The structure of antidesmone, an alkaloid from Antidesma membranaceum Müll. Arg. and A. venosum E. Mey. (Euphorbiaceae), was revised to be (S)-4,8-dioxo-3-methoxy-2-methyl-5-n-octyl-1,4,5,6,7,8-hexahydroquinoline [(S)-2], not the isoquinoline derivative 1, as assumed previously. The revision was initiated by biosynthetic feeding experiments of one of our groups.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called “Nonconventional Yeasts.” The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the “cell factory” concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Transient influx of Ca2+ constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae–derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca2+]cyt), which peaked at ∼1 μM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca2+]cyt signature was achieved by elicitor concentrations sufficient to stimulate Ca2+ influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca2+]cyt but not the rapidly induced [Ca2+]cyt transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca2+ influx across the plasma membrane or of Ca2+ release from internal stores suggests that the elicitor-induced sustained increase of [Ca2+]cyt predominantly results from the influx of extracellular Ca2+. Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca2+]cyt, and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca2+]cyt is causally involved in signaling the activation of pathogen defense in parsley.

The isolation of five known phenanthrenes and a mixture phytosterols from roots of Eulophia petersii is reported.

Phytochemical investigations of Aloe sabaea afforded a new chlorinated amide, N-4′-chlorobutylbutyramide, and the toxic piperidine alkaloids coniine, γ-coniceine and the quarternary N,N-dimethylconiine. This is the first report of the occurrence of a chlorinated compound in the Aloeaceae family.

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that γ-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of γ-tubulin in nuclei increased during the G2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. γ-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear γ-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of γ-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

From the acetone extract of the North American toadstool Lepiota americana 2-aminophenoxazin-3-one (1) and a novel amino-1,4-benzoquinone derivative, lepiotaquinone (2), were isolated. The structure of 2 was confirmed by its preparation from 2-aminophenol and amino-1,4-benzoquinone.

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2′:3′-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970–976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2′:3′-cyclic NMP to 3′-NMP and the 3′:5′-cyclic isomers to a mixture of 3′-NMP and 5′-NMP. Specific activities of the enzyme are 2-fold higher for 2′:3′-cyclic NMP than for 3′:5′-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared −Pi culture medium as a source of 3′-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.

Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1–9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1–9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp2-Tat(1–9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1–9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1–9) induces the production of the immunosuppressive cytokine transforming growth factor-β1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.