Über den Einfluß von Trenimon und Endoxan auf die Meiose der männlichen Maus

Mutagenic activity of alkylating agents has previously been studied in our group by the dominant lethal method (Röhrborn et al.). These investigations have now been supplemented by cytogenetic studies on the influence of trenimon and cytoxan in the same dosages as used by Röhrborn applied in single i.p. injektions. The stages of spermatogenesis treated were estimated using the time table established by Oakberg. The cytogenetic procedures used have been described in the preceeding paper (Schleiermacher, 1966). Relative frequency of meiosis was calculated from the number of meiotic prophases counted in relation to number of interphase nuclei.Relative frequency of meiotic prophases varied considerably following treatment. After maturation depletion of the tubules marked diminution but never complete disappearence of meiotic prophases occured. This observation corresponds in a certain manner to the sterile period known after X-irradiation. In breeding experiments, however, no definite decrease of fertility has been noted (Röhrborn). As soon as the spermatids disappear as result of lacking regeneration, the relative frequency of meiotic prophases must increase. Other factors also could account for this phenomenon. Change in frequency of meiosis indicates that regeneration of germ cells is slowed down after treatment with trenimon and cytoxan, which is at variance with the results reported of irradiation experiments. The sensible stages, in which retardation of regeneration occurs, are detectable by histological analysis only. This will be attempted in subsequent experiments. Differences in results of experiments with trenimon and cytoxan could be due to dosage effects.In all phases of spermatogenesis treatment results in increased frequency of univalents. The highest incidence of univalents has been found after treatment of preleptotene with trenimon. This could presumably be caused by invisible structural changes in chromosomes, most likely inversions. The expected occurrence of aneuploid second metaphases could not be demonstrated. Our results indicate that univalents do not play a role in causation of dominant lethals.Chromosome aberrations in diakinesis, mainly chromosome breaks and translocations, occured after treatment of all phases. In second metaphases breaks, dicentric chromosomes and translocation chromosomes have been seen very rarely. Less chromosome aberrations have been found in either M I or M II than have been expected from the results of irradiation experiments (Ashwood-Smith et al.). Chromosome aberrations even occured in stages of spermatogenesis in which no dominant lethals have been detected by Röhrborn. Whether in our experiments the chromosome aberrations may have been masked by loss of damaged cells cannot yet be decided.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin

Spheroplasts of the unstable l-form of Proteus mirabilis with fragile, shape defective cell walls grown in medium containing 120 mg/l penicillin G and then killed and permeabilized by ether treatment, were capable of in vitro synthesis of peptidoglycan from the precursors UDP-GlcNAc and UDP-MurNAc-l-Ala-d-Glu(ms-A₂pm-d-Ala-d-Ala). The in vitro peptidoglycan was extensively peptide-crosslinked, indicating a continuing function of peptidoglycan transpeptidase in the spheroplasts. The seven penicillin-binding proteins (PBPs) of P. mirabilis with their functions as multiple peptidoglycan transpeptidases were shown to be saturated in the spheroplasts and thereby functionally inactivated by the penicillin of the growth medium to a very different degree. Complete or almost complete saturation occurred with the PBPs 1A, 1B, and 3, for which functions as indispensible transpeptidases in Escherichia coli have been postulated. In contrast, PBPs 5 and 6 were not saturated in the l-form spheroplasts. Transpeptidase function has been described previously in PBP 5 of P. mirabilis. The working hypothesis is proposed that synthesis of the functionally defective peptidoglycan of l-form spheroplasts in the presence of penicillin takes place with transpeptidase function of PBP 5.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Function and presumed molecular structure of Na+-D-glucose cotransport systems

Functional characterization of Na⁺-d-glucose cotransport in intestine and kidney indicates the existence of heterogeneous Na⁺-d-glucose cotransport systems. Target size analysis of the transporting unit and model analysis of substrate binding have been performed and proteins have been cloned which mediate (SGLT1) and modulate (RS1) the expression of Na⁺-d-glucose cotransport. The experiments support the hypothesis that functional Na⁺-d-glucose cotransport systems in mammals are composed of two SGLT1-type subunits and may contain one or two RS1-type proteins. SGLT1 contains up to twelve membrane-spanning -helices, whereas RS1 is a hydrophilic extracellular protein which is anchored in the brush-border membrane by a hydrophobic -helix at the C-terminus. SGLT1 alone is able to translocate glucose together with sodium; however, RS1 increases the V _max of transport expressed by SGLT1. In addition, the biphasic glucose dependence of transport, which is typical for kidney and has been often observed in intestine, was only obtained after coexpression of SGLT1 and RS1.     

A comparative study of the chromosomes in the incipient species of the Drosophila paulistorum complex

Summary Drosophila paulistorum Dobzhansky et Pavan is a complex of six races or incipient species. The races are mostly allopatric, but they are reproductively isolated sufficiently to permit them to exist also sympatrically in some places. The gene arrangements in the chromosomes of the races have been compared by means of examination of the giant chromosomes in the larval salivary glands; 28 strains of all races, and about an equal number of interracial hybrids have been studied.Chromosomal inversion polymorphism has been discovered in all races, even in the Guianan race of which only a single strain is available. Inversion heterozygotes are found in every one of the five chromosomal strands which the species has. Interracial hybrids tend to be heterozygous for more inversions than are present in the strains of the parental races. The Transitional race has however much the same gene arrangements as the widespread Andean — South Brazilian race.With the exception of the Transitional race, and of three other possible exceptions, each race has a collection of its own race-specific inversion polymorphs, not found in the other races. This very striking finding is discussed in connection with the hypothesis which envisages the origin of new species from marginal colonies at the periphery of the geographic distribution area of the ancestral species.The work reported in this article has been carried under Contract No. AT-(30-1)-1151, U.S. Atomic Energy Commission, mostly at the Department of Zoology, Columbia University, New York.     

Scytinostroma galactinum as a pathogen of woody plants

Scytinostroma galactinum (Fr.)Donk is the fungus commonly known asCorticium galctinum (Fr.)Burt. Although it occurs as a saprobe on woody plants and plant debris, it also has been considered by several authors as an active pathogen that causes a white root and butt or collar rot. During the summer of 1970, it was found near Baltimore and also in Montgomery County, Maryland, under circumstances that seemingly provide additional records of pathogenicity. Several other Maryland records are cited, and many Canadian studies have been reviewed.S. galactinum has a distinctive dextrinoid mycelium, which is characteristic enough to permit a tentative identification even in the absence of other features. Outstanding elements of the basidiocarp are the dextrinoid hyphae and slender gloeocystidia. In culture, dextrinoid fiber hyphae and papillate oil-bearing hyphae with clamp connections are characteristic. These and other features of the basidiocarp and culture serve in the characterization of this fungus, which is economically significant and widely distributed.     

Urea and thiourea transport in Aspergillus nidulans

Wild-type Aspergillus nidulans has an active transport system specific for urea which concentrates urea at least 50-fold relative to the extracellular concentration. It is substrate concentration dependent, with an apparent K _m of 3×10^–5 m for urea. Competition studies and the properties of mutants indicate that thiourea is taken up by the same system as urea. Thiourea is toxic at 5mm to wild-type cells of Aspergillus nidulans. Mutants, designated ureA1 to ureA16, resistant to thiourea have been isolated, and transport assays and growth tests show that they are specifically impaired in urea transport. The mutant ureA1 has a higher K _m value than the wild type for thiourea uptake. The ureA locus has been assigned to linkage group VIII. ureA1 is recessive for thiourea resistance while semidominant for the low uptake characteristic. The urea uptake system is under nitrogen regulation, with l-glutamine as the probable effector. The mutants, meaA8 and gdhA1, which are insensitive to ammonium control of many nitrogen-regulated metabolic systems, are also insensitive to ammonium control of urea uptake, but both are sensitive to l-glutamine regulation.Formerly at the Department of Genetics, University of Glasgow, Glasgow, Scotland.     

Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe–4S] protein

The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mössbauer spectroscopy after reconstitution with ⁵⁷FeCl₃, Na₂S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.     

Biochemical properties of yeast l-asparaginase

Only a single l-asparaginase has been found in the yeast Saccharomyces cerevisiae. The enzyme is synthesized constitutively, and its functioning is not controlled by the products of its activity. The apparent K_m for the yeast l-asparaginase reaction is 2.5×10^–4 m. Activity is greatest at pH 8.5 and is unaffected by the ionic strength of reaction mixtures. l-Asparagine can serve as the sole nitrogen source for cell metabolism but cannot serve as the sole supply of carbon. Active l-asparaginase is necessary for the use of l-asparagine as a nitrogen donor for cell growth. This requirement suggests a possible way in which l-asparaginase-deficient strains of yeast or other organisms might easily be selected.G.E.J. was supported by U.S. Public Health Service Predoctoral Fellowship No. 5 F01 GM36,437.     

Symplast domains in extrastelar tissues of Egeria densa Planch.

A set of hydrophilic fluorescent dyes of known molecular weight has been used to determine the molecular exclusion limit and the extent of apical, epidermal and cortical symplasts in the root, stem and leaf of Egeria densa. These dyes are unable to pass the plasmalemma, so that any cell-to-cell movement of injected dye must occur via the symplast. The shoot-apex symplast has a high molecular exclusion limit, excluding dyes with a molecular weight of 749 dalton (fluorescein hexaglycine) and greater but allowing dyes of up to 665 dalton (fluorescein diglutamic acid) to pass. The leaf epidermal symplast is similar to that in the apex: fluorescein pentaglycine (674 dalton) moves to a limited extent, but fluorescein hexaglycine is immobile. Stem and root epidermal cells have a lower molecular exclusion limit, only the dye 6-carboxyfluorescein (376 dalton) is able to move from cell-to-cell. Cortical and epidermal tissues in both the stem and the root have similar symplast permeabilities. However, a barrier to dye (6-carboxyfluorescein) movement is found between the epidermis and the cortex in both organs. Barriers are also found at the nodes between expanded internodes. The stem barriers are not found in the unexpanded nodes near the shoot tip; apparently they are formed early during internode expansion. In the root tip, a barrier to the movement of dye is found between the root cap and the remainder of the root. Plasmodesmata are found linking all cell types studied, even cells where barriers to dye movement occur. Thus, the plant, far from being one uniform symplast, consists of a large number of symplast domains, which may or may not differ in molecular exclusion limit.Abbreviations F fluorescein isothiocyanate isomer I - Glu l-glutamic acid - (Glu)₂ l-glutamylglutamic acid - (Gly)₅ l-pentaglycine - (Gly)₆ l-hexaglycine     

The effect of carbon dioxide on reduction of methylene blue by microorganisms

Summary and Conclusions The experiments ofHes which indicate that CO₂ is essential for the reduction of methylene blue by microorganisms presumably because it is required for the functioning of the cellular hydrogen transport mechanisms, have been repeated and results have been obtained that seemingly differ from those ofHes. We could not demonstrate any effect of CO₂ on the rate of methylene blue reduction by various bacteria in the presence of utilizable compounds such as glucose, sucrose, lactate and pyruvate. UsingE. coli and other bacteria we found, however, that the time required for the reduction of methylene blue was greatly increased by CO₂ removal when no substrates were added. This effect of CO₂ on the endogenous metabolism was observed only when the cells were depleted of endogenous reserves and of CO₂ by aeration or dilution.     

Über die Induktion der Zellteilung in synchronen Chlorella-Kulturen

Zusammenfassung Durch vergleichende Analysen der Xanthophylle von: Taraxacum officinale, Helianthus annuus, Ranunculus acer, Impatiens noli tangere, Tussilago farfara und Sarothamnus scoparius ließ sich beweisen, daß Taraxanthin und Luteinepoxid identisch sind. Taraxanthin ist damit der gültige Trivialname für das 5,6-Epoxid des Luteins. Die Trennung der Isomeren wird auf Dünnschichten von Mg(OH)₂CO₃ erreicht. Violaxanthin und Lutein kommen ebenfalls in cis-und trans-Formen vor. In den Objekten wurden ausschließlich 5,6-Epoxide beobachtet, die erst während unvorsichtiger Aufbereitung in 5,8-Epoxide umgelagert werden, die daher als Artefakte zu werten sind.

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The identity of Taraxanthin and Luteinepoxid

Summary Taraxanthin was first isolated from flowers of Taraxacum officinale by Kuhn and Lederer (1931). The elementary analysis was in agreement with the formula C₄₀H₅₆O₄. Later, Eugster, Karrer and Jaeger (1957, 1963) showed that it might be a hydroxylated derivative of lutein epoxide.In a comparative investigation of the main sources of Taraxanthin (Taraxacum officinale, Helianthus annuus, Ranunculus acer, Impatiens noli tangere, Tussilago farfara and Sarothamnus scoparius) we have now found that the pigment is identical with lutein epoxide. Taraxanthin and luteine poxide are synonyms, an dtaraxanthin is the correct trivial name. It occurs partly as the trans, partly as the cis isomer. These two isomers are separated on thin-layers of Mg₂ (OH)₂ CO₃. With this chromatographic system naturally occurring cis-isomers of lutein and violaxanthin have been detected. The flowers investigated contain only 5,6 (±5,6)-epoxides. The corresponding furanoid 5,8-epoxides, which have been found by other workers, may be regarded as artifacts due to the isolation procedures.

     

Single Taste Stimuli Elicit Either Increases or Decreases in Intracellular Calcium in Isolated Catfish Taste Cells

Taste cells are specialized epithelial cells that respond to stimulation with release of neurotransmitters onto afferent nerves that innervate taste buds. In analogy to neurotransmitter release in other cells, it is expected that neurotransmitter release in taste cells is dependent on an increase in intracellular Ca²⁺ ([Ca²⁺] _i ). We have studied changes in [Ca²⁺] _i elicited by the taste stimuli l- and d-arginine in isolated taste cells from the channel catfish (Ictalurus punctatus). In a sample of 119 cells, we found 15 cells responding to l-arginine, and 12 cells responding to d-arginine with an increase in [Ca²⁺] _i . The response to l-arginine was inhibited by equimolar d-arginine in cells where d-arginine alone did not cause a change in [Ca²⁺] _i , which is consistent with mediation of this response by a previously characterized l-arginine-gated nonspecific cation channel antagonized by d-arginine [31]. However, we also found that these taste stimuli elicited decreases in [Ca²⁺] _i in substantial number of cells (6 for l-Arg, and 2 for d-Arg, n= 119). These observations suggest that stimulation of taste cells with sapid stimuli may result in simultaneous excitation and inhibition of different taste cells within the taste bud, which could be involved in local processing of the taste signal. Received: 25 May 1995/Revised: 29 September 1995     

Methionine-repressible homoserine dehydrogenase of serratia marcescens: Purification and properties

Summary Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case ofEs-cherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD^– or NADP⁺ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1mm methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, K_m for substrate and cofactors), it resembles its counterpart inE. coli K₁₂. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8m urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity ofS. marcescens unlike inSalmonella typhimurium andE. coli K₁₂ where it is a minor or a negligible component.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

Preparation and reactivity studies of synthetic microperoxidases containing<Emphasis Type="Italic"> b</Emphasis>-type heme

In order to create a heme environment that permits biomimicry of heme-containing peroxidases, a number of new hemin–peptide complexes—hemin-2(18)-glycyl-l-histidine methyl ester (HGH), hemin-2(18)-glycyl-glycyl-l-histidine methyl ester (HGGH), and hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) (H2GGH)—have been prepared by condensation of glycyl-l-histidine methyl ester or glycyl-glycyl-l-histidine methyl ester with the propionic side chains of hemin. Characterization by means of UV/vis- and ¹H NMR spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of five-coordinate complexes in the case of HGH and HGGH, with histidine as an axial ligand. In the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the iron center appears to be formed. However, ¹H NMR of H2GGH reveals the existence of an equilibrium between low-spin six-coordinate and high-spin five-coordinate species in solution. The catalytic activity of the hemin–peptide complexes towards several organic substrates, such as p-cresol, l-tyrosine methyl ester, and ABTS, has been investigated. It was found that not only the five-coordinate HGH and HGGH complexes, but also the six-coordinate H2GGH, catalyze the oxidation of substrates by H₂O₂. The longer and less strained peptide arm provides the HGGH complex with a slightly higher catalytic efficiency, as compared with HGH, due to formation of more stable intermediate complexes.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0532-5.Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) - DCC dicyclohexylcarbodiimide - HGH hemin-2(18)-glycyl-l-histidine methyl ester - HGGH hemin-2(18)-glycyl-glycyl-l-histidine methyl ester - H2GGH hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) - HOBt N-hydroxybenzotriazole     

5-HT receptors in mammalian brain: receptor autoradiography andin situ hybridization studies of new ligands and newly identified receptors

Summary In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combinedin situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT_1A receptors in monkey brain, labelled with the agonist³H-8-OH-DPAT and the antagonist³H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT_1A receptors in monkey brain. The labelling conditions to visualize 5-HT_1F receptors in guinea pig brain, namely³H-sumatriptan in the presence of 10⁻⁸ m 5-CT to block 5-HT_1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed byin situ hybridization. By using³H-ketanserin and³H-mesulergine in parallel within situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT_2A and 5-HT_2C binding sites in monkey brain. 5-HT₄ receptors were localized in the brain of several species including humans by using¹²⁵I-SB 207710.In situ hybridization experiments performed in guinea pig confirmed that 5-HT₄ receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT₇ binding sites were labelled in rat and guinea pig brains by incubating with³H-5-CT in the presence of 100 μm WAY 100135 and 250 μm GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate

Summary The production of l-phenylalanine from the racemate d,l-phenyllactate in an enzyme membrane reactor has been examined. In a first step the racemate is dehydrogenated to the prochiral intermediate phenylpyruvate by the enzymes d-and l-hydroxyisocaproate dehydrogenase. In a second step phenylpyruvate is reductively aminated to l-phenylalanine by l-phenylalanine dehydrogenase. Both steps are dependent on coenzyme, the first one requires NAD, the second one NADH in stoichiometric amounts; in this way the coenzyme is regenerated and only required catalytically. The coenzyme is covalently bound to polyethylene glyco-20 000 and can thus be retained in the reactor analogously to the three enzymes. In order to optimize the continuous production of l-phenylalanine from d,l-phenyllactate, models of the reaction kinetics and of the reactor system have been set up. By means of the reactor model, we can calculate the optimum ratio of the three enzymes, the optimum coenzyme concentration and the optimum phenylpyruvate concentration in the feed.In this process, at a substrate concentration of 50 mM d,l-phenyllactate we reached a spacetime-yield of 28 g l-Phe/(l*d).Abbreviations PEG polyethylene glycol - d-HicDH d-hydroxyisocaproate dehydrogenase - l-HicDH l-hydroxyisocaproate dehydrogenase - PheDH l-phenylalanine dehydrogenase - V _max maximum velocity - K _M Michaelis-Menten constant - K _l inhibition constant - R₁ reaction rate of the d-HicDH forward reaction - R₂ reaction rate of the d-HicDH reverse reaction - R₃ reaction rate of the l-HicDH forward reaction - R₄ reaction rate of the l-HicDH reverse reaction - R₅ reaction rate of the PheDH forward reaction - R₆ reaction rate of the PheDH reverse reaction - d-PLac d-phenyllactate - l-PLac l-phenyllactate - PPy phenylpyruvate - l-Phe l-phenylalanine - NH₄ ammonium - residence time     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.