Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.     

Der Turnover der Proteine in den Organen des Flußkrebses Orconectes limosus

Zusammenfassung Aus dem exponentiellen Abfall der spezifischen Aktivität nach einmaliger Injektion von L-Leucin-¹⁴C wurden die Halbwertszeiten der Gesamtproteine in den meisten Organen des Mußkrebses zu 8,5–19 Tagen bestimmt, in Leber und Niere der Maus zu 1,6–1,7 Tagen. Muskel- und Hämolymphproteine des Krebses zeigten weit längere Halbwertszeiten. Berücksichtigt man die Temperaturdifferenz von 25° unter Annahme eines Q ₁₀ von 2,0–2,5, so ergeben sich für Maus und Krebs etwa übereinstimmende Geschwindigkeiten des Proteinturnovers.

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Protein turnover in the tissues of the crayfish, Orconectes limosus

Summary The exponential decay of protein radioactivity after injection of L-Leucin-¹⁴C was measured in the different tissues of the crayfish, and in liver and kidney of the mouse. The half life time of tissue proteins was calculated to be 8.5 to 19 days in most crayfish tissues, 1.6 to 1.7 days in the mouse liver and kidney. Proteins of muscle and hemolymph of crayfish had much longer half life values. Taking into consideration the temperature difference of 25° C and assuming a Q ₁₀ of 2.0 to 2.5, the speed of protein turnover corresponds in the mouse and the crayfish.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Gesamtbestand an organischen Substanzen der Spinne Tegenaria atrica im Vergleich zu Protophormia terrae novae (Diptera) und Orconectes limosus (Crustacea,Decapoda)

Zusammenfassung Die Pulsationen der Lymphherzen von Normaltieren von Triturus vulgaris L. und T. alpestris Laur. sind unregelmäßig und verändern sich bei Erregung der Tiere. Die Lymphherzfrequenzen sind temperaturabhängig und folgen der R-G-T-Regel. Der Q ₁₀ beträgt bei T. vulgaris 2,5, bei T. alpestris 2,1. In Narkose oder bei Spinaltieren sinkt die Lymphherzfrequenz gegenüber der von Normaltieren ab, der Rhythmus wird regelmäßig. Bei Spinaltieren tritt homolaterale Synchronie auf. Jedes Lymphherz hat ein eigenes motorisches Zentrum im Rückenmark; die Impulse werden über Spinalnerven geleitet. Die Ergebnisse werden mit den von Anuren bekannten verglichen.

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Physiological investigations on the lymph hearts of urodelans

Summary The heart beats of normal individuals of Triturus vulgaris L. and T. alpestris Laur. are irregular and can be altered by excitation of the animal. The frequencies vary with alterations in temperature according to the rule of van t'Hoff. The Q ₁₀ was measured as 2.5 for T. vulgaris and 2.1 for T. alpestris. In anesthetized and in spinal animals the frequencies are strongly reduced and the pulsations become regular. Spinal animals show homolateral synchrony of the lymph heart beats. The motor centres, separated for each lymph heart, are situated in the spinal cord; the impulses are mediated by spinal nerves. The results are compared with those of the Anurans.

     

Absorption and long distance transport by isolated stele of maize roots in relation to the dual mechanisms of ion absorption

Summary Ion absorption and transport by intact roots, isolated cortex and isolated stele were compared shortly after tissue isolation and after aging. Absorption isotherms in the low and in the high concentration range show that in stripped-stele, which absorbs at a very low rate immediately after isolation, the capacity of system 1 but not system 2 is built up with aging. In agreement with this result analysis of individual fluxes across plasmamembrane and tonoplast reveals that only the influx from the medium into the cytoplasm increases considerably with aging of stele. Changes observed in aging excised roots and in isolated cortex are much less significant. In spite of the increase of absorption with aging by isolated stele, long distance transport, which is essentially passive through freshly stripped stele, decreases with aging. The reported results reflect the marked permeability of the plasmamembrane of fresh isolated stele, and demonstrate the importance of the cortex as a tissue collecting ions for long distance transport. New evidence for the theory of symplasmatic transport of ions into the xylem vessels is thus provided.

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Zusammenfassung Die Ionenaufnahme durch intakte Wurzeln, isolierte Rinde und isolierte Zentralzylinder und der Ferntransport durch intakte und entrind ete Wurzeln wurden verglichen, und zwar kurz nach der Isolierung und nach Altern der Gewebe. Frisch isolierte Zentralzylinder akkumulieren Io nen nur in ganz geringem Maße oder überhaupt nicht. Von den beiden Systemen der metabolischen Ionenaufnahme, die in einem niedrigen (System 1: bis 0,5 meq/l) und in einem hohen Konzentrationsbereich (System 2: 1-50 meq/l) die Geschwindigkeit der Ionenaufnahme durch intakte Wurzeln und isolierte Wurzelrinde bestimmen, entwickelt sich während des Alterungsprozesses in isolierten Zentralzylindern System 1, nicht aber System 2.In Übereinstimmung mit diesem Befund zeigt die Analyse der Einzelfluxe am Plasmalemma und am Tonoplasten, daß nur der Influx aus der Außenlösung in das Cytoplasma beim Altern der Zentralzylinder beträchtlich ansteigt. Veränderungen beim Altern von abgeschnittenen, intakten Wurzeln und isolierter Rinde sind viel weniger ausgeprägt.Obwohl die Ionenaufnahme beim Altern isolierter Zentralzylinder steigt, verringert sich der Ferntransport, der bei frisch isolierten Zentralzylindern rein passiv ist. Die mitgeteilten Ergebnisse zeigen die ausgeprägte Permeabilität frisch isolierter Zentralzylinder und demonstrieren die Bedeutung der Wurzelrinde als ein Gewebe, das Ionen für den Ferntransport sammelt. Auf diese Weise werden neue Anhaltspunkte für die Theorie des symplasmatischen Transportes der Ionen in die Gefäße gewonnen.

     

A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis-B surface antigen (Anti-HBsAg)

A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3U/I.     

Characterization of the genetic system of the xylose-fermenting yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Received: 29 February 1996 / Accepted: 29 March 1996