Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

ABA and Low Temperature Induce Freezing Tolerance via Distinct Regulatory Pathways in Wheat

The role of ABA in the induction of freezing tolerance was investigatedin two wheat (T. aestivum L.) cultivars, Glenlea (spring var)and Fredrick (winter var). Exogenous application of ABA (5x10^–5M for 5 days at 24°C) increased the freezing tolerance ofintact plants by only 3°C (LT₅₀) in both cultivars. Maximalfreezing tolerance (LT₅₀ of –9°C for Glenlea and –17°Cfor Fredrick) could only be obtained with a low temperaturetreatment (6/2°C; day/night) for 40 days. These resultsshow that exogenously applied ABA cannot substitute for lowtemperature requirementto induce freezing tolerance in intactwheat plants. Furthermore, there was no increase in the endogenousABA level of wheat plants during low temperature acclimation,suggesting the absence of an essential role for ABA in the developmentof freezing tolerance in intact plants. On the other hand, ABAapplication (5x10^–5 M for 5 days at 24°C) to embryogenicwheat calli resulted in an increase of freezing tolerance similarto that achieved by low temperature. However, as in intact plants,there was no increase in the endogenous ABA level during lowtemperature acclimation of calli. These results indicate thatthe induction of freezing tolerance by low temperature is notassociated with an increase in ABA content. Using an antibodyspecific to a protein family associated with the developmentof freezing tolerance, we demonstrated that the induction offreezing tolerance by ABA in embryogenic wheat calli was correlatedwith the accumulation of a new 32 kDa protein. This proteinis specifically induced by ABA but shares a common antigenicitywith those induced by low temperature. These results suggestthat ABA induces freezing tolerance in wheat calli via a regulatorymechanism different from that of low temperature. (Received June 15, 1993; Accepted September 16, 1993)     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Neurocircuitry of the basal ganglia studied by monitoring neurotransmitter release

The neurocircuitries of the basal ganglia are studied with in vivo microdialysis, with special consideration to dopamine transmission and its interaction with other neurotransmitter systems. The aim is to develop experimental models to study the pathophysiology and therapy of neurodegenerative disorders of the basal ganglia, as well as to develop models to study the short- and long-term consequences of perinatal asphyctic lesions. A main goal of these studies is to find and to characterize new treatments for these disorders.     

TCA-100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.     

Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain

Abstract: A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca²⁺, a temperature optimum at ～50°C, and two K_mvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher K_mhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]₂₀₀. At 5°C, the [θ]₂₀₀of the deiminated protein was -70 × 10³ compared with -30 × 10³ deg cm²/dmol for the native protein. When the temperature was increased to 70°C, the [θ]₂₀₀ was -44 × 10³ for the deiminated protein and -20 × 10⁷ deg cm²/ dmol for the native C-1. When plotted as a function of temperature, [θ]₂₀₀ decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.