1,5-diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases.

1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.     

Inclusion volume solid-phase synthesis

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin beads [inclusion volume synthesis]. This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.     

Remote sensing of chlorophyll in Lake Kinneret using highspectral-resolution radiometer and Landsat TM: spectral features of reflectance and algorithm development

High-resolution reflectance spectra in the range of 400–850nm were obtained from Lake Kinneret during a period when densepopulations of the dinoflagellate Peridinium gatunense dominatedthe phytoplankton. Chlorophyll (Chl) concentrations ranged from5.1 to 185 mg m^–3 and from 2.4 to 187.5 mg m^–3 inthe samples of two independent experiments. The most prominentfeatures of the reflectance spectra were: (i) a wide minimumfrom 400 to 500 nm; (ii) a maximum at 550–570 nm, whichdid not surpass 3% in samples with high Chl concentration (>20mgm^–3), indicating a strong absorption by pigments in thegreen range of the spectrum; (iii) a minimum at 676 nm; thiswas {small tilde}1% and was almost insensitive to variationin Chl concentration >10 mg m^–3; (iv) a maximum reflectanceshowed near 700 nm; its magnitude and position were highly dependenton chlorophyll concentration. High-spectral-resolution datawere used as a guideline for selection of the most suitablespectral bands for chlorophyll remote sensing. Models were devised,based on the calculation of the integrated area above the baselinefrom 670 to 850 nm and the reflectance maximal height withinthis range. Some algorithms already used m previous studieswere tested and showed a plausible degree of accuracy when appliedto the current data base. However, novel models devised in thisstudy improved substantially the accuracy of Chl estimationby remotely sensed data, by reducing the estimation error from>11 to 6.5 mg m^–3 Those models were validated by anindependent data set where Chl concentration ranged over twoorders of magnitude. The use of three relatively narrow spectralbands was sufficient for Chl mapping in Lake Kinneret. Therefore,a relatively simple sensor, measuring only a few bands willbe employed in future applications for Chl monitoring in inlandwaters. Radiometric data were also used to simulate radiancesin the channels of TM Landsat and to find the algorithm forChl assessment. The ratio of channel 4 to channel 3 was usedand enabled Chl estimation with an error of <15mg m^–3This algorithm was employed to map Chl in the entire area ofLake Kinneret with 10 gradations.     

SNARE Proteins-Why So Many,Why So Few?

Abstract: Both trafficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a small number of proteins, i.e., the exocytic core complex. In nerve terminals, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP-25 at the plasma membrane. In this survey, different secretory systems that depend on the exocytic core proteins are considered. The possibility that specificity in membrane recognition and fusion is achieved by the numerous variants of proteins of the exocytic core is discussed. Variability of the core complex proteins is determined by the complexity of gene families, isoform-specific localization, and posttranslational modifications. Basic biochemical properties depend on specific isoforms, and the possible protein-protein interactions are determined, in turn, by the compatibility of different isoforms. A correlation between specific variants and distinct biochemical or cellular properties is shown. The outcome of this survey is that heterogeneity in secretion may be dictated by the large number of possible combinations of variants of only a few proteins.     

Intervening sequences in the ribosomal RNA genes of Ascaris lumbricoides: DNA sequences at junctions and genomic organization

An rDNA size class in the genome of the nematode Ascaris lumbricoides is described which is interrupted by a 4.5-kb long intervening sequence located in the 26S coding region. This molecular form occurs in approximately 15 copies per haploid genome and amounts to approximately 5% of the total nuclear rDNA. Intervening sequences are present only in the 8.8-kb rDNA, but not in the 8.4-kb rDNA repeating units of A. lumbricoides. Cloning of the interrupted rDNA units revealed, in addition to the main 4.5-kb insertion, shorter intervening sequences of 4-kb and 119-bp length. Both shorter rDNA forms are present in the single copy range of the haploid genome. Sequence analyses of the intervening sequence/rDNA junctions show an identical right-hand junction for all of the three different rDNA forms. The two shorter intervening sequences are a coterminal subset of the right-hand end of the main 4.5-kb insertion, whereas all three insertions have a different left-hand junction with the coding region of rDNA. Each intervening sequence is flanked by a short direct repeat of variable length, being only once present in the uninterrupted rDNA. The intervening sequences of A. lumbricoides show striking similarity to the organization of type I insertion family in dipteran flies, even though they are inserted at different positions in the 26S coding region. Additional rDNA intervening sequences may be present outside of the rDNA cluster, but in not more than 15-20 homologous copies per haploid genome.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

Role of IAA conjugates in inducing ethylene production by tobacco leaf discs

Indole-3-acetic acid (IAA) labeled in its carboxyl group was metabolized by tobacco leaf discs (Nicotiana tabacum L. cv. Xanthi) into three metabolites, two of which were preliminarily characterized as a peptide and an ester-conjugated IAA. Reapplication of each of the three metabolites (at 10 M) resulted in a marked stimulation of ethylene production and decarboxylation by the leaf discs. Similarly, these three IAA metab olites could induce elongation of wheat coleoptile segments, which was accompanied by decarboxylation. Both the exogenously supplied esteric and peptidic IAA conjugates were converted by the leaf discs into the same metabolites as free IAA. (1-¹⁴C)IAA, applied to an isolated epidermis tissue, was completely metabolized to the esteric and peptidic IAA conjugates. This epidermis tissue showed much higher ethylene production rates and lower decarboxylation rates than did the whole leaf disc.The results suggest that the participation of IAA conjugates in the regulation of various physiological processes depends on the release of free IAA, which is obtained by enzymatic hydrolysis of the conjugates in the tissue. The present study demonstrates biological activity of endogenous IAA conjugates that were synthesized by tobacco leaf discs in response to exogenously supplied IAA.Contribution No. 952-E, 1983 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.     

Specific localization and quantification of biotin transport components in yeast by use of a biotin-conjugated,impermeant, electron-dense label

Summary Two approaches are described for the localization and quantification of biotin transport components in yeast cells. One approach is based on tracing the fate of a radioactive affinity label for the biotin transport system, [¹⁴C]-biotinyl-p-nitrophenyl ester (pBNP), through various stages of subcellular fractionations. A complementary method involves the use of a biotinderivatized, impermeant, electron-dense, affinity-cytochemical label (ferritin-biotin conjugates) for subsequent visualization by electron microscopy. Values of approximately 8,000 and 4,000 sites/cell, respectively, were achieved by the two methods. Complicating factors, future perspectives and the relevance of the two methods to the isolation of transport components are discussed.