The isolation of demolybdo xanthine oxidase from bovine milk.

It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.     

C-reactive protein antigenicity on the surface of human peripheral blood lymphocytes. Characterization of lymphocytes reactive with anti-neo-CRP

Previously, we have shown that antibodies specific for C-reactive protein determinants, not present on the native molecule, termed neo-CRP, also react with a significant percentage of PBL. In the present study, cells were evaluated by flow cytometry using alpha-neo-CRP antisera and mAb specific for lymphocyte subsets. With use of either monocyte-depleted PBL or Percoll-enriched large granular lymphocytes, we observed an overlap between cells reactive with alpha-neo-CRP and cells bearing the surface markers CD16, CD11b, Leu-7, and/or Leu-19, which are expressed on NK cells. In addition, we showed co-expression of the neo-CRP antigen with CD19, CD20, and HLA-DR, cell surface markers which are expressed on B lymphocytes. The major proportion of CD3+ cells failed to exhibit co-expression of neo-CRP. Single parameter flow cytometric analyses demonstrated that cells reactive with alpha-neo-CRP exhibited a bimodal staining pattern based on fluorescence intensity: high intensity neo-CRPbright and low intensity neo-CRPdim. Two-color analysis revealed that neo-CRPbright cells co-expressed CD19, CD20, and HLA-DR, whereas neo-CRPdim cells co-expressed CD16, CD11b, Leu-7, and Leu-19. Anti-neo-CRP also reacted with PBL obtained from patients with CD16+ lymphoproliferative disorders and from patients with chronic lymphocytic leukemia of B cell origin, but not with cells from patients with T cell or myeloid leukemias. The alpha-neo-CRP cells from patients with NK cell expansions showed dim fluorescence, whereas patients with B cell expansions showed bright fluorescence, consistent with the staining patterns observed with normal PBL. In addition, cell lines of T cell, B cell, NK cell, myeloid, and erythroid origin were evaluated for reactivity with alpha-neo-CRP. The cloned NK cell line NK 3.3 reacted as neo-CRPdim, but the B cell lines BL41, BL41/95, T1, T2, and CESS all reacted as neo-CRPbright. The cell lines K562, Molt-4, Hut-78, HL-60, U-937, and THP-1-0, which lack characteristic NK and B cell markers, did not react with alpha-neo-CRP. Additional study of the two-color histograms revealed a distinct diagonal staining pattern that was observed only when cells were co-stained with alpha-neo-CRP and either alpha-CD16 (alpha-Fc gamma RIII) or antibody IV3 (alpha-CDw32; alpha-Fc gamma RII). This finding suggests a 1:1 relationship between Fc gamma R on both NK and B cells and determinants recognized by alpha-neo-CRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

The molybdenum iron-sulphur protein from Desulfovibrio gigas as a form of aldehyde oxidase.

The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.     

Organ-Specific and Environmentally Regulated Expression of Two Abscisic Acid-Induced Genes of Tomato : Nucleotide Sequence and Analysis of the Corresponding cDNAs

The cDNAs, pLE4 and pLE25, represent mRNAs that accumulate in response to water deficit and elevated levels of endogenous abscisic acid in detached leaves of drought-stressed tomato (Lycopersicon esculentum Mill., cv Ailsa Craig) (A Cohen, EA Bray [1990] Planta 182: 27-33). DNA sequence analysis of pLE4 and pLE25 showed that the deduced polypeptides were 13.9 and 9.3 kilodaltons, respectively. Each polypeptide was hydrophilic, cysteine- and tryptophan-free, and found to be similar to previously identified proteins that accumulate during the late stages of embryogenesis. pLE4 and pLE25 mRNA accumulated in a similar organ-specific pattern in response to specific abiotic stresses. Yet, expression patterns of the corresponding genes in response to developmental cues were not similar. pLE25 mRNA accumulated to much higher levels in developing seeds than in drought-stressed vegetative organs. pLE4 mRNA accumulated predominantly in drought-stressed leaves. The similarities and differences in the accumulation characteristics of these two mRNAs indicates that more than one mechanism exists for the regulation of their corresponding genes.     

Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

The Lepocreadiidae (Digenea) of fishes from the north-east Atlantic: Profundivermis intercalarius n. g., n. sp. from the marine fish Coryphaenoides (Nematonurus) armatus (Hector) (Macrouridae) from the Porcupine Abyssal Plain

A new genus and species, Profundivermis intercalarius, is described from the deep-sea fish Coryphaenoides (Nematonurus) armatus from 4,850 m deep on the Porcupine Abyssal Plain, NE Atlantic. The genus is characterized by an inter-testicular ovary and is the only member of the subfamily Lepidapedinae to show this feature. The relationships of the members of the subfamily are analysed phylogenetically. Members of the subfamily are often found in deep-sea gadiforms.     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

Oschmarinella albamarina (Treshchev, 1968) n. comb., a liver fluke from the killer whale Orcinus orca (L.) off the British coast

Oschmarinella albamarina n. comb. is described from the bile-ducts of a killer whale Orcinus orca stranded on the British coast. The systematic position of the species is discussed in relation to the genera Campula Cobbold, 1858, Orthosplanchnus Odhner, 1905 and Oschmarinella Skrjabin, 1947, and the species is transferred from Orthosplanchnus to Oschmarinella. Campula rochebruni (Poirrier, 1886) is transferred to Oschmarinella, as O. rochebruni n. comb., and Oschmarinella mascomai Raga, 1986 is considered its synonym.