Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti     

Backbone dynamics of the cytotoxic ribonuclease alpha-sarcin by 15N NMR relaxation methods

The cytotoxic ribonuclease -sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of -sarcin through measurement of longitudinal relaxation rates R₁, off resonance rotating frame relaxation rates R₁, and the ¹⁵N¹HNOE of the backbone amide ¹⁵N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. -Sarcin behaves as an axial symmetric rotor of the prolate type (D/D=1.16 ± 0.02) which tumbles with a correlation time _m of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that -sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of -sarcin in the process of recognition of its ribosomal target.     

Die Intravitale Karyorrhexis Der Exokrinen Pankreaszelle im Elektronenmikroskopischen Bild

Zusammenfassung Die intravitale Karyorrhexig wird am exokrinen Pankreas der Maus nach Osmium- und Formalinfixierung licht- und elektronenmikroskopisch untersucht. Im Verlaufe der Karyorrhexis kommt es zu einer Trennung des Karyoplasmas in 'Chromatin und 'Karyolymphe (Interchromatin-Substanz). In fortgeschrittenen Stadien ist das Bild solcher Zellkerne — im Gegensatz zu normalen Intermitosekernen — nach beiden Fixierungsmethoden fast identisch. Das Chromatin ist in dichten, membranständigen Kappen konzentriert. Der übrige Kernraum wird — abgesehen vom Nukleolus — von der hellen Karyolymphe ausgefüllt. In ihrem Bereich treten die sog. Interchromatingranula (200–250 Å), einzeln oder in dichten Haufen, meist deutlich hervor. Der Nukleolus, dessen Feinstruktur nicht auffallend verändert ist, liegt entweder frei oder den Chromatinkappen unmittelbar an. Die Kernhülle ist bei fortgeschrittener Karyoplasmaentmischung unversehrt oder über kürzere oder längere Strecken geschwunden. Im Endstadium der Karyorrhexis sind mehrere, von Kernmembranfragmenten unvollständig begrenzte Chromatinbrocken wahllos im Zelleib verstreut. Die cytoplasmatischen Strukturen können auch bei ausgeprägten Kernveränderungen noch weitgehend normal aussehen.Die karyorrhektischen Kernveränderungen werden als Folge einer 'Entmischung des Karyoplasmas gedeutet, für die eine Dehydratation der DNS-tragenden Chromosomenstrukturen verantwortlich gemacht wird. Auch das Sichtbarwerden und das Verklumpen der 'Interchromatingranula wird auf eine Dehydratation zurückgeführt.

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Summary The intravital caryorrhexis in the exocrine pancreas of the mouse was studied by means of light- and electron microscopy after fixation with osmium and formaldehyde. During caryorrhexis the caryoplasm segregates into chromatin and caryolymphe (interchromatinic substance). In contrast to normal intermitotic nuclei both fixations result in a very similar picture of the typical caryorrhectic nuclei. The chromatin is concentrated in dense caps associated with the nuclear envelope. Except those dense caps and apart from the nucleolus the whole nucleus is occupied by the light caryolymphe. In this area the so-called interchromatinic granules (200–250 Å) become clearly visible, isolated or in dense clumps. The nucleolus, the fine structure of which has not remarkably changed, can be found either isolated or in touch with the chromatin caps. During advanced segregation of the caryoplasm the nuclear envelope was found to be either unimpaired or had disappeared for variable lenghths. In the final stage of caryorrhexis several chromatin clumps — partly confined by fragments of the nuclear envelope — are distributed irregularly within the cell. Even in the case of pronounced nuclear changes the cytoplasmic structures may appear almost unaffected.It is assumed that the nuclear changes during caryorrhexis are due to a segregation of the caryoplasm, caused by dehydration of the DNA-carrying chromosome fibrils. Also dehydration is made responsible for the appearance and clumping of the interchromatinic granules.

     

Elektronenmikroskopische Untersuchungen über Bildung und Struktur der Zygotenwand beiMicrasterias papillifera (Desmidiaceae)

Zusammenfassung Die Feinstruktur von Mesospor und Endospor reifer Zygoten vonMicrasterias papillifera wurde untersucht. Das für die Resistenz der Zygoten gegenüber ungünstigen Umweltbedingungen wichtige Mesospor besteht aus vier Schichten von unterschiedlicher Elektronendichte. Es ist insgesamt 460–500 nm dick. Die Schichten Mes a und Mes c bestehen aus akkrustierten, dicht gepackten globulären Elementen eines Stoffes, der dem Sporopollenin ähnlich ist. Die Schicht Mes b zeigt ein fibrilläres Grundgerüst, wahrscheinlich aus Zellulose, in das Stoffe inkrustiert sind, die nicht mit denen der Schichten Mes a und Mes c identisch, aber gegen Abbauversuche ähnlich résistent sind.Die Schicht Mes d ist eine Übergangsschicht zum Endospor. Zwischen die Zellulose-Mikrofibrillen in Streutextur sind isolierte Partikel des Materials der Schicht Mes c eingestreut. Das etwa 650 nm dicke Endospor ist eine Zelluloseschicht mit Streutextur. Es wird als Wand der sogenannten Keimblase bei der Zygotenkeimung nach Sprengung von Exospor und Mesospor stark gedehnt.

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Electron microscopical investigations on the structure and formation of the zygote wall inMicrasterias papillifera (Desmidiaceae) II. The structure of the mesospore and the endospore

Summary The mesospore (460–500 nm thick) which is responsible for the resistance of the zygote against desiccation during its resting period, consists of four layers of different electron density. The layers Mes a and Mes c are composed of densely packed amorphous globular elements of a substance resembling sporopollenine. The layer Mes b has a fibrillar, probably cellulosic frame. It is incrusted by a substance which is not identical with that of Mes a or Mes c but which shows a comparable resistance against degradation.The layer Mes d contains isolated particles such as in Mes c and cellulose microfibrils of the endospore. The endospore (650 nm thick) has foliate texture. This layer surrounds the protoplast after it has escaped from the ruptured exospore and mesospore during zygospore germination.

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Der Deutschen Forchungsgemeinschaft dank ich f:ur Sachbeihilfe.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways.     

Methodological problems in evolutionary biology VIII. Biology and culture

Biology cannot accommodate all aspects of culture. Aspects of culture that a biological approach can take into account can be covered by the biological categories of phenotype and environment. There is no need to treat culture as a separate category. Attempts to elaborate biological explanations of cultural variation will meet with success only if biologists expand theories of development, and integrate them in evolutionary biology. The alternative — elaborating the idea of so-called cultural inheritance — makes little sense from a biological point of view.     

Contextualizing Images in Dreams and Daydreams

A contextualizing image (CI) is a powerful central image of a dream which appears to contextualize (provide a picture-context for) the dreamer's emotion. For instance, dreamers who have experienced any serious traumatic event sometimes dream, I was overwhelmed by a tidal wave. This appears to picture their feeling of terror and/or vulnerability.A scoring system for CIs is examined here and is applied to dreams and daydreams supplied by 40 students. Two raters scoring dreams on a blind basis showed good inter-rater reliability. Recent dreams were shown to have more as well as more intense CIs than recent daydreams; likewise, dreams that stand out had more intense CIs than daydreams that stand out. Students with thin boundaries had more and more intense CIs than students with thick boundaries in their recent dreams and nightmare, but not so clearly in dreams and nightmares that stand out. The emotions judged as contextualized by the powerful images tended towards fear/terror and helplessness/vulnerability in dreams (especially in dreams that stand out) whereas emotions contextualized by images in daydreams showed a wide range with no clusters.     

Assignment of human ferritin genes to chromosomes 11 and 19q13.3→19qter

Summary Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.319qter produced human L type ferritin. This confirms the previous assignment of the ferritin gene to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human H type ferritin suggesting that the gene for the H subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either H or L type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.     

Bilan nutritionnel au cours du développement de l'ectoparasite grégaire Dinarmus vagabundus et du solitaire Dinarmus basalis

Résumé Nos méthodes expérimentales permettent l'isolement d'une larve de sexe déterminé par hôte de l'ectoparasite grégaire Dinarmus vagabundus et du solaitire, D. basalis. Des hôtes porteurs de 3 à 8 larves par hôte de D. vagabundus sont aussi isolés. Dans ces conditions la quantité de nourriture disponible est la même pour toutes les densités larvaires étudiées.Les larves élevées en solitaire des deux espèces assimilent une quantité de nourriture significativement supérieure à celle assimilée par les . Ceci conduit à des adultes de poids moyen supérieur à celui des . Le poids moyen des et des de D. vagabundus diminue significativement aux fortes densités larvaires. L'intensité de la liaison entre la quantité de nourriture assimilée et la biomasse produite s'affaiblit au fur et à mesure que la densité larvaire par hôte augmente.Les de D. vagabundus de poids moyen (0,42 mg) engendrent deux fois et demi plus de descendants que les lilliputiennes (0,20 mg) émergées d'hôtes à forte densité larvaire. Celles de D. basalis (0,65 g) sont moins prolifiques que les de D. vagabundus.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Die kerngrössenverhältnisse in der larvenentwicklung verschiedener kasten bei formiciden

Zusammenfassung Zur Klärung des Problems der Kastendetermination bei Formiciden konnte durch die Untersuchung der endomitotischen Polyploidisierung im Verlauf der Larvenentwicklung beigetragen werden. Endomitosen können hierbei nicht direkt beobachtet werden, die Polyploidisierung ist nur aus dem Wachstum der Kerne zu erschließen.Die Polyploidisierung sieben verschiedener Gewebe von Myrmica- wurde untersucht. Alle Tiere wachsen unter ständiger Polyploidisierung bis zum Puppenstadium heran. Während der Metamorphose werden alle hochpolyploiden Gewebe abgebaut. Besonders hohe Polyploidiegrade erreichen Gewebe der Stoffwechselorgane, wie Mitteldarm und Malpighische Gefäße. Oenocyten zeigen sehr unübersichtliche Verhältnisse. Die Spinndrüse wird im Zusammenhang mit dem Sekretionszyklus hochpolyploid. Fettzellen, Epidermis und Ganglien zeigen dagegen nur geringe Polyploidiegrade.Die Unterschiede in den verschiedenen Kasten werden festgestellt. Es zeigte sich, daß a anfänglich haploid sind and Geschlechtstiere einen Endomitoseschritt mehr ausführen als .Die Polyploidisierung entsprechender Gewebe von Lasius niger zeigt die gleiche Entwicklungstendenz. Futter- ud Temperatureinflüsse konnten festgestellt werden. Zwerg- zeigten Polyploidiegrade, die von denen der Normal- abweichen und dadurch auf blastogene Determination schließen lassen.-Brut gibt bei Ausschluß der Nestbegattung stets , die sick in ihren Kerngrößen nicht von den aus weiselrichtigen Nestern unterscheiden.Alle untersuchten Formicidenarten weisen die gleiche Entwicklungstendenz auf.Beobachtungen über Entwicklungsdauer, Eiablage und -Brut-Entwicklung werden angefügt.Auf Grund der Ergebnisse wurde zu Fragen der endomitotischen Polyploidisierung Stellung genommen. Die Gründe, die zur Annahme eines Polyploidisierungsvorganges in der Larvenentwicklung der Formiciden führen, werden diskutiert. Polyploidie wird in Beziehung gesetzt zur Körpergröße der Tiere, zur phylogenetischen Entwicklungshöhe und zur Gewebsfunktion (Deutung als Sparsamkeitsmaßnahme). Hypothesen zur Kastendetermination werden durch die Ergebnisse unterstützt.     

Intraorbital cerebrospinal fluid outflow and the posterior uveal compartment of the hamster eye

Summary An ultrastructural and tracer study was undertaken to determine normal outflow pathways of cerebrospinal fluid (CSF) at the terminal subarachnoid space (SAS) of the optic nerve. In the morphological studies, the optic nerve dura and arachnoid were found to be continuous with the sclera of the eye beyond the optic nerve SAS. The pia mater is continuous with the inner sciera and the lamina fusca of the eye. Montages and serial sections demonstrated that the distal SAS is divided into numerous tortuous channels to form an arachnoidal trabecular meshwork. Spaces of this meshwork continue into microcanals which bypass the outer arachnoid barrier layers of the optic nerve meninges to reach the sclera and posterior intraorbital connective tissue. Ferritin infused into the cisterna magna entered the optic nerve SAS within 1 min and reached arachnoidal trabecular meshwork channels and the microcanals within 8 min. It then passed into intraorbital connective tissue spaces at the posterior pole of the eye. Ferritin appeared to be blocked by the lamina fusca and a newly discovered posterior compact zone which together prevented its entrance into the choroidal interstitium. These observations suggest that a subarachnoidal-scleral-orbital outflow pathway provides a route for CSF drainage from the optic nerve SAS to intraorbital connective tissue. The previously described posterior uveal compartment in the hamster eye (Kelly et al. 1983) appears to be relatively isolated from this subarachnoidal-scleral-orbital CSF outflow.Parts of this work have been presented at the 1984 meetings of the American Association of Anatomists (Shen 1984).     

Cryptomonad biliproteins — an evolutionary perspective

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as 50000 kDa '₂ complexes which carry one bilin on the and three on the subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The subunit is encoded on the chloroplast genome, whereas the subunits are encoded by a small nuclear multigene family. The subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is primitive and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein and subunits.Abbreviations Chl chlorophyll - CER chloroplast endoplasmic reticulum - SSU rRNA small subunit ribosomal RNA     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Über das Verhalten von Champignonstämmen in Mischkultur

Zusammenfassung Mischungen aus dem Mycel sweier weißer oder zweier brauner Stämme des Kulturchampignons hatten keinen Einfluß auf den Fruchtkörperetrag.Bei Mischungen aus einem weißen und einem braunen Stamm was dagegen der Ertrag immer niedriger als erwartet und das Mycel verschwand verhältnismäßig schnell. Der Ertragsausfall beruht in der Regel auf einer Unterdrückung des braunen Stammes.Herrn Prof. von Sengbusch zum 65. Geburtstag in Dankbarkeit und Verehrung gewidment.     

Effect of Intercalators on the Properties of Liquid-Crystalline Dispersions of Nucleic Acid–Chitosan Complex

Molecules of deoxyribonucleic acid and synthetic polydeoxyribonucleotides (NA) in the particles of liquid-crystalline dispersions resulting from interaction with chitosan are accessible to interaction with intercalators. The intercalation is accompanied by alteration in the direction of spatial twist of cholesterics of NA–chitosan complexes. This effect is absent in the case of classical cholesterics produced from NA molecules via phase exclusion, i.e., the cholesteric structure of NA–chitosan complex is very labile as distinct from classical cholesteric NA.     

Microbial metabolism of alkylbenzene sulphonates the oxidation of key aromatic compounds by a Bacillus

A Bacillus species originally elected for growth at the expense of alkylbenzene sulphonate detergents was found to metabolise a wide range of aromatic compounds. p-Hydroxybenzoate (PHB) was initially hydroxylated to protocatechuate (PCA) i.e. 3,4-dihydroxybenzoate, which was oxidatively cleaved to succinate and acetyl-CoA by a classical ortho cleavage pathway initiated by a substrate-specific 3, 4-oxygenase: no evidence of an alternative meta cleavage pathway was detected. Several key enzymes of this ortho cleavage pathway were induced by growth of the Bacillus on either PHB or PCA. Both PHB and PCA were able to act as sole source of carbon for energy and overall growth of the microorganism.In strict contrast, the higher homologue p-hydroxyphenylacetate (PHPA), after initial hydroxylation to 3, 4-dihydroxyphenylacetate (DHPA), was oxidatively cleaved to 4-carboxymethyl-2-hydroxymuconic semialdehyde (CMHMS) by a meta cleavage catalysed by a substrate-specific 2, 3-oxygenase: no evidence of an alternative ortho cleavage was detected. Several lines of evidence suggested that CMHMS was not further metabolised by the Bacillus and accumulated in the growth medium. Both PHPA and DHPA were unable to act as sole source of carbon for energy and overall growth.The implication of the occurrence in a single bacterium of two separate oxidative pathways catalysing the cleavage of different aromatic nuclei have been discussed.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine