Die Bedeutung der “feedback”-Hemmung der Threonin-Desaminase für die Synthese von Valin und Leucin

Zusammenfassung In Arthrobacter Stamm 23 führte die durch Mutation verursachte allosterische Unempfindlichkeit der Threonin-Desaminase zur dereprimierten Bildung der Enzyme im Isoleucin-Valin-Leucin-Biosyntheseweg. Derepression erfolgte auch, wenn Wildtypzellen in Gegenwart von -Ketobuttersäure inkubiert wurden. In beiden Fällen wurde Isoleucin überproduziert und ins Kulturmedium ausgeschieden. Wie aus Wachstumsexperimenten hervorging, verursachte der Überschuß an -Ketobuttersäure im Medium primär einen Valin- und Leucin-Mangel, der zu einer vorübergehenden Wachstumshemmung führte. Durch die dereprimierte Bildung der Enzyme im Isoleucin-Valin-Biosyntheseweg konnte die Wachstumshemmung überwunden werden.Der vorübergehende Hemmeffekt der -Ketobuttersäure ließ sich auf eine Konkurrenz der Substrate am ersten gemeinsamen Enzym im Isoleucin-Valin-Biosyntheseweg, der Acetohydroxysäure-Synthase, zurückführen. Wegen des niedrigen K _m-Wertes für -Ketobuttersäure wird dieses Substrat vom Enzym bevorzugt umgesetzt. Durch gaschromatographische Bestimmungen der Acetoin- und Acetyläthylcarbinol-Bildung in Enzymtests mit variierten Substrat-Konzentrationen konnte nachgewiesen werden, daß relativ geringe Konzentrationen an -Ketobuttersäure genügen, um die -Acetolacetat-Bildung vollständig zu unterdrücken. Diese Ergebnisse erklären die durch -Ketobuttersäure verursachte vorübergehende Wachstumshemmung bei Bakterien.

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The effect of the feedback inhibition of threonine deaminase on valine-leucine biosynthesis

In Arthrobacter strain 23 the allosteric insensitivity of threonin deaminase caused by mutation resulted in derepressed formation of the enzymes of the isoleucine-valine-leucine pathway. Derepression was also observed, when wild type cells were incubated in the presence of -oxobutyrate. In both cases isoleucine was overproduced and excreted. As growth experiments indicated the excess of -oxobutyrate in the medium caused endogenous valine and leucine deficiency and a transient inhibition of growth. Derepressed formation of the isoleucinevaline biosynthetic enzymes resulted in relief of growth inhibition.The transient inhibitory effect of -oxobutyrate has been traced back to substrate competition at the first enzyme common to the isoleucine and valine pathway, acetohydroxy acid synthase. Due to the low K _m of the enzyme for -oxobutyrate this substrate is preferentially converted. As proven by gaschromatographical measurements of acetoin and acetylethyl carbinol produced in enzyme (acetohydroxy acid synthase) assays with varied substrate concentrations, relatively low concentrations of -oxobutyrate are able to suppress the formation of -acetolactate completely. These results explain the transient inhibitory effect of -oxobutyrate on the growth of bacteria.

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Abkürzungen -KBS -Ketobuttersäure - FAD Flavin-adenin-dinucleotid - AHS Acetohydroxysäure - IPM Isopropylmalat - TPP Thiaminpyrophosphat     

Zur Pyruvat- und α-Ketoglutaratausscheidung durch Streptomyces rimosus

Zusammenfassung Submerskulturen von Streptomyces rimosus scheiden auf glucosehaltigen Medien in Gegenwart von Casaminoazids Pyruvat und -Ketoglutarat aus, deren Konzentration durch Zugabe von puffernden Carbonaten erheblich erhöht werden kann.N-freie Versuchsansätze enthalten geringere Ketosäuremengen, zeigen jedoch innerhalb der späteren Entwicklungsabschnitte ein antagonistisches Verhalten von Pyruvat und -Ketoglutarat. Von der 24. Std bildet sich -Ketoglutarat unter Abnahme des ausgeschiedenen Pyruvates. Die Umbildung erfolgt stöchiometrisch.Zusätze von Succinat oder -Ketoglutarat verzögern lediglich die Wiederaufnahme der extracellularen Brenztraubensäure, sie beeinflussen nicht die Anstiegswerte der Pyruvatbildung.Versuchsansätze mit Glycerin anstelle von Glucose zeigen, daß die Pyruvatstauung keinem Glucoseeffekt im Sinne spezieller Enzymrepressionen zugeordnet werden kann.Aus den Ergebnissen der NH₄ ^.-Versuche geht hervor, daß Wachstumsvorgänge und Pyruvatstauung Antagonisten sind. Eine NH₄ ^.-bedingte Pyruvatanhäufung erfolgt nur bei Anwesenheit von Succinat.Die Ketosäureausscheidung der Streptomyceten wird auf zwei ungleiche Stoffwechselstörungen zurückgeführt: die anfängliche gehemmte Pyruvatoxydation und die anschließende Konkurrenzbeziehung der Pyruvat-bzw. -Ketoglutaratoxydation auf der Basis eines früher formulierten Kontrollprinzipes.

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Excretion of pyruvic and -oxoglutaric acid by Str. rimosus

Summary Submerged cultures of Str. rimosus excrete pyruvic and -oxoglutaric acid in glucose-containing mediums in the presence of casaminoacids. After buffering with carbonates the amounts of keto acids increase about threefold.N-free cultures show smaller amounts of keto acids, but after 24 h we can see an antagonistic behaviour of both curves: pyruvic acid is converted stoichiometrically to oxoglutaric acid.Succinate and -oxoglutarate delay the secondary reception of pyruvate without changes in primary excretion.Replacement of glucose by glycerine-containing cultures shows that pyruvate excretion is not due to a glucose-effect in the sense of enzyme-repression.NH₄-ions depress, pyruvate-excretion and induce a very good growth. A NH₄-dependent pyruvate peak appears only in the presence of succinate.The excretion of keto acids by Streptomycetes is caused by two unequal metabolic disturbances: the initially inhibited pyruvate-oxidation with failed pyruvate dehydrogenase, and the associated competition from pyruvate and/or -oxoglutarate oxidation based on a previously formulated regulation principle.

     

αB-Crystallin is Associated with Intermediate Filaments in Astrocytoma Cells

B-crystallin, a major protein of the vertebrate lens and a member of the small heat shock protein family, is expressed in non-lenticular tissues, including the central nervous system, where it is found mainly in glia. In Rosenthal fibers (RF), astrocytic inclusions that accumulate in Alexander's Disease, B-crystallin is found with hsp27 and skeins of intermediate filaments (IF) of the GFAP and vimentin types. We have investigated the association between IF and B-crystallin in a human astrocytoma cell line, U-373MG, which expresses B-crystallin. Cytoskeletal preparations contained B-crystallin, and a filamentous pattern in which B-crystallin co-localized with GFAP and vimentin by double label immunofluorescence. Immuno-electronmicroscopy confirmed the localization to IF. GFAP isolated from bovine brain and re-assembled, was associated with B-crystallin. Thus, a proportion of B-crystallin in astroglia is associated with IF, and this association may be critical in the formation of RF.     

Physico-chemical and immunological properties of allophycocyanins

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure ()₃. Representative native allophycocyanins and their and subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties.Renatured and subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein.Trimeric allophycocyanin was readily reconstituted from the purified and subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques.The results support the view that allophycocyanins are a highly conserved group of proteins.Abbreviation Used SDS sodium dodecyl sulfate     

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor <Emphasis Type="Italic">α</Emphasis>-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the -subunit 1-205 residues (₂₀₅) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR₂₀₅ as the template and inserted into vector pMAL-c2X. The constructed pMAR₂₀₅ was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR₂₀₅ protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR₂₀₅ could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR₂₀₅ and AChR₂₀₅ were similar to that of AChR -subunit from Torpedo.Revisions received 23 September 2004     

A new α2HS-glycoprotein typing by isoelectric focusing

Summary Isoelectric focusing (pH 4.0–5.0) of serum ₂HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, ₂HS 1-1, ₂HS 2-1 and ₂HS 2-2, and showed that ₂HS types are determined by two autosomal codominant alleles, ₂ HS ¹ and ₂ HS ². The method is simple, fast and easy to perform. Results of typing for the two alleles, ₂ HS ¹ and ₂ HS ², are described for a Japanese population sample (n=1003).     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3′ genes

In sheep as in man and most other mammals, there are two -globin genes (^I and ^II), which are expressed at different levels, the upstream gene being the most efficient. In -globin gene triplication and quadruplication, this trend is confirmed, i.e., the -chain output of the downstream genes progressively decreases. In this study, we have determined the complete sequence of the cDNAs and of both the introns in a triple- haplotype in which each gene could be recognized for the presence of distinct alleles. The sequence analysis reveals that the bodies of the three -globin genes are essentially identical (99.9% homology) and moreover indicates that the down-regulation of additional -globin genes in sheep is not the effect of sequence variation from the Cap to the Poly(A) addition sites. This striking similarity among -genes is higher than that seen in other mammals and is probably sustained by particularly efficient mechanisms of gene conversion and cross-over fixation. Correspondence to: Dr. M.S. Ristaldi     

Autoradiographische Untersuchungen zum Eiweiss- und Melanin-Stoffwechsel der verschiedenen Zellarten des Melanoms der Maus (Typ Harding-Passey)

Zusammenfassung Mäusen mit einem transplantierbarem Melanom, Typ Harding-Passey, wurde H-3-markiertes Dl-DOPA-, -T₂, Dl-DOPA-2,5,6-T₃, Dl-Prolin-2-T und L-Ty-rosin-3-T' injiziert und die H-3-Inkorporation in verschiedenen Zellarten des Melanom autoradiographisch zu verschiedenen Zeiten untersucht.H-3-DOPA wird selektiv in Melanin eingebaut. Dieses H-3-Melanin findet sich 1,5–24 Std nach Gabe des H-3-DOPA in Bindung an feine Melaningranula im Cytoplasma melaninarmer Melanocyten. Macrophagen dagegen enthalten zu dieser Zeit kaum H-3-Melanin. Nach 5 Tagen aber ist das H-3-Melanin fast ausschließlich in den Macrophagen nachzuweisen. Die Menge des synthetisierten H-3-Melanin war unabhängig davon, ob DOPA-, -T₂ oder DOPA-2,5,6-T₃ als H-3-Melaninvorstufe diente.Der Eiweißstoffwechsel wurde mit H-3-Prolin und H-3-Tyrosin untersucht. Die Eiweißneubildung in Melanoblasten und jugendlichen Melanocyten lag in der gleichen Größenordnung wie die der Leberparenchymzellen, während sie in Melaninspeicherzellen etwa zehnmal geringer war. Wurden die Versuchstiere erst 7 Tage nach Injektion der H-3-Aminosäure getötet, so war das H-3-Eiweiß aller Zellen zu 70–90% bereits wieder abgebaut, nur bei den Macrophagen stieg der Gehalt an H-3-Eiweiß auf das Doppelte an. Dies wird durch Phagocytose H-3-markierter Eiweiß-Melanin-Granula erklärt.Die H-3-Markierung des Tyrosin-3-T wurde nur zu einem autoradiographisch im Vergleich zur H-3-Markierung des Eiweißes nicht mehr faßbaren Bruchteil in Melanin eingebaut.Die Arbeit wurde durch Mittel der Deutschen Forschungsgemeinschaft und des Bundesministeriums für Atomkernenergie unterstützt.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Zur Pigmentarchitektonik der Großhirnrinde des Menschen

Zusammenfassung Mit Hilfe einer neu entwickelten Methode zur Darstellung der Neurolipofuscine werden die am Aufbau der Regio entorhinalis beteiligten Zellschichten elektiv hervorgehoben. Bei einem solchen Vorgehen werden die Unterschiede zwischen den einzelnen Zellarten stärker betont als im Nisslbild, weil nur eine Cytoplasmakomponente dargestellt wird. Diese Beschränkung erlaubt zugleich die Verwendung sehr dicker Schnitte (bis zu 800 ), die — aufgehellt — unter dem Stereomikroskop analysiert werden. Auf diese Weise lassen sich Verfugungen aneinandergrenzender Rindenregionen und Kantenbildungen einzelner Rindenschichten sicher erfassen.Die Schichten des Allocortex unterscheiden sich im Pigmentbild deutlich von denen des Isocortex. Sie gehen nicht kontinuierlich ineinander über. Die Rinde der Regio entorhinalis läßt sich in eine Lamina principalis externa (Pre) und eine Lamina principalis interna (Pri) gliedern. Die äußere und innere Hauptschicht sind meist durch einen zellarmen Faserstreifen (Lamina dissecans) voneinander getrennt. Beide Schichten lassen sich weiter unterteilen (Pre- Pre-, Pre-, Pri-, Pri-, Pri-).In der Regio entorhinalis des Menschen werden 16 Felder pigmentarchitektonisch voneinander unterschieden. Davon bestehen 11 Felder ausschließlich aus allocorticalen Schichten, während die restlichen Areae, welche den Übergang zum Isocortex bilden, aus einer wechselnden Zahl allo- und isocorticaler Zellschichten zusammengesetzt sind.Im Bereich des Gyrus parahippocampalis lassen sich 7 rein allocorticale Felder voneinander abgrenzen. Die Areae gruppieren sich ringartig mit stufenweise abnehmender Organisationshöhe um ein hoch differenziertes Zentrum, das im oralen und lateralen Bezirk der Regio entorhinalis liegt. Das kennzeichnende Merkmal für die zentralen Felder ist eine Aufspaltung von Pri- in drei Schichten (Pri-, Pri-, Pri-). In dem Feld e _{centr. lat.} sind alle drei Unterschichten der Lamina principalis externa enthalten, während in e _{centr. med.} Pre- fehlt. Die angrenzenden Felder mit einheitlichem Pri- lassen sich wieder in Arae mit Pre- (e _{interpol.lat.} , e _caud. ) und ein Gebiet ohne Pre- (e _{interpol. med.} ) gliedern. In den rostralen und medialen Abschnitten verschmelzen Pri- und Pri- zu einer einheitlichen Zellschicht und bilden damit das Feld e _oral. . An der Grenze zum Gyrus ambiens in Nähe des Sulcus rhinencephali inferior findet sich ein schmaler Rindenstreifen, in dem die Schichten der Lamina principalis externa nur mangelhaft ausgebildet sind. Diese limitrophe Zone setzt sich nach caudal in das Grenzfeld zum Praesubiculum (e _{marg. caud.} ) fort.Eine ähnliche areale Gradation wie im Gyrus parahippocampalis findet sich auch unter den vier Feldern des Gyrus ambiens. Das am höchsten organisierte Feld (ga _centr. ) liegt im caudalen und medialen Abschnitt und ist durch eine dreischichtige Lamina principalis interna und eine deutliche Lamina cellularis profunda ausgezeichnet. Im angrenzenden Feld ga _lat. ist Pri- stark reduziert. In ga _oral. findet sich nur eine einschichtige Lamina principalis interna. Der Grenzstreifen zum Mandelkernkomplex (e _{marg. oral.} ) besteht nur aus Teilen der äußeren Hauptschicht.Der breite Übergangsbereich von den rein allocorticalen Feldern der Regio entorhinalis bis zum Isocortex wird in vier Areae unterteilt, in denen allo- und isocorticale Schichten fugenartig ineinandergreifen. Die Stufungen ergeben sich dadurch, daß die einzelnen Zellamellen unterschiedlich weit vordringen. Eine modifizierte äußere Körnerschicht reicht bis in das Feld e _{trans. med.} ; zugleich wird Pre- in tiefer gelegene Rindenschichten verlagert. An der Grenze zu e _{trans. intermed.} endet Pre-. Die Spindelzellschicht beteiligt sich als ein weiteres isocorticales Element am Aufbau des intermediären Übergangsfeldes. Die seitlichen Kanten von Pre- und Pri- bilden die lineare Grenze zum lateralen Übergangsfeld, e _{trans. lat.} , dessen Struktur durch das Hinzutreten einer äußeren und inneren Pyramidenschicht bereits weitgehend dem Isocortex gleicht. Im Feld e _{trans. caud.} findet sich sowohl die Spindelzellschicht als auch Pre-. Es bildet damit eine Stufung zwischen dem medialen und intermediären Übergangsfeld, die jedoch nur im caudalen Abschnitt der Regio entorhinalis am Übergang zum Praesubiculum vorhanden ist.

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Pigmentarchitecture of the human cortex cerebriI. Regio entorhinalis

Summary By means of a newly developed method demonstrating neurolipofuscines the cellular layers constituting the regio entorhinalis are stained selectively. The differences between the individual cell types show up more clearly than in ordinary Nissl-preparations since by the new technique only one cytoplasmic component is stained. This limitation allows at the same time to use rather thick sections (up to 800 ), which — after clearing — are studied under the stereoscopic microscope. Thus indentations of neighbouring regions of the cortex and the edgelike formations of individual cortical layers can be demonstrated with certainty.The pigmentarchitecture of the allocortical layers differs clearly from that of the isocortex. The layers of the allocortex are not continuous with those of the isocortex. Within the regio entorhinalis the cortex can be divided into a lamina principalis externa (Pre) and a lamina principalis interna (Pri), which are separated by a narrow zone of fibers (lamina dissecans). The two main layers can be further subdivided (Pre-, Pre-, Pre-, Pri-, Pri-, Pri-).In the regio entorhinalis of man 16 areas can be distinguished by their pigmentarchitecture. 11 of these areas consist exclusively of allocortical layers, whereas the other areas which form the transitory part to the isocortex consist of various numbers of allo- and isocortical layers.In the region of the gyrus parahippocampalis 7 purely allocortical areas can be separated from each other. These areas are grouped in gradually decreasing levels of organisation round a highly differentiated center, which lies in oral and lateral parts of the regio entorhinalis. The characteristic feature of the central areas is a splitting of Pri- into three layers (Pri-, Pri-. Pri-). The area: e _{centr. lat.} contains all three sublayers of the lamina principalis externa, whereas in e _{centr. med.} Pre- is lacking. The neighbouring areas with uniform (not subdivided) Pri- can again be separated in areas with Pre- (e _{interpol. lat.} , e _caud. ) and a field without Pre- (e _{interpol. med.} ). In the rostral and medial parts Pri- and Pri- fuse forming an uniform cellular layer constituting the area: e _oral. . At the border of the gyrus ambiens near the sulcus rhinencephali inferior a narrow strip of cortex is to be found, in which the layers of the lamina principalis externa are only poorly developed. This limitrophic zone continues caudally into the border area to the praesubiculum (e _marg.caud. ).A similar areal gradation as in the gyrus parahippocampalis can be found in the four fields of the gyrus ambiens. The area with the highest organisation (ga _centr. ) is situated in the caudal and medial part of the gyrus ambiens and is characterised by a three layered lamina principalis interna and a clearly recognisable lamina cellularis profunda. In the neighbouring field ga _lat . Pri- is considerably reduced. In ga _oral. only an one layered lamina principalis interna is to be found. The border field to the amygdala (e _marg.oral. ) consists only of parts of the lamina principalis externa.The broad transitory region from the exclusively allocortical fields of the regio entorhinalis to the isocortex can be subdivided into four areas, in which allo- and isocortical layers meet in a zone of mutual indentations. The subdivision of the area is based on the different distances of penetration of the individual cellular layers. A modified lamina granularis externa extends into the field e _{trans. med.} ; at the same time Pre- is translocated into deeper cortical regions. At the border to e _{trans. intermed.} Pre- terminates. The lamina multiformis (VI) takes part as a further isocortical element in the construction of the area: e _{trans. intermed.} . The lateral edges of Pre- and Pri- form the linear border to the lateral transitory area (e _{trans. lat.} ), the structure of which resembles considerably that of the isocortex by additional appearance of a lamina pyramidalis externa and interna. In the area e _{trans. caud.} a lamina multiformis as well as the cellular layer Pre- is to be found, thus constituting a gradation between e _{trans. med.} and e _{trans. intermed.} , which, however, is present only in the caudal portions of the regio entorhinalis at the border to the praesubiculum.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human 1 and constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by¹H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (1 6)-flucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc12, Gal1 4GlcNAc1 2 or Gal1 3G11 4GlcNAc1 2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (1 2)-linked GlcNAc residues. Galactosylation of the GlcNAc1 2Man1 6 branch occurs four times more frequently than that of the GlcNAc1 2Man1 3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carryingN-acetylneuraminic acid (Neu5Ac) orN-glycolylneuraminic acid (Neu5Gc), exclusively (2 6)-linked to Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.     

Über die Wirkung von Röntgenstrahlen auf Methionin

Zusammenfassung Durch Untersuchung der Einwirkung von Röntgenstrahlen (50–200 kR) auf Carboxyl-¹⁴C-, 2-¹⁴C- und¹⁴CH₃-markiertes Methionin sowie auf nicht radioaktivmarkiertes Methionin konnte festgestellt werden, daß folgende Umwandlungsprodukte entstehen: Methioninsulfoxyd (MSO),-Hydroxy--methylmercaptobuttersäure (HMMB),-Methylmercaptopropionsäure (MMP) und Homocystein (HO).MSO und die als Zwischenstufe vermutete-Methylmercapto--ketobuttersäure konnten nicht nachgewiesen werden. Außerdem wurde in keinem Versuch eine Abspaltung die S-CH₃-Gruppe (z. B. zu-Aminobuttersäure) und eine Bildung von Methioninsulfon gefunden, die von anderen Autoren nach Einwirkung hoher Dosen beschrieben wurde. Bei einer Dosis 50 kR. konnte als Umwandlungsprodukt nur MSO festgestellt werden.Die Einwirkung von H₂O₂ auf Methionin liefert qualitativ die gleichen Umwandlungsprodukte, jedoch unterscheiden sich die Mengenverhältnisse der gebildeten Produkte deutlich von den Werten, die nach Bestrahlung erhalten werden.Der Deutschen Forschungsgemeinschaft danken wir für eine Sachbeihilfe.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.     

Veränderungen einfacher aminostickstoffhaltiger Substanzen in Mycel und Kulturmedium während des Wachstums von Streptomyces spec.

Zusammenfassung Bei einem in synthetischer Nährlösung mit Glycerin und Nitrat in Schüttelkultur wachsenden Streptomyces-Stamm wurden nach 2 bis zu 15 Tagen Kulturdauer fortlaufende qualitative und halbquantitative papierchromatographische Analysen der freien Amino-N-haltigen Substanzen des Mycels, des Kulturmediums und der Proteinaminosäuren des hydrolysierten Mycels vorgenommen. Die halbquantitativen Werte wurden gesichert durch Gesamt--Amino-N-Bestimmungen nach van Slyke u. Mitarb. Die Ergebnisse sind zusammen mit weiteren Analysendaten besprochen: Trockenmycelgewichte, alkoholische Mycelextraktgewichte, Ph-Werte, Gesamt-N-Gehalte extrahierten Mycels, Amino-N-Gehalte hydrolysierten, extrahierten Mycels, Amid- und NH₄ ⁺–N-Gehalte des Kulturfiltrates. Vorherrschende freie Aminosäuren im Mycel—bei maximaler Gesamtmenge am 3. Tag—waren in absteigenden Mengen: Glutaminsäure, Glykokoll, -Alanin, Asparaginsäure und Valin. Häufigste Substanzen im Kulturmedium—bei maximaler Abgabemenge des Mycels am 2. Tag—waren neben 8 nur am 2. und 3. Tag vorhandenen unbekannten Substanzen: Glykokoll, Serin, -Alanin, Glutaminsäure, -Aminobuttersäure. Am 7. Tag kamen dazu: Prolin, Leucin-Isoleucin, Phenylalanin und -Aminobuttersäure. In der Autolyse am 12.–15. Tag erschienen weiterhin: Oxyprolin, Tyrosin, Tryptophan, Lysin, Histidin und Arginin.Auf Grund einer zusammenfassenden Darstellung der Literatur des Gebietes wurden die Analysen besprochen und mit entsprechenden Ergebnissen anderer Autoren verglichen. Die im Wachstumsverlauf sich ändernde qualitative und quantitative Zusammensetzung der untersuchten Fraktionen als Zwischensubstanzen des Proteinauf- und-abbaus weist hin auf dessen charakteristische, tiefgehende Unterschiede in den verschiedenen Entwicklungsstadien.Fräulein Diemut Schwarz danke ich für verständnisvolle Assistenz.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.