Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes [9]. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.     

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3 end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)_n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, and estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1–4 million years ago.     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

Filtration rates of mosquito larvae in suspensions of latex microspheres and yeast cells

Filtration rates of fourth instars of Aedes aegypti L., Anopheles albimanus Wiedemann, Anopheles quadrimaculatus Say and Culex quinquefasciatus Say (Diptera: Culicidae) were determined by quantifying removal rates of suspended latex microspheres or yeast cells. Average filtration rates were 33–34 l/larva/h (An. quadrimaculatus), 49–55 (An. albimanus), 490–590 (C. quinquefasciatus) or 590–690 l/larva/h (Ae. aegypti) for larvae exposed to latex beads suspended in phagostimulant yeast extract solutions. In suspensions of yeast cells, filtration rates of Ae. aegypti and C. quinquefasciatus were not significantly different from filtration rates in latex bead suspensions. Larval density, ranging from 0.3 to 2.4 individuals/ml in tests with Ae. aegypti and C. quinquefasciatus and up to 4.8 larvae/ml in tests with Anopheles, did not influence filtration rates.

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Zusammenfassung Die Filtrierraten von Viertlarven der Stechmückenarten Aedes aegypti, Anopheles albimanus, Anopheles quadrimaculatus und Culex quinquefasciatus wurden in Laborversuchen bestimmt. Dabei wurde die Filtrierrate definiert als ein Wasservolumen, welches pro Stunde von einer Larve von den Testpartikeln (Hefezellen oder Latexkugeln mit einem Durchmesser von 2 m) befreit wurde. Nach Exposition der Larven in Dichten zwischen 0.15 und 2.4 Larven/ml (Ae. aegypti und C. quinquefasciatus), oder zwischen 0.6 und 4.8 Larven/ml (Anopheles) wurde der Partikelgehalt der Suspensionen in einem elektronischen Partikelzähler bestimmt. Die Filtrierraten wurden über die Verringerung der Partikeldichte mit zunehmender Larvendichte entsprechend einer in der Literatur angegebenen Formel berechnet.Suspensionen von Hefezellen wurden von Ae. aegypti Larven mit einer Leistung von 680±220 l/Larve/h gefiltert. Bei Larven von C. quinquefasciatus wurden Filtrierraten von 600±120 l/Larve/h gemessen. Die Filtrierraten beider Arten waren unabhängig von der Larvendichte. In Suspensionen von Latexpartikeln (zur Phagostimulation der Larven wurden diese Partikel in Lösungen von Hefeextrakt angeboten) wurden die folgenden Filtrierraten festgestellt: An quadrimaculatus: 33–34, An. albimanus: 49–55, C. quinquefasciatus: 490–590, und Ae. aegypti: 560–690 l/Larve/h. Die Larvendichte hatte auch hier keinen Einfluss auf die Filtrierrate. Hefezellen und Latexpartikel wurden von Ae. aegypti und C. quinquefasciatus mit statistisch nicht signifikant verschiedenen Filtrierraten aufgenommen. Die Filtrierraten der Anopheles Larven waren um mehr als eine Zehnerpotenz kleiner als die Filtrierraten von Culex und Aedes, und auch untereinander signifikant verschieden. Der Einfluss der Filtrierraktivität von Stechmückenlarven auf das Seston der Brutgewässer wird diskutiert.

     

The refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.     

1-Methylguanine and 7-methylguanine increase cell agglutinability

Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10^-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10^-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.