The ultrastructural ionic fixation technique localizing acetylcholinesterase activity,reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

Summary A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous ionic fixation of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ acetylcholine phosphomolybdate and copper thiocholine phosphomolybdate. Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

Effects of high ambient temperatures on the metabolism of West African dwarf goats. I

Thirty-two West African dwarf goats were exposed to temperature treatments: 20, 25, 30, 35, 35, 35, 30, 25, 20°C each lasting three days.Sixteen goats were kept in individual pens (I), the others in two group pens (G). Heat production (HP) and activity were recorded during 48 hours in each temperature treatment.Mean HP and ME intake were similar for G and I animals, but I animals had lower values than G animals at low chamber temperatures and higher values than G animals at high temperatures. Upper critical temperature was between 25°C and 30°C under an increasing T and between 30 and 35°C under a decreasing T. Adaptation of heat production and ME intake to a change in temperature of 5°C required at least six and possibly more than nine days.Diurnal variation in HP was large, up to 44% between extremes. This was largely due to variation in activity.     

The taxonomic significance of the trunk limbs of the chydoridae (Cladocera)

Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.

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Chydoridae (Cladocera)

Chydoridae (. ), , .

     

Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

Computational constraints that may have favoured the lamination of sensory cortex

At the transition from early reptilian ancestors to primordial mammals, the areas of sensory cortex that process topographic modalities acquire the laminar structure of isocortex. A prominent step in lamination is granulation, whereby the formerly unique principal layer of pyramidal cells is split by the insertion of a new layer of excitatory, but intrinsic, granule cells, layer IV. I consider the hypothesis that granulation, and the differentiation between supra- and infra-granular pyramidal layers, may be advantageous to support fine topography in their sensory maps. Fine topography implies a generic distinction between where information, explicitly mapped on the cortical sheet, and what information, represented in a distributed fashion as a distinct firing pattern across neurons. These patterns can be stored on recurrent collaterals in the cortex, and such memory can help substantially in the analysis of current sensory input. The simulation of a simplified network model demonstrates that a non-laminated patch of cortex must compromise between transmitting where information or retrieving what information. The simulation of a modified model including differentiation of a granular layer shows a modest but significant quantitative advantage, expressed as a less severe trade-off between what and where. The further connectivity differentiation between infra-granular and supra-granular pyramidal layers is shown to match the mix of what and where information optimal for their respective target structures.     

Changes in density of chromatin in the meristematic cells ofSinapis alba during transition to flowering

Summary Changes in the density of nuclear chromatin in the shoot apical meristem ofSinapis alba L. during floral transition (floral evocation) are described using Feulgen-stained 2 m thick semi-thin sections and scanning cytophotometric techniques. In both G1 and G2 nuclei the chromatin becomes less heterogeneous and less dense in evoked meristems compared to vegetative meristems. When chromatin is resolved into two fractions the dispersed fraction increases relative to the condensed fraction at evocation. This decondensation process occurs earlier in G1 than in G 2 nuclei. These chromatin changes are presumably closely related to the dramatic stimulation of biosynthetic activity and cell division during floral transition.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.     

Media from Rhabdomyosarcoma and Neuroblastoma Cell Cultures Stimulate in Vitro Aggregation and Fibrillization of Amyloid Beta-Protein

In vitro aggregation and fibrillization of synthetic amyloid beta-protein A 1–40 was assessed in the conditioned media from rhabdomyosarcoma (CRL 1598, HTB 82, HTB 153, CCL 136), adenocarcinoma (CCL 218), neuroblastoma (SY5Y), and COS cells cultured in the absence and presence of 10% heat-inactivated fetal bovine serum (FBS). The aggregation and formation of cross -pleated sheet structures in A was quantitated by Thioflavin T (ThT) fluorescence spectroscopy, while the morphology of A fibrils was examined in negative staining in the electronmicroscope (EM). In cultures supplemented with 10% FBS, the conditioned media from CRL 1598, HTB 82, CCL 218, and SY5Y cell cultures stimulated A aggregation in a time-dependent manner as compared to that of control (serum-containing medium that had not been exposed to cells). The order of stimulation was SY5Y > CRL 1598 HTB 82 > CCL 218, and the stimulation was higher in 2 week cultures than in 1 week cultures. Similar studies using media from HTB 153, CCL 136 and COS cell cultures showed no effect on A 1–40 aggregation. In serum-free cell cultures, only media from SY5Y and CRL 1598 could promote significant aggregation of A 1–40. Negative staining in EM revealed A fibril formation only with conditioned media from SY5Y and CRL 1598 cultured under serum free conditions; no A fibrils were noticed in media from cell cultures supplemented with 10% FBS. We propose that both the SY5Y neuroblastoma cell line and the CRL 1598 rhabdomyosarcoma cell line may serve as experimental models for in vitro studies of extracellular aggregation and fibrillization of A-protein in cell cultures, while rhabdomyosarcoma HTB 82 and adenocarcinoma CCL 218 may be models for study of A aggregation only.     

Die Hypokotylfarbe als Markierungsfaktor von Genomstufen der Zuckerrübe

Zusammenfassung In zunehmendem Maße werden anisoploideBeta-Rübensorten angebaut, deren zytologische Kontrolle zwecks Feststellung der Genomstufenprozentanteile recht arbeitszeitaufwendig ist. Übereinstimmend mit polnischen Autoren wurde festgestellt, daß die Hypokotylfarbe ein geeigneter Markierungsfaktor für die einzelnen Genomstufen darstellt. Kreuzt man tetraploide Pflanzen, die ein grünes Hypokotyl besitzen, mit diploiden Pflanzen, die ein rosa Hypokotyl aufweisen, so erhält man von dem tetraploiden Partner tetraploide grüne und triploide hellbraune, von dem diploiden Partner diploide rosa und triploide hellbraune Nachkommenschaften. Die in bezug auf die Hypokotylfarbe heterozygoten Pflanzen kann man demnach von den homozygot grünen und homozygot rosa Individuen unterscheiden. Die Kreuzung diploid grünxtetraploid rosa ist für diese Zwecke nicht brauchbar, da sich die triploiden Heterozygoten mit einem grünen und zwei rosa Allelen in der Hypokotylfarbe nicht deutlich von den homozygoten rosa Pflanzen abheben. Auf die Bedeutung dieser Markierungsmöglichkeit für bestimmte Forschungsprobleme, die Züchtung und die Saatgutkontrolle wird hingewiesen.     

Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Isoform-Specific Membrane Translocation of Protein Kinase C After Ischemic Preconditioning

Mild cerebral anoxic/ischemic/stress insults promote tolerance and thereby protect the brain from subsequent lethal anoxic/ischemic insults. We examined whether specific activation of PKC , , , or isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKC, , , and . PKC showed a significant translocation to the membrane fraction from 30 min to 4 h and PKC at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKC showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKC was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKC and PKC may be associated with IPC-induced tolerance in the rat hippocampus.