The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro6 with Leu and Asp5 with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional 1H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L6-Tat(1–9) and I5-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.

The repertoire of secondary metabolism (involving the production of compounds not essential for growth) in the plant kingdom is enormous, but the genetic and functional basis for this diversity is hard to analyse as many of the biosynthetic enzymes are unknown. We have now identified a key enzyme in the ornamental plant Gerbera hybrida (Asteraceae) that participates in the biosynthesis of compounds that contribute to insect and pathogen resistance. Plants transformed with an antisense construct of gchs2, a complementary DNA encoding a previously unknown function1,2, completely lack the pyrone derivatives gerberin and parasorboside. The recombinant plant protein catalyses the principal reaction in the biosynthesis of these derivatives: GCHS2 is a polyketide synthase that uses acetyl-CoA and two condensation reactions with malonyl-CoA to form the pyrone backbone of thenatural products. The enzyme also accepts benzoyl-CoA to synthesize the backbone of substances that have become of interest as inhibitors of the HIV-1 protease3,4,5. GCHS2 is related to chalcone synthase (CHS) and its properties define a new class of function in the protein superfamily. It appears that CHS-related enzymes are involved in the biosynthesis of a much larger range of plant products than was previously realized.

Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 μM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response. We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes.

From a cDNA library generated from mRNA of white leaf tissues of the ribosome‐deficient mutant ‘albostrians' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine‐5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313–319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross‐reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477–486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.

The biosynthesis of complex alkaloids in plants involves enzymes that, due to high substrate specificity, appear to have evolved solely for a role in secondary metabolism. At least one class of these enzymes, the oxidoreductases, catalyze transformations that are in some cases difficult to chemically mimick with an equivalent stereo‐ or regiospecificity and yield. Oxidoreductases are frequently catalyzing reactions that result in the formation of parent ring systems, thereby determining the class of alkaloid that a plant will produce. The oxidoreductases of alkaloid formation are a potential target for the biotechnological exploitation of medicinal plants in that they could be used for biomimetic syntheses of alkaloids. Analyzing the molecular genetics of alkaloid biosynthetic oxidations is requisite to eventual commercial application of these enzymes. To this end, a wealth of knowledge has been gained on the biochemistry of select monoterpenoid indole and isoquinoline biosynthetic pathways, and in recent years this has been complemented by molecular genetic analyses. As the nucleotide sequences of the oxidases of alkaloid synthesis become known, consensus sequences specific to select classes of enzymes can be identified. These consensus sequences will potentially facilitate the direct cloning of alkaloid biosynthetic genes without the need to purify the native enzyme for partial amino acid sequence determination or for antibody production prior to cDNA isolation. The current state of our knowledge of the biochemistry and molecular genetics of oxidases involved in alkaloid biosynthesis is reviewed herein.

On the basis of a model of the pharmacophore conformations of agonist of the δ-opioid receptor the corresponding δ-antagonist conformations were determined by means of force field calculations. The results explain the unusual behavior of several cyclic β-casomorphin analogues on the molecular level. Thus, for instance, the model helps to understand why Tyr-c[D-Orn-2-Nal-D-Pro-Gly] is a mixed μ-agonist and δ-antagonist. Furthermore, the model is consistent with low energy conformations of other δ-antagonists such as Tyr-Tic-Phe, Tyr-Tic-Phe-Phe, naltrindole and BNTX. The occupation of a special spatial area by bulky groups close to the protonated N-terminus of opioid peptides is assumed to be highly critical for the switch from agonist to antagonist behavior.

In seedlings of Arabidopsis thaliana the thionin gene Thi2.1 is inducible by methyl jasmonate, wounding, silver nitrate, coronatine, and sorbitol. We have used a biochemical and genetic approach to test the signal transduction of these different inducers. Both exogenously applied jasmonates and jasmonates produced endogenously upon stress induction, lead to GUS expression in a Thi2.1 promoter-uidA transgenic line. No GUS expression was observed in a coi1 mutant background which lacks jasmonate perception whereas methyl jasmonate and coronatine but not the other inducers were able to overcome the block in jasmonic acid production in a fad3-2 fad7-2 fad8 mutant background. Our results show conclusively that all these inducers regulate Thi2-1 gene expression via the octadecanoid pathway.

Monoclonal antibodies raised against a phylogenetically conserved peptide from the C‐terminal domain of γ‐tubulin molecule were used for immunofluorescence detection of γ‐tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos‐methyl, oryzalin and colchicine, γ‐tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophos‐methyl treatment, γ‐tubulin was localized with the re‐growing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ‐tubulin co‐localized with α‐tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ‐tubulin might be directly or indirectly involved in modulation and/or stabilization of kinetochore–microtubule interactions.

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An account of the most commonly used reagents for the introduction of the benzeneselenyl (phenyl seleno) group is given. The review focuses on the various methods of its introduction as auxiliary, modifying or protective entity, and its subsequent removal, thereby often promoting other reactions as cyclizations or double bond formation. Less emphasis is laid on reactions of the phenylselenenylated intermediates with the PhSe‐group left intact utilizing its stabilizing properties on charged intermediates, on reagents with a modified phenyl group, e.g. chiral derivatives, or on reactions not involving intermediate CSe‐bond formation.