Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Human monocytes selectively bind to cells expressing the tumorigenic phenotype

Summary The characteristics of the binding of human monocytes to tumor cells were studied by a newly developed microassay. First, we determined the kinetics and optimal conditions of the binding. Monocytes recognized and bound to tumor cells very rapidly within 10–20 min of cellular interaction. Binding was also more efficient at 37°C suggesting that active metabolism of monocytes is required. Second, we determined that selective binding of monocytes to cells with tumorigenic phenotypes occurs. For this purpose, lymphocytic leukemia cell lines versus normal lymphocytes, and tumorigenic versus nontumorigenic hybrids from the same parental lines were compared as the targets of the binding assay. In both cases, neoplastic cells were selectively bound by monocytes. Although tumor cells were bound rapidly and selectively by monocytes, initial recognition and binding did not necessarily lead to subsequent tumor cell lysis. This is based on the observation that some tumorigenic parental and hybrid lines were avidly bound by monocytes yet not subsequently killed in a cytotoxicity assay.This work was supported in part by a grant from the National Institutes of Health CA42992 and a grant from the Kleberg foundation Abbreviations used: [¹²⁵I]IdUrd [¹²⁵I]iododeoxyuridine; rIFN-, recombinant human interferon ; IL-1, interleukin 1; rTNF, recombinant human tumor necrosis factor     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

Circular DNA is excised by immunoglobulin class switch recombination

We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart.

PAF decreases cardiac contractility and blood pressure. To characterize the cardiac PAF receptor, we screened a human ventricular cDNA library in a low stringency condition, using a PCR product derived from guinea pig lung PAF receptor as a probe. Four clones were obtained and named HV1-4. In Xenopus oocytes injected with cRNA derived from HV3 or 4 but not from HV1 or 2, PAF elicited a Ca(2+)-activated Cl- current. HV3 and HV4 were duplicate clones, encoding a 342 amino-acid polypeptide which was identical to that of the human leukocyte PAF receptor. However, a portion of the 5' untranslated region of HV3 (or 4) was different from that of the leukocyte receptor cDNA. Northern blotting of human ventricles and atria using the HV3 insert showed a single band of approximately 4 kb. These results suggest a tissue-specific translational mechanism responsible for regulation of the expression of the PAF receptor mRNA in these tissues.