Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Influence of temperature on energetics of hydrogen metabolism in homoacetogenic,methanogenic, and other anaerobic bacteria

Hydrogen consumption by various thermophilic, mesophilic and/or psychrotrophic homoacetogens and methanogens was measured at temperatures between 4 and 80°C. Within the tolerated temperature range H₂ was consumed until a final H₂ threshold partial pressure was reached. H₂ thresholds generally decreased with temperature, parallel to the values calculated from the thermodynamics prevailing under culture conditions, i.e. the Gibbs free energy (G) of H₂ oxidation corrected for temperature by both the free-energy form of the Nernst equation and the Van't Hoff equation. The difference between the observed and the calculated H₂ partial pressures gives the minimum energy required for H₂ utilization being about-5 to-6 kJ/mol H₂ for the homoacetogenes and-9 to-12 kJ/mol H₂ for methanogens. The temperature dependence of the standard Gibbs free energy (G⁰) as described by the Van't Hoff equation apparently became the more important for thermodynamics as well as H₂ thresholds the more the temperature deviated from standard conditions (i.e. 25°C). Correction factors for calculation of temperature-corrected G _infT ^sup0 are presented for various H₂-producing and H₂-consuming reactions.     

The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

A theoretical investigation into the lipid interactions of m‐calpain

The protease, mcalpain, has been implicated in a number of pathological conditions. The enzyme is a calciumdependent heterodimer whose activity appears to be modulated by membrane interaction involving a segment, TAMRIL, located in domain V of the protein's small subunit. Based on a sequence analysis of mcalpain, using DWIH and hydrophobic moment plot based methodologies, we have shown that this segment may contribute to a lipid interactive, oblique orientated, helical region. Our results could form a basis for future studies on the postulated lipid modulation of mcalpain activity.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Polymerization of the Actin Component of the Cytoskeleton and Formation of the Geometry of a Cultured Nerve Cell (a Model Study)

Using mathematical modeling, we studied the biophysical aspects of the growth of the cell membrane and the growth of the actin network of the cytoskeleton of a neuron cultured on the rigid sublayer and the correlation between these processes. To describe the dynamics of the growth of the cytoskeleton limited by the cell membrane, we used the model of the thermal ratchet. Using the approaches of theoretical biophysics, we obtained a simple biophysical criterion that governs the selection of an alternative scenario of the formation of the cell, either growth of a single neurite or growth of a number of neurites. This criterion depends on the value of the adhesion between the cell and the substrate, the dimension of the actin monomer, and the thermal energy determining the frequency of thermal fluctuations of the cell membrane.     

“Gomori-positive” neurosecretion in the rat after deafferentation of the medial basal hypothalamus

A portion of the "Gomori-positive" peptidergic neurosecretory (NS) cells in the paraventricular and especially in the supraoptic and postoptic nuclei degenerate three weeks after deafferentation of the medial basal hypothalamus. Most of the remaining NS cells show signs of high activity. Regenerating NS fibres form "muffs" around the blood vessels laterally from the lesion; some of them enter the "isolated" area or persist there if a thin layer of the brain tissue is left somewhere untouched under the basal end of the cut. The regenerating NS fibres are also found outside the nervous tissue: within the scar tissue, in the proliferating connective tissue of the brain sheet below the basal end of the cut and in the mantel plexus area. The NS fibres make close contact with blood vessels invading or penetrating the vascular wall. It is suggested that peptide neurohormones discharged from the "Gomori-positive" NS terminals enter the general blood circulation as well as the portal blood at the site of these newly formed axovasal contacts. It is supposed that under these conditions monoaminergic terminals do not discharge monoamines because no stimulation of monoamine-producing NS cells occurs with deafferentation.     

Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts

Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Apical and lateral shoot apex-specific expression is conferred by promoter of the seed storage protein β-phaseolin gene

A previous analysis with deletion mutants of the native -phaseolin gene demonstrated that removal of a negative element 5 upstream of–107 permitted phaseolin expression in stem cortex and secondary root (Burowet al., 1992). Here we employed the -glucuronidase (GUS) reporter gene to visualize, by histochemical staining, the cell type-specificity of phaseolin expression in stem and root, and to understand further the spatial control of the -phaseolin gene. The 782 bp 5 upstream promoter and its deletion mutants were fused to the GUS gene, and these chimaeric genes were used to transform tobacco. Histochemical staining for GUS activity demonstrated that phaseolin promoters truncated downstream of –227 conferred cell-type specific expression in internal/external phloem and protoxylem of mature stem. Surprisingly, GUS staining was prominent in both apical and lateral shoot apices of plants that contain the full-length –782 promoter and mutant promoters deleted up to –64. GUS expression was extended to all cell types of shoot tips, including epidermis, cortex, vasculature, procambium and pith. Expression in vasculature of petioles was limited to plants with promoters truncated to –106 and –64. The current results are in agreement with our previous findings with the native phaseolin gene: that the major positive element (–295/–228) is sufficient for seed-specific late-temporal expression of the phaseolin gene. We conclude that the 5 upstream sequence of the -phaseolin gene directs spatially- and temporally-controlled gene expression in developing seeds during the reproductive phase, but also confers expression in shoot apices during the vegetative phase of plant development.     

DNA polymorphisms in the ψβ1- and β-globin gene regions in asian macaques

DNA polymorphisms in the ₁--globin gene region in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several restriction endonucleases and the human ₁, IVS2, and IVS2 probes. TheBamHI site 3 to the -globin gene was polymorphic inM. fuscata andM. mulatta, while the HincII site and the EcoRI site in the ₁-globin gene region was highly polymorphic inM. fuscata andM. mulatta, respectively. These polymorphic sites also seem to be present in other Asian macaques. The present study of the polymorphism at theBamHI site 3 to the -globin gene in Asian macaques supports, at the nuclear DNA level, the idea that thefascicularis group includingM. fuscata, M. mulatta, M. cyclopis, andM. fascicularis is different from other Asian macaque groups.This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.