X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Cellular fatty acid composition of six fastidious,gramnegative, xylem-limited bacteria from plants

Composition of cellular fatty acids was determined for strains of fastidious, Gramnegative, xylem-limited bacteria causing or associated with Pierce's disease of grapevine, phony disease of peach, plum leaf scorch, stunt of ragweed, elm leaf scorch, and periwinkle wilt. The most abundant fatty acids were straight-chain 150, 160, 170, and 180, unsaturated 161, 181, and unsaturated 17-and 19=carbon homologs. Minor fatty acids included straightchain 120, 140, 190, and 200; an unsaturated 15-carbon homolog; hydroxy-substituted 2-OH 120, 3-OH 120, and 3-OH 140; and branched chain iso-140 and iso-200. Cyclopropane acids were not detected. Physiological age had no effect on fatty acid composition. Class analysis of data indicated relative uniformity within the group. Saturated even-carbon chains comprised 31%–42%, unsaturated acids 41%–52%, saturated odd-carbon chains 10%–18%, hydroxysubstituted chains 2%–7%, and branched-chains 1%–4% of total fatty acids. The ratio of saturated-unsaturated acids ranged from 0.8 to 1.2.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

Transfection of epithelioma papulosum cyprini (EPC) carp cells

The variables involved in the transfection of epithelioma papulosum cyprini (EPC) cells (a representative carp fish cell line) with the genes for -galactosidase from E. coli, for luciferases from firefly or renilla, for the G protein of viral haemorrhagic septicemia virus or for green fluorescent protein under the cytomegalovirus, the SV40 or the T7 polymerase promoters have been studied. Fugene was selected among 10 transfection different reagents because it is simpler to use and it induced maximum efficiences of transfection of 37% equivalent to 10–15 ng -galactosidase per 500000 EPC cells.     

Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea

Embryo suspensor masses (ESMs) were induced by culture of isolated mature zygotic embryos of balsam fir [Abies balsamea (Mill.)] on media containing 10 M cytokinin [6-(--dimethylallylamino)purine (2iP), 6-benzyladenine (BA), or thidiazuron (TDZ)]. Once induced, ESMs proliferated on media containing 2iP, BA or TDZ (10 M) or on 4.5 M BA in combination with 10 M naphthyl-1-acetic acid. When ESMs were transferred to media containing 5–80 M abscisic acid, cotyledonary-stage embryos were formed. Embryos were readily germinated on medium lacking growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - ESM embryo-suspensor mass - 2iP 6-(--dimethylallylamino)purine - NAA naphthyl-1-acetic acid - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl urea (thidiazuron)     

Methodological problems in evolutionary biology III. Selection and levels of organization

Apparently factual disagreement on the level(s) at which selection operates often results from different interpretations of the term selection. Attempts to resolve terminological problems must come to grips with a dilemma: a narrow interpretation of selection may lead to a restricted view on evolution; a broader, less precise, definition may wrongly suggest that selection is the centre of a unified, integrated theory of evolution. Different concepts of selection, therefore, should carefully be kept apart.     

Reversal of a reflex to a single motoneuron in the stick insect Çarausius morosus

In inactive stick insects ramp-wise stretching of the femoral chordotonal organ excites the slow extensor tibiae motoneuron. In active animals the same stimulus decreases the firing rate of this motoneuron. The time-course of increased and decreased activity of this motoneuron can be seen with triangular stimulation.Supported by the Deutsche Forschungsgemeinschaft     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

Localization of glycosidases with naphthyl substrates

Synopsis Unsubstituted naphthyl substrates were found to be superior to substituted naphthyl, indolyl and hydroxyquinoline substrates for the histochemical demonstration of -mannosidase, -galactosidase, hetero--glycosidase, glucoamylase and sucrase-isomaltase, equivalent for -N-acetylglucosaminidase and lactase--glucosidase, and inferior for -glucuronidase and acid -galactosidase. Aldehyde fixation is necessary for the localization of lysosomal glycosidases with naphthyl substrates.i-naphthyl substrates are suitable for the detection of acid glycosidases in lysosomes and hetero--glycosidase in the cytoplasm of animal cells, and 2-naphthyl substrates can be employed for the demonstration of microvillous glycosidases and for the evaluation of the total activity of soluble glycosidases with semipermeable membranes. When naphthyl substrates are used coupling should be carried out simultaneously and hexazotized pararosaniline is the coupling reagent of choice.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Isolation of cobamides from Methanothrix soehngenii: 5-methylbenzimidazole as the α-ligand of the predominant cobamide

Methanothrix soehngenii was found to contain five different cobamides when grown on vitamin B₁₂ supplemented as well as vitamin B₁₂ free media. In both cases, it was shown by HPLC-chromatography and UV/VIS spectroscopy, that -5-methylbenzimidazolyl--cyanocobamide was the predominant cobamide, accounting for 27% and 23%, respectively, of the total corrinoid content. Vitamin B₁₂ and -5-hydroxybenzimidazolyl--cyanocobamide could also be detected in both cell batches in varying amounts. Cells grown on vitamin B₁₂ free medium contained significantly more baseless cobamides, indicating biosynthesis of cobamides.Abbreviations (5-MeBza)CNCba -5-methylbenzimidazolyl--cyanocobamide - (5-HOBza)CNCBa -5-hydroxybenzimidazolyl--cyanocobamide (factor III) - (5-MeOBza)CNCba -5-methoxybenzimidazolyl--cyanocobamide (factor III_m) - (Bza)CNCba -benzimidazolyl--cyanocobamide     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Rna-mediated regulation of Receptor-Ck gene in human platelets

The study addressed to understand the regulation of Receptor-C_k gene atthe translational level revealed that exogenous cholesterol has the inherentcapacity to regulate the endogenous synthesis of Receptor-C_k by initiatingintracellular targeting of the Receptor-C_k to the mRNP pool within humanplatelets and this effect could be reversed when the platelets wereincubated with cholesterol coupled with either dB cAMP or dB cGMP. Basedupon these observations, we propose that Receptor-C_k initiated signalling,which involves second messengers like PA, cAMP and cGMP, may be responsiblefor the autoregulation of Receptor-C_k gene expression at the translationallevel.     

Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

Die Feinstruktur der Zirbeldrüse normaler,trächtiger und experimentell beeinflußter Meerschweinchen

Zusammenfassung In der Meerschweinchenzirbeldrüse lassen sich elektronenmikroskopisch helle und dunkle Pinealzellen sowie einzelne Gliazellen nachweisen. In den bei weitem überwiegenden hellen Pinealzellen zeichnet sich ein Teil der vesicle-crowned rodlets (VCR) durch lokale Auftreibungen aus. Von VCR deutlich abzugrenzen sind die vesicle-crowned balls (VCB). Erstmalig beschrieben wird das Vorkommen von sog. Zylindern, die als Vorstufen von VCB aufgefaßt werden. In den relativ seltenen dunklen Pinealzellen, die sich durch chromatinreiche Kerne und elektronendichtes Zytoplasma auszeichnen, sind Vesikel, VCR, VCB und Zylinder seltener als in hellen Pinealzellen. Die reichlich vorhandenen marklosen Nervenfasern finden sich vor allem in perivasculären Räumen, seltener im Parenchym. Synapsen zwischen Nerven und Pinealzellen wurden nicht beobachtet. In den Zirbeldrüsen trächtiger Meerschweinchen zeichnen sich in der 2. Hälfte der Tragzeit die hellen Pinealzellen durch stärkere Lappung der Kerne, gehäuftes Auftreten von laktiven Zonen, Vermehrung von Mitochondrien, glattem ER, agranulären Vesikeln, VCR, VCB und Zylindern aus. Die dunklen Pinealzellen nehmen während der Tragzeit an Zahl zu. Post partum bilden sich diese Veränderungen innerhalb einer Woche zurück. Längerer Aufenthalt der Tiere in Dunkelheit führt zu einer Aktivierung der hellen Pinealzellen mit auffallender Vermehrung der VCR und zu einer Zunahme der dunklen Zellen. Unter Dauerbelichtung kommt es in den hellen Zellen zu einer Abnahme fast aller Zellorganellen und zu einer starken Vermehrung der VCR, die nach 70 Tagen auch Formveränderungen aufweisen. Nach Reserpinbehandlung beobachtet man eine Verminderung und degenerative Veränderungen der VCR. Es wird diskutiert, daß die VCR als prae- bzw. postsynaptische Strukturen der Erregungsübertragung von Nerven zu Pinealzellen bzw. von Pinealzellen untereinander dienen könnten.

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The fine structure of the pineal gland of normal, pregnant and experimentally affected guinea-pigs

Summary By means of electron microscopy light and dark pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the light pinealocyte. the most frequent cell type, some vesicle-crowned rodlets (VCR) show circumscribed thickenings. From these structures vesicle-crowned balls (VCB) have to be clearly distinguished. Furthermore cylinders occur, which, it is suggested, are precursors of VCB. Dark pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and cylinders than light pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The light cells show many nuclear indentations and an increase of active zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and cylinders respectively. The dark cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the light cells accompanied by an increase of the VCR and to an increase in number of the dark cells. Under constant illumination the light cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Untersuchung unter Leitung von Univ.-Doz. Dr. L. Vollrath.     

The adaptive significance of cultural behavior: Comments and reply

Summary Fundamentally, theoretically, there is only one process underlying genetic and cultural evolution: natural selection. Organism fitness-enhancement (adaptive significance) is one of its practical mechanisms; group formation and maintenance is another, often but not always through fitness-enhancement; and need-fulfillment is still another. If Durham can accept that formulation, and switch from organism-thinking to instruction-thinking (Cloak, 1975: 178), he will free himself from two handicaps: First, he can forget his worries about reductionism and determinism (1976a: 100, 101). Under this general theory of natural selection, cultural evolutionis biological evolution, continued by other (nongenetic) means. Second, he will spare himself the appearance of anthropomorphism, mentalism, and wishy-washiness attendant on his discussion of kinds of significance, other than adaptive significance, of cultural behaviors (1976a: 102–106, 115).     

Glycoconjugates of the Rat Ciliary Body Epithelium: A Lectin Histochemical and Protein Blotting Study

The present study sought to identify and partially characterize the glycoconjugates specific to the double-layered ciliary body epithelium of the rat eye by lectin histochemistry and lectin blottings. Hydrated paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with a panel of 21 different biotinylated lectins, followed by streptavidin-peroxidase and the glucose oxidase-diaminobenzidine-nickel staining procedure. The results of lectin histochemistry revealed that the inner epithelial layer was rich in GlcNAc(1,4)GlcNAc, -Gal, Gal(1,3)GalNAc, GalNAc(1,3)GalNAc/Gal, GalNAc(1,6)Gal, Fuc(1,2)Gal(1,4)GlcNAc and Gal(1,4)GlcNAc(1,2)Man(1,6) sugar residues as shown by its positive reactivities with S-WGA, PWA, DSA, GS-I-B4, PNA, DBA, SBA, WFA, UEA-I, LTA and PHA-E. The reactivities of GS-I-B4, PNA, DBA and SBA were restricted to the inner layer at the tips of the ciliary processes. On the other hand, the outer epithelial layer was stained evenly by DSA and Jacalin, and partly by MAA, showing that this epithelial layer was rich in GlcNAc(1,4)GlcNAc, Gal(1,3)GalNAc and NeuAc(2,3)Gal disaccharides. These lectin binding patterns of the ciliary body epithelium suggest a topographical and functional difference in this double cell-layered epithelium. Their possible roles in the secretion of aqueous humour and production of ciliary zonule are discussed. Some identified lectin markers specific to these two cell layers may be useful for further experimental studies. Glycoproteins extracted from the dissected ciliary body were separated by SDS-PAGE electrophoresis and analyzed by protein blottings with 8 different lectins. The results showed that at least 10 major membrane-bound glycoproteins, with molecular weights ranging from 30 to 150kD, rich in -GlcNAc, -Gal, /-GalNAc and NeuAc(2,6)Gal residues, were present in the microsomal fraction.