Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic biasamidine

By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (₁₁, ₂₁) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the ₂₁ isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the ₂ subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the ₂₁ isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.     

Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes

Summary Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary (reverse) banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.     

New near-infrared optical probes of cardiac electrical activity

Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been widely and successfully used as probes for mapping membrane potential changes in cardiac cells and tissues. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450- to 550-nm range. Longer excitation/emission wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering and lower absorption by endogenous chromophores, improve recording from deeper tissue layers. In this article, we report efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra extending to 900 nm. Three dyes for cardiac optical mapping were investigated in depth from several hundred dyes containing 47 variants of the styryl chromophores. Absorbance and emission spectra in ethanol and multilamellar vesicles, as well as voltage-dependent spectral changes in a model lipid bilayer, have been recorded for these dyes. Optical action potentials were recorded in typical cardiac tissues (rat, guinea pig, pig) and compared with those of di-4-ANEPPS. The voltage sensitivities of the fluorescence of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because of molecular engineering of the chromophore, the new dyes provide a wide range of dye loading and washout time constants. These dyes will enable a series of new experiments requiring the optical probing of thick and/or blood-perfused cardiac tissues.     

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, C and C, to yield a C-aldehyde structure, which was further oxidized to the C-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between C and C, and oxidation of the C-alcohol to an aldehyde group. Further oxidation of C to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific C-C epoxidation followed by non-enzymatic hydrolysis of the epoxide.Abbreviations TLC thin layer chromatography - LDA lithium diisopropyl amide - DDQ 2,3-dichloro-5,6-dicyanobenzoquinone - MS mass spectrometry - UV ultraviolet spectroscopy     

Determination of the backbone torsion angle ɛ in nucleic acids

Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of ³J_cp coupling constants.     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Interpretation and Optimization of Absorbance and Fluorescence Signals from Voltage-sensitive Dyes

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density (OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.     

Oligomerization of deoxyguanosine 5′-phosphoro-2-methylimidazolide on a polycytidylate template

The oligomerization of deoxyguanosine 5-phosphoro-2-methylimidazolide on a polycytidylate template is much less efficient than the oligomerization of the corresponding activated ribonucleotide. Nonetheless oligomers containing up to eight nucleotide residues are detected. The products are 3–5-linked oligodeoxyribonucleotides capped at the 5-terminus with a pyrophosphate-linked monomer.     

The effects of mitochondrial energetics inhibitors on the fluorescence of potential-sensitive dyes rhodamine 123 and DiS-C3-(5) in lymphocyte suspensions

The effects of uncouplers (FCCP, DNF), oligomycin, and rotenone on the fluorescence of potential-sensitive dyes, rhodamine 123 and diS-C₃-(5), in lymphocyte suspensions were compared. The fluorescence of these optical probes gradually increased at higher FCCP concentrations. The dependences of fluorescence intensities and FCCP concentrations were similar for both dyes, and only diS-C₃-(5) fluorescence started increasing at lower FCCP concentrations. Rotenone (1 µM) significantly increased rhodamine 123 fluorescence. TMPD-induced and uncoupler-induced diS-C₃-(5) fluorescence changes increased 1.5- to 2-fold if the incubation mixture was supplemented with oligomycin (0.1–0.2 µg/ml). The fluorescence responses of the dyes in the lymphocyte suspension correlate with the effects of mitochondrial energetics inhibitors on _m in isolated mitochondria. The results suggest the possibility of using these dyes for estimating the direction of the _m changes in the lymphocyte suspension.Abbreviations _m difference in electrical potential across the mitochondrial inner membrane - _p difference in electrical potentials across the plasma membrane - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DNP 2,4-dinitrophenol - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - diS-C₃-(5) 3,3-dipropylthiodicarbocyanine - MOPS morpholinopropane sulfonic acid     

Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum

Summary The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca²⁺ transport. The absorbance and fluorescence responses of 3,3-diethyl-2,2-thiadicarbocyanine, 3,3-dimethyl-2,2-indodicarbocyanine and oxonol VI during ATP-dependent Ca²⁺ transport are influenced by the effect of accumulated Ca²⁺ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca²⁺ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca²⁺, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulumin vivo andin vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca²⁺ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca²⁺ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca²⁺ translocation. The potential generated during Ca²⁺ transport is rapidly dissipated by passive ion fluxes across the membrane.     

PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: A highly sensitive new tool in basic and applied photosynthesis research

A newly developed modulation fluorometer is described which employs repetitive 1 s Xe-flashes for excitation light. Similar to the standard PAM Chlorophyll Fluorometer, which uses 1 s LED pulses for measuring light, the integrated measuring light intensity is sufficiently low to monitor the dark-fluorescence level, F_o. The maximal fluorescence yield, F_m, can be determined with high selectivity upon application of a saturating light pulse. The Xe-PAM displays exceptionally high sensitivity, enabling quenching analysis at chlorophyll concentrations as low as 1 g/l, thus allowing to assess photosynthesis of phytoplankton in natural waters like lakes, rivers and oceans. Due to high flexibility in the choice of excitation and emission wavelengths, this system also provides the experimental basis for a thorough study of fluorescence and photosynthesis properties of various algae classes with differing antenna organisation. By appropriate modifications, the instrument may as well be used to measure with great sensitivity and selectivity other types of fluorescence (e.g. NADPH-fluorescence), as well as light-scattering and absorbance changes.     

Characterization of recurrent somatic embryogenesis of alfalfa on auxin-free medium

Callus cultures from 300 genotypes of alfalfa (Medicago sativa L.) were initiated from leaf, petiole, and internode explants placed on Blaydes medium containing 10.74 M -naphthaleneacetic acid, 11.42 M indole-3-acetic acid, and 9.29 M kinetin. Five genotypes produced somatic embryos. Upon transfer of these embryos to growth regulator-free Murashige and Skoog medium with B₅ vitamins, new somatic embryos repeatedly formed directly on older somatic embryos without an intervening callus phase in a cycle lasting about 30 days. These cultures have been maintained for two years, during which time their embryogenic capacity has remained stable. New embryogenic cultures could be started repeatedly from these genotypes. The elimination of sugars from the medium could stop recurrent embryogenesis. Glucose, maltose, and fructose stimulated recurrent embryogenesis more effectively than sucrose. Sucrose was superior to lactose, while sorbitol and mannitol did not stimulate recurrent somatic embryogenesis. The absence of nicotinic acid in the medium, as long as sucrose was present, was lethal to embryos of three of the five tested genotypes. The ability of this system to propagate embryos exponentially offers potential for development of new gene transfer systems and application to artificial seed technology.Abbreviations NAA -naphthaleneacetic acid - RSE recurrent somatic embryogenesis     

A critical evaluation of the specificity of aniline blue induced fluorescence

Summary Commercial aniline blue dyes are heterogeneous and variable. We have isolated and purified the fluorochrome from water soluble aniline blue. This fluorochrome fluoresces weakly with a maximum emission around 455 nm but the fluorescence is shifted to longer wavelengths (500–506 nm) when complexed with isolated -1,3-glucans, cellulose or mixed-linked glucans. A similar intense fluorescence is observed in sieve plates, new cell walls and around pitfields in the presence of the fluorochrome. The fluorescence induced by the aniline blue fluorochrome does not specifically indicate the presence of -1,3-glucans. Indeed most wall features are induced to fluoresce to some extent by the fluorochrome. However, fluorescence is modified by lignins and phenolics. Furthermore the intense fluorescence induced in the traditional callose sites, sieve plates, around pitfields and in new cell walls is probably related to localized differences in the structural packing of wall polymers.     

Complete assignment of 1H, 13C and 15N chemical shifts for bovine β-lactoglobulin: Secondary structure and topology of the native state is retained in a partially unfolded form

Although -lactoglobulin (-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine -LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of -LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a¹³ C,¹⁵N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric -LG A. It includes eight antiparallel -strands arranged in a barrel, flanked by an -helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth -strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of -LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.     

Biochemical and spectroscopic analysis of UV effects in the marine flagellate Cryptomonas maculata

The effects of solar and artifical ultraviolet radiation on the marine cryptoflagellate, Cryptomonas maculata, were studied. Even after short exposure to UV the accessory photosynthetic pigment phycoerythrin is bleached; likewise the fluorescence undergoes significant changes both in amplitude and in the maximal peak wavelength. In parallel, the photosynthetic oxygen production decreases rapidly during exposure. Gel electrophoresis and FPLC of membrane proteins show a significant decrease in chromoproteins after 2 h UV, which is confirmed by fluorescence excitation and emission spectra of the FPLC fractions.Abbreviations APS ammonium persulfate - DCMU 3-(3,4dichlorophenyl)1,1-dimethylurea; Emulphogen, polyoxyethylene 10 tridecyl ether - FPLC fast protein liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - SDS PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - TEMED NN NNtetramethylethylene diamine - UV-A wavelength range between 320 nm and 400 nm - UV-B wavelength range between 280 nm and 320 nm Dedicated to the 60th birthday of Professor Dr. W. Wehrmeyer     

Cyanine dye structural and voltage-induced variations in photo-voltages of bilayer membranes

Summary Flash illumination alters the voltage across bilayer lipid membranes in the presence of certain cyanine dyes. The waveforms of the photo-voltage vary systematically with dye structure and imposed transmembrane voltage. Experimental results are reported for 27 positively charged cyanine dyes, primarily oxazole derivatives, using lecithin/oxidized cholesterol bilayer membranes and 10-mm sodium chloride solutions. Several dyes do not induce any photo-voltages. Examples are 3,3 diethyl 9 ethyl 2,2 oxacarbocyanine iodide, 3,3 diethyl 2 oxa 2 thiacyanine iodide, and 3,3 dimethyl 2,2 indocarbocyanine iodide. Several dyes, when added to one side of the membranes, induce monophasic waveforms. Examples are 3,3 dimethyl 2,2 oxacarbocyanine chloride, and 3,4,3,4 tetramethyl 2,2 oxazalinocarbocyanine iodide. Other dyes induce a photo-voltage only if transmembrane voltages are imposed. These waveforms are biphasic with some dyes (3,3 diethyl 2,2 oxacarbocyanine iodide, for example) and monophasic with other dyes (3,3 dibutyl 2,2 oxacarbocyanine iodide, for example).The photo-voltage waveforms are explained by models that consider the movement of charged dye molecules within the membrane, following optical excitation. The dye movements are probably induced through charge rearrangements in the dye associated with long-lived triplet states, isomerization, or through excimer formation. These results provide information on the location and orientation of the dye molecules within bilayer membranes. The variations which occur in the waveforms with applied voltage indicate that these membranes are fluid in the direction perpendicular to the membrane plane.     

Potential-sensitive response mechanism of diS-C3-(5) in biological membranes

Summary The potential-sensitive response mechanism of 3,3-dipropylthiodicarbocyanine iodide (diS-C₃-(5)) was examined based on our previous model of diS-C₃-(5) interaction with brush border membrane vesicles (BBMV) in the absence of a membrane potential. The model contained binding (6 msec), reorientation (30 msec), dimerization (<10 nsec), and translocation (1 sec) reaction steps (Cabrini & Verkman, 1986.J. Membrane Biol. 90:163–175). Transmembrane potentials () were induced in BBMV by K⁺ gradients and valinomycin. Steady-state diS-C₃-(5) fluorescence (excitation 622 nm, emission 670 nm) increased linearly with . The reorientation and translocation reaction steps were resolved by the stopped-flow technique as a biexponential decrease in fluorescence following mixture of diS-C₃-(5) with BBMV at varying . The fractional amplitude of the faster exponential increased from 0.36 to 0.73 with increasing (–17 to 87 mV); the time constant for the faster exponential (30–35 msec) was independent of . There were single exponential kinetics (0.5–1.5 sec) for diS-C₃-(5) fluorescence response to a rapid (<2 msec) change in in the absence of a diS-C₃-(5) concentration gradient. These results, and similar findings in placental brush border vesicles, red cell vesicles, and phosphatidylcholine vesicles, support a translocation mechanism for diS-C₃-(5) response, where induced membrane potentials drive diS-C₃-(5) redistribution between sites at the inner and outer membrane leaflets, with secondary effects on diS-C₃-(5) dimerization and solution/membrane partitioning. Fluorescence lifetime and dynamic depolarization measurements showed no significant change in diS-C₃-(5) rotational characteristics or in the polarity of the diS-C₃-(5) environment with changes in . Based on the experimental results, a mathematical model is developed to explain the quantitative changes in diS-C₃-(5) fluorescence which accompany changes in at arbitrary dye/lipid ratios.     

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.     

Somatic embryogenesis from ovaries,developing ovules and immature zygotic embryos,and improved embryo development of <Emphasis Type="Italic">Castanea sativa</Emphasis>

Somatic embryogenesis of European chestnut (Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 M 2,4-dichlorophenoxyacetic acid plus 0.5 M 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 M BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 M BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.