[Role of adhesion molecules in acute ischemic renal insufficiency].
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Acute Kidney Injury↗
Biomedical subjects
J Bonventre
Publications and source records attributed to J Bonventre.
Expression, purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site.
We have overexpressed the type II restriction endonuclease PvuII (R-PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2(1)2(1)2(1) with cell constants a = 95.8 A, b = 86.3 A, c = 48.5 A, and diffract X-rays to at least 2.7 A. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit.
Base Sequence↗
Defects in signal transduction caused by a T cell receptor beta chain substitution.
An antigen-specific T-T hybridoma was mutagenized with ethylmethane sulfonate and negatively selected by anti-Ly-6 antibody-induced growth inhibition. One of the mutants generated, M4/8, had lost surface expression of a T cell receptor (TcR) V beta 8 epitope detected on the surface of the parental cell line. However, the mutant cell line did express high levels of TcR heterodimer as detected with a pan-specific anti-TcR antibody. CD3 epsilon, Ly-6 and Thy-1 were expressed at levels similar to the wild-type parental cell line. Analysis of the surface TcR/CD3 complexes by immunoprecipitation and two-dimension gel electrophoresis confirmed that the major discernable difference between the wild-type and mutant TcR/CD3 complexes resided in the TcR beta chain. The parental cell line had the potential to express two TcR heterodimers, V alpha V beta 1 and V alpha V beta 8, as determined by Northern blot analysis. Co-modulation experiments suggested that both types of receptors were expressed. However, the V alpha V beta 8 receptor was the predominant form. In contrast, the mutant M4/8 cell line did not synthesize V beta 8 mRNA and, thus, only the V alpha V beta 1 TcR was synthesized. Despite the normal surface expression of TcR/CD3 complex, the M4/8 mutant cell line did not produce interleukin 2 (IL 2) in response to antigen or soluble anti-CD3 epsilon monoclonal antibody (mAb). Furthermore, it responded poorly to concanavalin A, phytohemagglutinin and anti-Ly-6 mAb. Cross-linking of the stimulatory antibodies partially restored the IL 2 response to anti-CD3 epsilon or anti-Ly-6 to wild-type levels. Phorbol ester and ionomycin stimulated a full IL 2 response in the M4/8 cell line, demonstrating that the defect in the decreased signaling in the mutant did not result from a defect in the IL 2 gene program. In conclusion, these data suggested that the pairing of alpha/beta heterodimer not only determined antigen/MHC specificity but also the signaling efficiency of the TcR/CD3 complex.
Antigens, Differentiation, T-Lymphocyte↗
The subunit composition of cerebellar tubulin: evidence for multiple beta tubulin messenger RNAs.
Cytoplasmic proteins were isolated from adult rat forebrain and cerebellum and analyzed by two-dimensional gel electrophoresis under conditions which the major subunits of tubulin were separated. Forebrain cytoplasmic tubulin consisted of two groups of alpha subunits (alpha 1 and alpha 2) and a minimum of two beta subunits (beta 1 and beta 2). However, the rat cerebellar cytoplasmic proteins contained greatly decreased amounts of the beta 1 tubulin subunit relative to the analysis of forebrain proteins. Messenger RNA (mRNA) was purified from cerebellum and forebrain and translated in wheat germ homogenate. Analysis of the translation products of cerebellar mRNA indicated only a trace amount of the beta 1 subunit, whereas the expected amount of beta 1 was found among the translation products of forebrain mRNA. This data is consistent with the conclusion that the beta 1 and beta 2 subunits of tubulin are synthesized from different mRNAs. A decrease in beta 1 mRNA relative to other tubulin mRNAs may be one of the factors responsible for the low steady state amounts of beta 1 tubulin in the cerebellum.
Animals↗
Chemical modulation of agonistic display in Betta splendens.
Mirror-induced aggressive display of male Siamese fighting fish was reduced by water-borne traces of (a) a pair of male combatants, (b) a nondisplaying fish of either sex, and (c) bits of torn cadual fin. These findings may indicate a mechanism whereby males tolerate the presence of conspecifics to permit nonagressive social interactions.
Aggression↗
Evaluation of ethanol analysis on brain and liver by head-space gas chromatography.
An evaluation of the reliability of ethanol analysis in brain by the head-space gas chromatographic method has shown the following. (a) This method may be used directly, without prior distillation, with homogenized brain tissue; (b) a good correlation exists with values obtained on blood (blood/brain ratio of 0.97 and 1.32 for blood ethanol concentrations of 0.1 and 0.2 g/dl respectively); (c) in non-preserved blood, there is a 15% deviance in the correlation resulting from changes in ethanol concentration; (d) the same evaluation applied to ethanol analysis in liver reveals a poor correlation between the results in liver and blood; this is believed to result mainly from natural oxidative losses of ethanol in liver, and also from the variable fat content of the liver samples analyzed. Caution is emphasized with regard to interpretations of blood/brain and blood/liver ethanol concentration ratios for the purpose of determining pre- and post absorption stages of intoxication where samples involved are not properly preserved.
Analysis of Variance↗
Improved detection and identification of basic drugs extracted from tissue using TLC.
Improved separation of methaqualone, lidocaine, cocaine, phencyclidine, phenacetin and methadone was achieved by a double development technique using dichloromethane:methyl ethyl ketone:ammonia (90:10:1.5 and 30:70:2). Improved identification of spots for some basic drugs was achieved after the traditional detection spray sequence with successive topical applications of: Marquis reagent, Mandelin reagent and a special ammonium molybdate fuming sulfuric acid reagent.
Chromatography, Thin Layer↗
How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?
In restriction-modification systems, cleavage of substrate sites in cellular DNA by the restriction endonuclease is prevented by the action of a cognate methyltransferase that acts on the same substrate sites. The PvuII restriction endonuclease (R.PvuII) has been structurally characterized in a complex with substrate DNA (Cheng et al., 1994) and as an apoenzyme (Athanasiadis et al., 1994). We report here a structure, determined to 1.9 A resolution by crystallography, of a complex between R.PvuII and iodinated DNA. The presence of an iodine at the 5-carbon of the methylatable cytosine results in the following changes in the protein: His84 moved away from the modified base; this movement was amplified in His85 and disrupts an intersubunit hydrogen bond; and the base modification disturbs the distribution of water molecules that associate with these histidine residues and the area of the scissile bond. Considering these observations, hypotheses are given as to why a similar oligonucleotide, where a methyl group resides on the 5-carbon of the methylatable cytosine, is slowly cleaved by R.PvuII (Rice et al., 1995).
Crystallography, X-Ray↗