Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.     

Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1

Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al. 1983), was shown to grow with naphthalene. After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene. The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al. 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene. These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.Abbreviations DHBP dihydroxybiphenyl - DHBPDO 2,3-dihydroxybiphenyl dioxygenase - DHDHNDH 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase - DHN 1,2-dihydroxynaphthalene - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBP cis-2-hydroxybenzalpyruvate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - PCB polychlorinated biphenyl - 2NS naphthalene-2-sulfonic acid     

Neuraxin corresponds to a C-terminal fragment of microtubule-associated protein 5 (MAP5)

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C-terminal region of mouse microtubule-associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C-terminal fragment of MAP5 that contains a low-affinity tubulin binding site.     

Alpha-globin gene markers identify genetic differences between Australian aborigines and Melanesians.

Australian aborigines exhibit a number of alpha-globin cluster rearrangements involving both alpha- and zeta-globin genes. alpha+-Thalassemia (-alpha/) in this population is heterogeneous and includes the 3.7 types I, II, and III gene deletions. The alpha alpha alpha/ and zeta zeta zeta/ rearrangements are each found in association with two haplotypes, indicating origins from at least two separate DNA crossover events. Differences in alpha-globin cluster rearrangements and in haplotypes between Australian aborigines, Papua New Guinea highlanders and island Melanesians, are consistent with multiple colonizing events into Australia.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 mm lactate with or without 2 mm glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 mm glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.     

Metabolic pathways involved in lipogenesis from lactate and acetate in bovine adipose tissue: Effects of metabolic inhibitors

Metabolic inhibitors were used in vitro in an attempt to elucidate the biochemical pathways by which lactate is converted to fatty acids by bovine adipose tissue. Subcutaneous adipose tissue samples were obtained by biopsy techniques from steers fed a high-energy ration. Kynurenate (α-2-diamino-γ-oxabenzenebutanoic acid) (5–10 mm), an inhibitor of acetyl-CoA carboxylase, and cerulenin (2,3-epoxy-4-oxo-7,10-dodecadienamide) (20–100 μg/ml), an inhibitor of the fatty acid synthetase enzyme complex, inhibited fatty acid synthesis from both acetate and lactate. The hydrogen acceptor, N-methylphenazonium methosulfate (10 μm) inhibited acetate but not lactate incorporation into fatty acids. α-Cyanohydroxycinnamate (5 mm) and phenylpyruvate (10 mm), which inhibit pyruvate entry into the mitochondria and pyruvate carboxylase, respectively, decreased lipogenesis from both acetate and lactate. The effects of phenylpyruvate on lipogenesis from acetate were greater in the presence of glucose plus insulin. Agaric acid (2-hydroxy-1,2,3-nonadecanetricarboxylic acid) (0.2 and 1.0 mm), which inhibits citrate efflux from the mitochondria also decreased lipogenesis from both acetate and lactate. Fluoroacetate (2.5 mm), an inhibitor of aconitate hydratase, had no effect on lipogenesis from acetate; but, in the presence of glucose or pyruvate, decreased lactate incorporation into fatty acids. n-Butylmalonate (5 mm), which blocks malate transport across the mitochondrial membrane, decreased lipogenesis from lactate but not acetate. Malate transport during lipogenesis is not associated with an operative malate:asparate shuttle in bovine adipose tissue, as indicated by the lack of effect of either 0.2 or 1.0 mm aminooxyacetate, a transaminase inhibitor, on lipogenesis from acetate or lactate. The results suggest a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine subcutaneous adipose tissue and that this pathway may be involved in lipogenesis from acetate as well as lactate.     

Fermentation of Bacillus thuringiensis for exotoxin production: Process analysis study

The exotoxin produced by certain serotypes of Bacillus thuringiensis was used as a means of microbiological control of the larval development of flies. The optimal batch cultivation conditions with respect to pH, temperature, aeration, agitation, and initial concentration of growth-limiting substrate were determined. A dynamic model describing the process was designed and fitted to the experimental data. The application of a method for estimating exotoxin and bacterial concentrations from on-line measurable quantities such as oxygen consumption and heat production is presented.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.