.