Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Decreases in Plasma Aβ1-40 Levels with Aging in Non-Demented Elderly with ApoE-ε4 Allele

This report examines plasma amyloid proteins A40 and A42 and apolipoprotein E (apoE) levels and their relationships with age in non-demented older adults with (N = 32) or without the apoE-4 allele (N = 94). A levels did not differ between the groups whereas the 4 allele was associated with a significant reduction in plasma apoE. In subjects with the 4 allele, increasing age was associated with significant reduction in plasma A40. Subjects without the 4 allele showed a significant positive correlation between A40 and A42 levels. There was also a significant correlation between plasma A40 and apoE levels in all subjects.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Die Wirkung von Actinomycin D auf die phytochromabhängigen Veränderungen des RNS-Gehaltes im Senfkeimling (Sinapis alba L.)

Summary Actinomycin D (10 g/ml) cancels completely the phytochrome-mediated RNA net synthesis in the cotyledons of the mustard seedling whereas RNA net synthesis in the cotyledons of the dark-grown seedling is only partially inhibited (Fig. 1). — In the hypocotyl Actinomycin D of the same concentration lowers the RNA contents in the light (i.e. far-red)-grown seedling as well as in the dark-grown seedling down to the same level (Fig. 2). In the presence of Actinomycin D phytochrome has no significant influence on the RNA contents neither in the cotyledons nor in the hypocotyl (Fig. 1,2).The data support the view that P₇₃₀, the active phytochrome, acts through differential gene activation in the cotyledons and predominantly through differential gene repression in the hypocotyl (cf. Mohr, 1966; Schopfer, 1967a, b). —The data further support the conception that active genes (as defined by Mohr, 1966 and Schopfer, 1967a, b) are much less sensitive towards Actinomycin D than potentially active and repressible genes (cf. Schopfer, 1967a; Mohr and Bienger, 1967).     

Regional distribution of membrane-bound γ-glutamyl transpeptidase activity in mouse brain

Activity of membrane-bound -glutamyl transpeptidase (-GTP) was examined in various regions of mouse brain, in capillaries of the cerebral cortex and in telencephalic choroid plexuses. The level of activity in the capillaries was double and that of the choroid plexus nine times that of the -GTP activity found in the brain, septum, hippocampus, hypothalamus, thalamus, cerebellum, frontal cortex, pons, medulla oblongata, and amygdala. Histochemically the -GTP activity was demonstrated in the surface membranes of choroidal cells and in the endothelium of small capillaries.The activities of -GTP of cerebral cortex, choroid plexus, and capillaries from rabbit were 5–17 times greater than those from corresponding areas of mouse brain. While 30 mM methionine stimulated (in vitro) the enzyme from mouse brain, no such effect was observed with the enzyme activity from rabbit brain. The -GTP activity from the capillaries of cerebral cortex of both mouse and rabbit was not effected by the presence of methionine.These findings suggest existence of differences in the specificity of -GTP activity in these two species.     

Against “Revolution” and “Evolution”

Those standard historiographic themes of evolution and revolution need replacing. They perpetuate mid-Victorian scientists history of science. Historians history of science does well to take in the long run from the Greek and Hebrew heritages on, and to work at avoiding misleading anachronism and teleology. As an alternative to the usual evo-revo themes, a historiography of origins and species, of cosmologies (including microcosmogonies and macrocosmogonies) and ontologies, is developed here. The advantages of such a historiography are illustrated by looking briefly at a number of transitions the transition from Greek and Hebrew doctrines to their integrations by medieval authors; the transition from the Platonist, Aristotelian, Christian Aquinas to the Newtonian Buffon and to the no less Newtonian Lamarck; the departures the early Darwin made away from Lamarcks and from Lyells views. Issues concerning historical thinking about nature, concerning essentialism and concerning classification are addressed in an attempt to challenge customary stereotypes. Questions about originality and influence are raised, especially concerning Darwins tree of life scheme. The broader historiography of Darwinian science as a social ideology, and as a worldview, is examined and the scope for revisions emphasised. Throughout, graduate students are encouraged to see this topic area not as worked out, but as full of opportunities for fresh contributions.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

Über das Wachstum der Pankreaszellen von Säugetieren als Monolayer Cultures

Zusammenfassung Fetale menschliche Pankreaszellen bilden sowohl spontan auf zuerst gewachsenem Zellrasen (self-aggregation) als auch in Rollröhrchen (48 rph) (rotation-mediated-aggregation) histotypische Aggregate, die A- und B-Zellen enthalten. In der Nährlösung dieser 6–17 Tage lang gewachsenen Gewebestückchen konnte, Insulin (=IRI=immunoreactive-insulin) nachgewiesen werden.

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Monolayer cultures of pancreatic tissueII. Formation of histotypic aggregates from cell suspensions of fetal human pancreas

Summary Fetal Pancreas cells of man, whether in self-aggregation or rotation-mediated-aggregation, form spontaneously histotypical aggregates containing A- and B-cells. It is possible by means of an immunological method to determine insulin (=IRI=immuno-reactive-insulin) in the nutrient medium of these 6 to 17 days old tissue aggregates.

     

In vivo effects of tunicamycin on the secretory processes of rat parotid glands

Summary The morphological and functional effects of tunicamycin were studied in rat parotid glands at the stage of the reformation of secretory granules following secretory stimulation by isoproterenol. Tunicamycin inhibited the incorporation of (³H)-mannose into the acid-insoluble fraction but had no effect on total protein synthesis as determined by the incorporation of (¹⁴C)-leucine. Thus the administration of tunicamycin in vivo inhibits the synthesis of mannose-rich glycoproteins in a manner similar to that in an in vitro system. The ultrastructure of the acinar cell showed little change following treatment with this drug, except that the number of reaccumulated secretory granules was greater than in the control. Amylase secretion stimulated by isoproterenol was inhibited in tunicamycin-treated cells, but did not decrease following treatment with N⁶,2-O-dibutyryladenosine 3-5-cyclic monophosphate, a secretory stimulator bypassing the -receptor. A radio-receptor assay using (³H)-dihydroalprenolol and direct localization using the fluorescent -adrenergic blocker 9-amino-acridin propranolol showed a marked reduction in the binding activity of -receptor following treatment with tunicamycin. Thus the inhibition of N-linked glycosylation appears to produce profound effects on the -adrenergic receptor-adenylate cyclase complex of acinar cells, although the steps of the transport and the exocytotic discharge of secretory materials are not affected.     

Role of the Na,K-ATPaseβ-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors

Summary No functional role could yet be established for the glycosylated -subunit of the Na,K-ATPase. In this study, we describe the intracellular processing of the -subunit as a glycoprotein in toad bladder cells and the consequences of its structural perturbation with glycosylation inhibitors on the cellular expression of the - and -subunits and on the structural and functional maturation of the enzyme. Controlled trypsinolysis of homogenates from pulse-labeled cells reveals that the -subunit is subjected to glycosylation-dependent structural rearrangements during its intracellular routing. Inhibition of correct terminal glycosylation of the -subunit with deoxynojirimycin or swainsonine has no effect on the trypsin sensitivity of the -subunit, its ability to perform cation-dependent conformation changes or the cellular Na,K-ATPase activity. Acquisition of core-sugars is sufficient for the enzyme to assume its catalytic functions. On the other hand, complete inhibition of glycosylation with tunicamycin leads to a destabilization of both the - and the -subunits as judged by their higher trypsin sensitivity. In addition, tunicamycin treatment results in a decrease of the amount of newly synthesized - and -subunit indicating that a glycoprotein, possibly the -subunit itself, plays a role in the efficient accumulation of the -subunit in the endoplasmic reticulum.     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

GM1 Inhibits Amyloid β-Protein-Induced Cytokine Release

The ganglioside GM1 is known to play a pivotal role in neuronal survival and/or regeneration. Recently it has been shown that GM1 binds tightly with membrane-bound amyloid protein (A) and prevents its conversion from a helical to a -sheet structure. To examine the potential physiological consequences of this binding, we studied the effect of GM1 on A-stimulated release of proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and TNF-, using the human monocytic cell line, THP-1, as a model system. Treatment of THP-1 cells with A 1–40 or A 25–35 resulted in an increased cytokine release from these cells. However, treatment of A-activated THP-1 cells with GM1 and several other complex gangliosides, but not hematosides and neutral glycosphingolipids such as asialo-GM1 (GA1), lactosylceramide, and globoside, significantly decreased the cytokine release. In contrast, this effect was not observed for lipopolysaccharide (LPS)-activated and thrombin-activated THP-1 cells, indicating that the ganglioside effect is specific for A-induced cytokine release. A direct interaction between GM1 and A was demonstrated using the surface plasmon resonance technique. We found that GM1 ganglioside exhibited higher affinity for A 1–40 than GA1, suggesting that the sialic acid moiety of GM1 is necessary for its interaction with A. We conclude that the inhibitory effect of GM1 on A-induced cytokine release may reflect pre-existing abnormalities in membrane transport at the stage of amyloid formation and that GM1 may induce conformational changes in A, resulting in diminished fibrillogenesis and prevention of the inflammatory response of neuronal cells in Alzheimer's disease.     

Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

Glutathione and γ-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats

Summary Components of the -glutamyl cycle, including thiols, glutathione (GSH) and -glutamyl transpeptidase (-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. -GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. Inrespiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, -GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the -glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

The new problem of genetics: A response to Gifford

Recently, Fred Gifford attempted to explicate the meaning of the term genetic as applied to phenotypic traits. He takes as his primary goal the explication of how the term is used and tries to avoid conclusions about how it should be used. He proposes two independent criteria (DF and PI) which together capture much of what biologists mean when they describe traits as genetic. Although Gifford's approach is extremely insightful in many ways, I argue that his analysis is not sufficiently critical concerning the adequacy of common usage.In particular, while DF is a perfectly legitimate and useful measure of heritability in populations, it is not necessarily a genetic one and should not be labeled as such. PI on the other hand, although very intuitive, depends on an extremely problematic distinction between causes and mere conditions (e.g., genes and epigenetic factors). Both criteria will be highly relative and both, via what I term the new problem of genetics, will inspire contradictory analyses based on the same data.Fortunately, as Gifford recognizes, it is not necessary to make sense of genetic at all in order to do biology. Quantitative genetics can do the kind of (heritability) analysis that DF embodies without making questionable claims about genes. Causal-mechanical or bottom-up biology can proceed perfectly well without postulating the priveleged role for genetic causes that PI entails. In short, talk of genetic traits, under either criteria, is unnecessary and misleading.     

Decrease of P-Glycoprotein Activity in K562/ADR Cells by MβCD and Filipin and Lack of Effect Induced by Cholesterol Oxidase Indicate That This Transporter Is Not Located in Rafts

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl--cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, light, heavy, and extra heavy fractions we show that no P-gp is found in the light fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.