On the method of glenner for the histochemical demonstration of the monoamine oxidase (MAO)

Summary The method of Glenner et al. for the histochemical demonstration of MAO activity was studied by means of scanning microdensitometry and discrete measurement of optical density (-590 nm) of the liver and brain tissues sections.The experiments indicated that: (1) The optimal time of incubation (the thickness of sections is 15 m) is 60–90 min. (2) The histochemical reaction proceeds with the following substrates: dopamine, noradrenalin, serotonin, and tryptamine. (3) Iproniazid is the best inhibitor for preincubation whereas for simultaneous inhibition the substrate semicarbazide is better. (4) The incubation under the anaerobic conditions caused a considerable decrease of the stain intensity of the sections. We consider these data to indicate that both the aldehydes and acids formed under oxidation may take part in direct reduction of NBT to diformazan. (5) The histochemical reaction for MAO without substrates testifies to the presence of endogenous amines or other redox reactions leading to the reduction of NBT.     

Polysaccharide synthesis in tissue sections

Summary Biochemical methods for demonstration of enzyme activity are test tube models outside the organization of cell. Their application to the complicated organization of the cell present problems to histochemistry. The morphological and chemical preservation of tissue which is desirable in histochemistry leads to a multiplicity of reactions when test tube methods are applied. For example, the histochemical phosphorylase and glycosyltransferase reactions rest on the assumption that one can distinguish between preexisting glycogen and newly formed polysaccharides. We used frozen dried canine myocardium and liver for examination of the authenticity of histochemical phosphorylase and glycosyltransferase (branching enzyme, UDPG-glycogen transglycosylase) reactions as described in histochemical reference books. We were unable to distinguish between preexisting glycogen and supposedly newly formed polysaccharides with methods presently used for this purpose (Iodine stain, differential digestion with amylases, acid hydrolysis). Tissue without PAS stainable glycogen remained so after substrate incubation. When preexisting glycogen was present, the amounts of stainable polysaccharides after incubation were invariably less. Therefore, we could not prove beyond doubt that any polysaccharide synthesis due to enzyme reaction had oceured. The prescribed controls, perhaps adequate for biochemical test tube reactions, have to be redefined for meaningful histochemical procedures.This work was supported by grant number HE-07605-07 from the National Heart Institute, National Institutes of Health, Bethesda, Md., U.S.A.     

Histochemistry of Alcian blue

Summary Two new cationic copper phthalocyanins, isomers of each other, have been synthesised, and compared with Alcian blue 8GX as stains for a variety of polyanions. Analysis of dye-substrate affinities has been carried out using the critical electrolyte concentration (CEC) approach. Recently acquired data on the complete structure of Alcian blue permit interpretations of the affinities in terms of clearly defined interactions with known features of the secondary structures of natural and synthetic nucleic acids. Molecular models and the CEC analysis complement each other in providing an integrated picture of the staining mechanism. A size criterion for the intra-helical binding of phthalocyanins has been established, which explains the histochemical staining behaviour of Alcian blue and its analogues. Evidence is produced to show that appropriate dyes may act as templates on which very flexible polyanions containing aromatic rings may take up conformations, in complexes which are very stable.One of the new phthalocyanins based on quinolinic acid, has been shown to have a very specific affinity for RNA in vitro and in tissues, in concentrated salt solutions.The general problem of the analysis of histochemical staining by dyes is discussed. The CEC analysis is obtained in a form which is immediately applicable to the investigation of tissue polyanions.Future developments of cationic phthalocyanin dyes, to perform specific histochemical tasks, are indicated.     

Lipochemistry of the progamic stage of a self-incompatible species: Neutral lipids and fatty acids of the secretory stigma during its glandular activity,and of the solid style,the ovary and the anther in Forsythia intermedia Zab. (Heterostylic species)

Chromatographic (thin-layer, gas column, column chromatography) analyses of neutral lipids and fatty acids of reproductive tissues of Forsythia intermedia Zab., a self-incompatible species, were performed with two objectives in mind: 1. To determine whether there is a qualitative evolution of the different classes of lipids and fatty acids that could be correlated with the three functional stages observed during previous histochemical and ultrastructural studies. The stigmatic exudate and intracellular accumulations consist mainly of neutral lipids. 2. To compare the lipid composition of the stigma (both thrum and pin forms) with that of the style, the ovary, and the anther, and to investigate the possible existence of a stigma-specific lipid compound. Stigmatic neutral lipids are found mostly in a glyceridic mixture probably containing hydrocarbons and terpenes. The fatty acids identified are between C:7 and C: 12, with the maximum unsaturated form being a C: 18. During the secretory process there is no great qualitative diference between the neutral lipids and fatty acids found in the stigmas of thrum and pin forms. Sterols are present in styles, ovaries, and anthers, but not in stigmas. They represent the only difference in the lipid composition of these various floral structures.     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

Capsella embryogenesis: The suspensor and the basal cell

Summary The suspensor and basal cell ofCapsella were examined with the electron microscope and analyzed by histochemical procedures. The suspensor cells are more vacuolate and contain more ER and dictyosomes, but fewer ribosomes and stain less intensely for protein and nucleic acids than the cells of the embryo. The end walls of the suspensor cells contain numerous plasmodesmata but there are no plasmodesmata in the walls separating the suspensor from the embryo sac. The lower suspensor cells fuse with the embryo sac wall and the lateral walls of the lower and middle suspensor cells produce finger-like projections into the endosperm. At the heart stage the suspensor cells begin to degenerate and gradually lose their ability to stain for protein and nucleic acids.The basal cell is highly vacuolate and enlarges to a size of 150 X 70. An extensive network of wall projections develops on the micropylar end wall and adjacent lateral wall. The nucleus becomes deeply lobed and suspended in a strand of cytoplasm traversing the large vacuole. The cytoplasmic matrix darkens at the late globular stage and histochemical staining for protein becomes very intense. The basal cell remains active after the suspensor cytoplasm has degenerated. It is proposed that the suspensor and basal cell function as an embryonic root in the absorption and translocation of nutriments from the integuments to the developing embryo.Research supported by NSF grant GB 3460 and NIH grant 5-RO 1-CA-03656-09.     

Tissue uptake of catechol and indole amino acids histochemical studies on the rat iris

Summary Rat irises exposed to DOPA in vitro and subsequently treated with the histochemical technique of Falck and Hillarp for catecholamines show accumulation of a fluorescent formaldehyde condensation product within the capillary endothelial cells. A similar accumulation was observed when rat irises were exposed to 5-OH-Tryptophan and -methyl-dopa but not after exposure to the corresponding amines. Fluorescent products were also observed in the same cells when control irises were treated with the trihydroxyindole histochemical reaction. It is concluded that certain catechol and indole amino acids accumulate within the endothelial cells of the rat iris capillaries in a manner similar to that observed in brain capillaries. Furthermore, small amounts of DOPA are probably present within these cells normally.     

Nutritional Requirements of <Emphasis Type="Italic">Allisonella histaminiformans,</Emphasis> a Ruminal Bacterium that Decarboxylates Histidine and Produces Histamine

A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium. The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids). These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T. ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events. XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues. Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the 2 × 6 helix paradigm. This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium.     

The histochemistry of complex carbohydrates in the ovarian follicles of adult mice

Summary In the ovarian follicles of adult mice, complex carbohydrate-containing structures have been studied by means of light microscopic histochemical methods. In the ovarian follicles, the zona pellucida of oocytes, follicular fluid and intercellular matrix of the granulosa layer are found to exhibit positive reactions for complex carbohydrates with 1,2-glycol and acidic groups and -D-mannosyl and -D-glucosyl residues. The present histochemical analyses have revealed that the complex carbohydrates common to the three types of the structures are hyaluronic acid, sulfated glycosaminoglycans other than isomeric chondroitin sulfates and neutral glycoproteins and that sialic acid is a particular moiety of the zona pellucida, whereas isomeric chondroitin sulfates being that of the follicular fluid and intercellular matrix of the granulosa layer. The histophysiological activities of the carbohydrate-containing structures have been discussed with special reference to their histochemical properties determined in the present study.     

Salinity dependence,uptake kinetics,and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved¹⁴C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to¹⁴C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,¹⁴C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S).     

Some features of the fine structure and histochemistry of planarian subepidermal gland cells

Summary The conspicuous subepidermal gland cells, namely eosinophiles, basophiles and rhabdite forming cells, in Planaria vitta, have been studied using a variety of histochemical methods for proteins, amino acids, polysaccharides, lipids, nucleic acids and calcium, in addition to common histological methods. Several fixation methods and freeze-drying have been employed. The eosinophilia of certain of the gland cells has been investigated by blocking amino acid end- and side-groups and by the staining capacity at different pH's. The fine structure of the secretion granules has been elucidated by means of electron microscopy.The eosinophilic granules are very coarse and up to 1.5 long and 0.8 wide. They are predominantly composed of protein and give positive reactions for tyrosine, arginine and cysteine-cystine and a negative reaction for tryptophan. They possibly also contain histidine. The eosinophilia is only slightly affected by acetylation or nitrosation and is only slightly decreased at pH 11.8, so the presence of arginine is probably the basis for the eosinophilia. The granules probably also contain phospholipid. The fine structure of the granules is rather unique for secretion granules. They are not surrounded by a membrane and are built up of light and dark striated structures, very regular in appearance and unperforated. The striation is mainly oriented transverse to the long axis of the granules. Parallel with this axis a much finer striation is found, bounding rectangular compartments. The coarse, dark striations are about 175–250Å wide.The basophilic cells may be divided into at last three types. The most commonly encountered contain granules 0.2–0.3 in diameter. They are selectively stained by aldehyde-fuchsin after permanganate oxidation. The granules are negative to tests for protein and amino acids. They are PAS-positive and become intensely metachromatic and stainable by alcian blue at pH 2.9 after sulfation. These and several other observations point to the presence of neutral and/or acid mucopolysaccharides. Glycogen is not present. Another type of basophilic cells contains RNA in great amounts. The basophilic granules appear homogeneous on the electron micrographs.The rhabdites are up to 5 long and 1 wide. They are negative to tests for lipid, nucleic acid and polysaccharides. They are intensely eosinophilic even at pH 11.8. They are probably composed of proteins, but only one amino acid was found reacting to a noteworthy degree: cysteine. It was not possible to elucidate the basis for the strong eosinophilia. The presence of ionic calcium was ruled out. The rhabdites are very electron-dense and on the electron micrographs appear homogeneous and not invested by a membrane.     

Formation of (n-9) and (n-7) cis-monounsaturated fatty acids in seeds of higher plants

The relative abundance of (n-9) and (n-7) isomers in the monounsaturated fatty acids of seed lipids has been determined for selected plants in order to assess the biosynthetic reactions involved in their formation. 9 Desaturation of stearic acid to (n-9) octadecenoic acid is almost exclusively operative in the formation of monounsaturated fatty acids in the seeds of Helianthus annuus, Glycine max and Brassica napus, cv. Quinta and Erglu, in which chain elongation of monounsaturated fatty acids terminates at the level of an 18 carbon chain. 9 Desaturation of palmitic acid is a minor yet significant pathway in the seeds of Sinapis alba and Brassica napus, cv. Rapol and Tira, in which chain elongation of monounsaturated fatty acids occurs extensively beyond the 18 carbon chain. In each of these seeds, both (n-9) and (n-7) octadecenoic acids formed are subsequently elongated to icosenoic acids. However, elongation of the (n-7) isomer is terminated at the level of a 20 carbon chain, whereas the (n-9) icosenoic acid is selectively elongated to docosenoic acid and even up to tetracosenoic acid in Sinapis alba. 9 Desaturation of palmitic acid followed by elongation to (n-7) octadecenoic acid occurs to a minor extent in the seeds of Tropaeolum majus. Only the (n-9) octadecenoic acid, and not its (n-7) isomer, is elongated to icosenoic and docosenoic acids.     

The morphology,innervation and neural control of the anterior arterial system of Aplysia californica

Summary The morphology, innervation, and neural control of the anterior arterial system of Aplysia californica were investigated. Immunocytochemical and histochemical techniques generated positive reactions in the anterior arterial system for several neuroactive substances, including SCP_B, FMRFamide, R151 peptide, dopamine and serotonin. Three neurons were found to innervate the rostral portions of the anterior arterial tree. One is the identified peptidergic neuron R15 in the abdominal ganglion, and the other two are a pair of previously unidentified neurons, one in each pedal ganglion, named pedal arterial shorteners (P_AS)- The endogeneously bursting neuron R15 was found to innervate the proximal anterior aorta. It also innervates a branch of the distal anterior aorta, the left pedal-parapodial artery. Activity in R15 causes constriction of the left pedal-parapodial artery. This effect is presumed to direct hemolymph towards the genital groove and penis on the right side in vivo. This vasoconstrictor action of R15 is mimicked by the R151 peptide. The P_AS neuron pair causes longitudinal contraction of the rostral anterior aorta and the pedal-parapodial arteries. In vivo, the pair is active during behaviors involving head withdrawal and turning. By adjusting the length of the arteries during postural changes, the P_AS neurons may prevent disturbances in blood flow due to bending or kinking of the arterial walls.     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat

Summary The segmentation of the proximal tubules of the male rat kidney was studied by means of enzyme histochemical reactions. Soluble oxidoreductases (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, NAD- and NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase) were demonstrated using methods which reduce enzyme diffusion (incubating in presence of polyvinyl alcohol) and eliminate interference from tissue tetrazolium reductases. Less soluble or insoluble enzymes (glucose 6-phosphatase, -hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases) were demonstrated by incubation in conventional watery media.Segmental differences were observed in respect to all enzymes studied, and most reactions clearly visualized the three segments known to exist from ultrastructural as well as previous histochemical studies: The pars convoluta includes the first (P₁) and most of the second (P₂) segment. The transition to the third segment (P₃) is in the beginning of the pars recta. Also these reactions revealed a difference between the first part of the P₃, which runs through the cortex in the medullary rays, and the terminal part transversing the outer stripe of the medulla. In most instances intensity of reaction decreased in the last portion of the P₃.A number of the enzymes studied were mainly or solely localized to the P₃ (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenases, -hydroxybutyrate dehydrogenase, 3-hydroxysteroid dehydrogenase, decarboxylating malate dehydrogenase and uridine diphosphate glucose dehydrogenase). Some possible functional implications of the findings are discussed.Supported by grants from Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — Mr. Kaj L. Pedersen is thanked for valuable photographic assistance.     

Enzymatic synthesis and characterization of N5-(carboxymethyl)-L-ornithine and N6-(carboxymethyl)-L-lysine

Summary This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N⁶-(carboxymethyl)-L-lysine and N⁵-(carboxymethyl)-L-ornithine. The two N -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and the- or-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N⁵-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.     

New aspects on factors determining the sensitivity of the formaldehyde and glyoxylic acid fluorescence histochemical methods for monoamines

Summary The fluorophore and fluorescence yield from tryptamine and 3-methoxytyramine in histochemical protein models have been compared in the standard formaldehyde reaction, the acid-catalyzed formaldehyde reaction, the formaldehyde-ozone reaction, and the aluminum-formaldehyde reaction. In the standard formaldehyde reaction both the fluorophore and fluorescence yields are low. However, the other reactions give a dramatic increase in fluorescence intensity (18–20 times) from tryptamine and 3-methoxytyramine whereas only minor changes (up to 100% increase) in fluorophore yield are observed. It is concluded that the relative fluorescence intensity of each fluorophore molecule formed in the three modifications of the formaldehyde reaction is much higher than that of the molecules formed in the standard formaldehyde reaction. It has previously been demonstrated that the fluorophores formed from dopamine in the gaseous formaldehyde and glyoxylic acid reactions have a much higher (10 times) relative fluorescence intensity than the synthetic fluorophores. The present experiments show that if the histochemical models are dissolved in buffer after the reaction and new models are made from this solution, the fluorescence intensity of the fluorophores formed in the reaction is drastically reduced and becomes comparable to that of the synthetic ones. The results of this and our previous studies indicate that hitherto unknown fluorescence enhancing mechanisms play a major role for the fluorescence yield, i.e. the sensitivity, in the various formaldehyde and glyoxylic acid methods. One possible explanation to the high relative fluorescence intensity of the fluorophores formed in the histochemical reactions could be an energy transfer between, e.g. the non-fluorescent intermediary reaction products (the tetrahydro derivatives) and the fluorophores (the dihydroisoquinolines and dihydro--carbolines). Such an energy transfer is probably attenuated in the dissolved models, where the distances between and orientations of the various molecules have been changed.Abbreviations DA dopamine - FA formaldehyde - GA glyoxylic acid - 3-MT 3-methoxytyramine - 4-MT 3-hydroxy-4-methoxyphenylethylamine - T tryptamine - DHC dihydro--carbolines - THC tetrahydro--carboline - 2-Carb.Me-DHIQ 2-carboxymethyl-3,4-dihydroisoquinolinium compound - THIQ-1-COOH tetrahydroisoquinoline-1 carboxylic acid     

The connection between spinal cord and notochord in Amphioxus (Branchiostoma lanceolatum)

Summary Silver impregnation of nerves, histochemical reactions for acetylcholinesterase, and electron microscopy reveal an efferent innervation of the notochord in amphioxus. Extensions of the notochordal lamellae end in groups (the notochordal horns) just below the ventro-lateral surface of the spinal cord where they are opposed to large nerve terminals originating as short collaterals of axons running longitudinally in the nerve cord. This neurochordal junction resembles an ordinary neuro-muscular junction in several respects and is interpreted as a part of the muscular system found in the notochord itself. Acknowledgement. The author is indebted to the staffs at the Marine laboratory in Plymouth and the Biological station at Helgoland for supply of material. The expert advices and criticism of Q. Bone, Ph. D., Plymouth, and Dr. U. Welsch, Kiel, are also greatly appreciated.     

Asymmetric imine-ene reactions: Diastereofacial selective reactions with chiral glyoxylate-derivedα-imino esters and asymmetric catalysis of enantiofacial selective reactions with prochiralα-imino esters

Summary The diastereofacial selective imine-ene reactions with-imino esters, prepared from (–)-8-phenylmenthyl glyoxylate, are shown to provide an efficient entry to the asymmetric synthesis of-amino acids. The feasibility study of the asymmetric catalysis is also reported on the enantiofacial selective ene reactions with prochiral-imino esters.