Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Hämatologische beobachtungen an regenbogenforellen (Salmo gairdneriRich.) VII. Färbeindex (FI), absoluter Hämoglobingehalt des Einzelerytrozyten (HbE) and mittlere Hämoglobinkonzentration des Einzelerythrozyten (MCHC)

180 rainbow trouts (Salmo gairdneriRich.), aged from 1 to 3 years, were examined for fluctuations, caused by age and season, by means of colour index (CI), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).CI and MCH behave similarly. Both are increasing until the 2nd year and stay relatively constant thereafter. If the gender is not considered — there are no significant differences in the values of males and females — the CI increases from 1,4 in the first year over 1,6 to 1,7 in the age of 3 years, and the MCH increases from 44,4 over 52,6 , 56,8 , 58,1 to 55,5 .A seasonal periodicity of both indices could not be indicated on not-matured animals (F₂) which were two summers of age. Only, the january values appeared increased — CI: 2, MCH: 68,3 — otherwise the CI varies between 1,8 and 1,7 and the MCH between 53,3 and 59,1 .The MCHC-values of the age groups examined vary between 24,4% and 27,3%. The values of the yearlings form an exception (19,8%). These values certainly are inexact and too low because of the small number of individuals checked (3).

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Mit finanzieller Unterstützung durch die DFG.Institut für Siedlungswasserbau und Wassergütewirtschaft der Universitat Stuttgart Fischtoxikologische Arbeitsgruppe     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Beziehungen zwischen Carnitinstoffwechsel und Fettsäureassimilation bei Pseudomonas putida

Zusammenfassung Der Carnitinstoffwechsel und einige Beziehungen zum Fettsäurestoffwechsel wurden mittels der Wachstumskontrolle, der Bestimmung von Metaboliten und des Nachweises von Enzymaktivitäten in Pseudomonas putida untersucht. Der Stamm wuchs auf -Butyrobetain, D,L-und L-Carnitin, Glycinbetain, Cholin, D,L-Norcarnitin, D,L--Amino--hydroxybutyrat und D,L--Hydroxybutyrat. Obwohl der Stamm unverzweigte Fettsäuren von 2–16 C-Atomen zu untzen vermag, konnte er nur auf O-Acyl-L-carnitinen von 10 oder mehr C-Atomen in der Acylgruppe wachsen. Zugabe von Carnitin stimulierte das Wachstum auf langkettigen Fettsäuren.Die Bildung von Trimethylamin stieg, wenn Carnitin oder -Butyrobetain nur C-Quellen waren, und sank, wenn diese Trimethylammoniumverbindungen sowohl C-als auch N-Quellen waren. L-Carnitin induzierte sowohl die Carnitindehydrogenase als auch die -Hydroxybutyratdehydrogenase. -Butyrobetain als C-und N-Quelle induzierte ebenfalls die Carnitindehydrogenase. Im Rohextrakt betrug die spezifische Aktivität der -Hydroxybutyratdehydrogenase entsprechend dem Wachstum auf L-Carnitin oder D,L--Hydroxybutyrat 0,7 oder 1,6 Mol · min^-1 · mg^-1. Glycinbetain, Glucose und langkettige Fettsäuren reprimierten die Synthese beider Enzyme. Abhängig von der N-Quelle wird L-Carnitin offensichtlich auf zwei unterschiedlichen Stoffwechselwegen abgebaut.

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Interrelationships between carnitine metabolism and fatty acid assimilation in Pseudomonas putida

The carnitine metabolism and some relations to the fatty acid metabolism were studied in Pseudomonas putida by means of control of growth, analysis of metabolites, and determination of enzyme activities. The strain grew on -butyrobetaine, D,L-and L-carnitine, glycinebetaine, choline, D,L-norcarnitine, D,L--amino--hydroxybutyrate, and D,L--hydroxybutyrate. Although the strain used straight-chain fatty acids of 2–16 C-atoms, it was only able to grow on O-acyl-L-carnitines of 10 or more C-atoms in the acylgroup. Addition of carnitine stimulated the growth on long-chain fatty acids.The formation of trimethylamine increased, if L-carnitine or -butyrobetaine were the only carbon sources, and decreased, if these trimethylammonium compounds were carbon as well as nitrogen sources. L-Carnitine induced the carnitine dehydrogenase as well as the -hydroxybutyrate dehydrogenase. -Butyrobetaine as carbon and nitrogen source induced the carnitine dehydrogenase, too. In the crude extract the specific activities of -hydroxybutyrate dehydrogenase were 0.7 or 1.6 moles·min^-1·mg^-1 after growth on L-carnitine and D,L--hydroxybutyrate, respectively. The synthesis of both enzymes was repressed by glycinebetaine, glucose and long-chain fatty acids. Dependent on the nitrogen source L-carnitine was catabolized via two different pathways.

     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.     

Solubilization and some properties of γ-glutamyltransferase from human brain microvessels

Detergents Triton X-100, sodium deoxycholate, and octyl--D-glucopyranoside, and proteinase papain proved to be excellent agents solubilizing the -glutamyl-transferase (-GT) from human brain cortex microvessels. Ficin also solubilized -GT but to a lesser extent than papain. The relative molecular mass of the detergent-solubilized enzyme form was greater than 200,000 (in the presence of Triton X-100). The relative molecular mass of the proteinase-solubilized form was slightly greater than that of albumine. -GTs of microvessels from five human brain regions and from the choroid plexus were tested for their specificity toward acceptors. The best acceptors were found to be (in decreasing order of activity)l-cystine, glycylglycine,l-glutamine,l-methionine, andl-alanine. The findings suggest that the main features of -GT of the human blood-brain barrier are very similar to those of -GTs from other human tissues.     

Separation of Monomerizing and Lysozyme Activities of Destabilase from Medicinal Leech Salivary Gland Secretion

Destabilase, endo--(-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of - and - fibrin chains bound by -(-Glu)-Lys-isopeptide bonds.     

The NGF complex from the African ratMastomys natalensis

The 7S NGF complex from the male mouse submaxillary gland consists of the , and subunits in the ratio ₂₂. The (NGF) subunit contains all the known biolocial activity of 7S NGF. The and subunits are both members of glandular kallikrein gene family, yet only subunit has protease activity. The subunit plays a role in the processing of preproNGF to its mature form, while the role of the subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the or subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the ratMastomys natalensis in order to more fully understand the roles of the and subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio ₂. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.Special issue dedicated to Dr. Lawrence Austin     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme ⁶-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding ⁶-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking ⁶-desaturase. Expression of the Synechocystis ⁶-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis ⁶-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a ¹²-desaturase gene linked to the ⁶-desaturase gene. Expression of Synechocystis ⁶-and ¹²-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid.     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid     

Lindane degradation by cell-free extracts of Clostridium rectum

For lindane degradation, a cell suspension of Clostridium rectum strain S-17 demands the addition of substrates such as leucine, alanine, pyruvate, a leucine-proline mixture, and molecular hydrogen. In the presence of leucine-proline mixture, lindane decomposed in parallel with isovaleric acid formation, and both lindane degradation and isovaleric acid formation were inhibited by monoiodoacetic acid, suggesting a close relation between lindane degradation and the Stickland reaction. Lindane was degraded by cell-free extracts of C. rectum in the presence of dithiothreitol (DTT). Radiogaschromatograms of n-hexane soluble metabolites from [¹⁴C] lindane showed the presence of monochlorobenzene and -3,4,5,6-tetrachlorocyclohexene, Leucine, NADH, and NADPH were somewhat less active than DTT for lindane degradation in cell-free extracts. Reductive dechlorination seemed the major route of lindane degradation in cell-free extracts as well as in the intact cells of C. rectum.Abbreviations Lindane (-HCH) -1,2,3,4,5,6-hexachlorocyclohexane - -HCH -1,2,3,4,5,6-hexachlorocyclohexane - -TCH -3,4,5,6-tetrachlorocyclohexene - -PCH -1,3,4,5,6-pentachlorocyclohexene - DTT 1,4-dithiothreitol     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.