The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Molecular neurophysiology of taste in <Emphasis Type="Italic">Drosophila</Emphasis>

The recent identification of candidate receptor genes for sweet, umami and bitter taste in mammals has opened a door to elucidate the molecular and neuronal mechanisms of taste. Drosophila provides a suitable system to study the molecular, physiological and behavioral aspects of taste, as sophisticated molecular genetic techniques can be applied. A gene family for putative gustatory receptors has been found in the Drosophila genome. We discuss here current knowledge of the gustatory physiology of Drosophila. Taste cells in insects are primary sensory neurons whereupon each receptor neuron responds to either sugar, salt or water. We found that particular tarsal gustatory sensilla respond to bitter compounds. Electrophysiological studies indicate that gustatory sensilla on the labellum and tarsi are heterogeneous in terms of their taste sensitivity. Determination of the molecular bases for this heterogeneity could lead to an understanding of how the sensory information is processed in the brain and how this in turn is linked to behavior.Received 12 May 2003; received after revision 9 June 2003; accepted 13 June 2003     

Unique evolution of Bivalvia arginine kinases

The clams Pseudocardium, Solen, Corbicula and Ensis possess a unique form of arginine kinase (AK) with a molecular mass of 80 kDa and an unusual two-domain structure, a result of gene duplication and subsequent fusion. These AKs also lack two functionally important amino acid residues, Asp⁶² and Arg¹⁹³, which are strictly conserved in other 40-kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. However, these AKs show higher enzyme activity. The cDNA-derived amino acid sequences of 40-kDa AKs from the blood clam Scapharca broughtonii and the oyster Crassostrea gigas were determined. While Asp⁶² and Arg¹⁹³ are conserved in Scapharca AK, these two key residues are replaced by Asn and Lys, respectively, in Crassostrea AK. The native enzyme from Crassostrea and both of the recombinant enzymes show an enzyme activity similar to that of two-domain clam AKs and at least twofold higher than that of other molluskan AKs. Although the replacement of Asp⁶² or Arg¹⁹³ by Gly in normal AK causes a considerable decrease in V_max (6–15% of wild-type enzyme) and a two- to threefold increase in K_m for arginine, the same replacement in Scapharca AK had no pronounced effect on enzyme activity. Together with the observation that bivalve AKs are phylogenetically distinct from other molluskan AKs, these results suggest that bivalve AKs have undergone a unique molecular evolution; the characteristic stabilizing function of residues 62 and 193 has been lost and, consequently, the enzyme shows higher activity than normal.Received 14 October 2003; accepted 1 November 2003     

Acetyl-coenzyme A synthetase (AMP forming)

Acetyl-coenzyme A synthetase (AMP forming; Acs) is an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. The physiological role of this enzyme is to activate acetate to acetyl-coenzyme A (Ac-CoA). The importance of Acs has been recognized for decades, since it provides the cell the two-carbon metabolite used in many anabolic and energy generation processes. In the last decade researchers have learned how carefully the cell monitors the synthesis and activity of this enzyme. In eukaryotes and prokaryotes, complex regulatory systems control acs gene expression as a function carbon flux, with a second layer of regulation exerted posttranslationally by the NAD⁺/sirtuin-dependent protein acetylation/deacetylation system. Recent structural work provides snapshots of the dramatic conformational changes Acs undergoes during catalysis. Future work on the regulation of acs gene expression will expand our understanding of metabolic integration, while structure/function studies will reveal more details of the function of this splendid molecular machine.Received 4 December 2003; received after revision 2 March 2004; accepted 16 March 2004     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

Olfactory receptors

Olfaction is an ancient sensory system allowing an organism to detect chemicals in its environment. The first step in odor transduction is mediated by binding odorants to olfactory receptors (ORs) which belong to the heptahelical G-protein-coupled receptor (GPCR) superfamily. Mammalian ORs are disposed in clusters on virtually all chromosomes. They are encoded by the largest multigene family (1000 members) in the genome of mammals and Caenorhabditis elegans, whereas Drosophila contains only 60 genes. Each OR specifically recognizes a set of odorous molecules that share common molecular features. In mammals, signal transduces through the G-protein-dependent signal pathway in the olfactory sensory neurons that synapse ultimately in the glomeruli of the olfactory bulb, and is finally processed in higher brain structures. The expression of a given OR conditions neuron and glomerulus choices. To date, the processes which monitor OR expression and axon wiring have emerged but are not completely elucidated.Received 9 July 2003; reiceived after revision 3 Ocotober 2003; accepted 22 Ocotober 2003     

Genomic sources of regulatory variation in cis and in trans

Regulatory variation results from genetic changes with both cis and trans acting effects on gene expression. Here I describe the types of genetic variants that alter cis and trans regulation and discuss differences in the potential for cis and trans changes among different classes of genes. I argue that the molecular function of the protein encoded by each gene and how the gene is wired into the genomic regulatory network may influence its propensity for cis and trans regulatory changes.Received 15 February 2005; received after revision 12 April 2005; accepted 26 April 2005     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.Received 26 June 2003; received after revision 29 July 2003; accepted 25 August 2003     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

PcF protein from <Emphasis Type="Italic">Phytophthora cactorum</Emphasis> and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato

The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline- containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.Received 22 February 2003; received after revision 25 March 2003; accepted 14 April 2003     

Molecular basis of homocysteine toxicity in humans

Because of its similarity to the protein amino acid methionine, homocysteine (Hcy) can enter the protein biosynthetic apparatus. However, Hcy cannot complete the protein biosynthetic pathway and is edited by the conversion to Hcy-thiolactone, a reaction catalyzed by methionyl-transfer RNA synthetase in all organisms investigated, including human. Nitrosylation converts Hcy into a methionine analogue, S-nitroso-Hcy, which can substitute for methionine in protein synthesis in biological systems, including cultured human endothelial cells. In humans, Hcy-thiolactone modifies proteins posttranslationally by forming adducts in which Hcy is linked by amide bonds to -amino group of protein lysine residues (Hcy-N-Lys-protein). Levels of Hcy bound by amide or peptide linkages (Hcy-N-protein) in human plasma proteins are directly related to plasma total Hcy levels. Hcy-N-hemoglobin and Hcy-N-albumin constitute a major pool of Hcy in human blood, larger than total Hcy pool. Hcy-thiolactone and Hcy-thiolactone-hydrolyzing enzyme, a product of the PON1 gene, are present in human plasma. Modification with Hcy-thiolactone leads to protein damage and induces immune response. Autoantibodies that specifically recognize the Hcy-N-Lys-epitope on Hcy-thiolactone-modified proteins occur in humans. The ability of Hcy to interfere with protein biosynthesis, which causes protein damage, induces cell death and elicits immune response, is likely to contribute to the pathology of human disease.Received 30 May 2003; received after revision 21 July 2003; accepted 15 August 2003     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Hydroxylation of menthols and cineoles withm-chloroperbenzoic acid

Summary Reaction of menthols and cineoles withm-chloroperbenzoic acid afforded tertiary, secondary, and primary alcohols, some of which were natural products having potent plant growth regulatory activity or were mammlianm metabolies.We thank Nippon Terpene Co. Ltd for their generous gift of the compounds used in this work and Drs M. Kido and Y. Fukuyama, Otsuka Pharmaceutical Co. Ltd, for measurement of high resolution mass spectra. The present work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare.     

Lipid transport in microorganisms

Summary Microorganisms are useful model systems for the study of intracellular transport of lipids. Eukaryotic microorganisms, such as the yeastSaccharomyces cerevisiae, are similar to higher eukaryotes with respect to organelle structure and membrane assembly. Experiments in vivo showed that transport of phosphatidylcholine between yeast microsomes and mitochondria is energy independent; transfer of phosphatidylinositol to the plasma membrane and the flux of secretory vesicles take place by different mechanisms. Linkage of transfer and biosynthesis of phospholipids was demonstrated in the case of intramitochondrial phospholipid transfer. A yeast phosphatidylinositol/phosphatidylcholine transfer protein, which is essential for cell viability, was isolated and characterized. Another phospholipid transfer protein present in yeast cytosol, which has a different specificity, is currently under investigation. Transfer of phospholipids between cellular membranes was also demonstrated with prokaryotes. The cytoplasm and the periplasma of the gram-negative facultative photosynthetic bacteriumRhodopseudomonas sphaeroides contain phospholipid transfer proteins; these seem to be involved in the biosynthesis of prokaryotic membranes.     

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, <Emphasis Type="Italic">Calyptogena</Emphasis>

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33–42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei. Received 13 May 2003; received after revision 5 June 2003; accepted 12 June 2003     

Mögliche Beziehungen der optischen Aktivität zum Problem des Alterns

Summary The solubilities and other properties of compounds made by combination of optically active components, e.g. the solubilities of the salts made from the dextro-form of an acid and the dextro- or the laevo-form of a base are in some instancesextremely different. It is a consequence of these differences that in a living organism, containing optically active substance,the degree of optical purity of these substances is of outmost importance. The presence of undesired antipodes of any kind might fundamentally disturb the metabolism. It is also known that living organisms at least partly and continuously produce the optically active substances needed bysynthesis from optically inactive material.A kinetical and thermodynamical study of the conditions under which optically active substances can, with use of optically active catalysers, be synthetized from inactive material, shows that the degree of optical purity of the material synthetized is, even under the most favorable conditions, maximum in the beginning of the synthesis andis bound to decay when the synthesis is, chemically speaking,completed and if the synthetized material is left in contact with the catalyzer. Thus a racemization of the synthetized active material occurs in which the catalyzer which has originally produced this active substance actively accelerates the deterioration of its state of optical purity.The means are discussed by which the organism is able to delay this loss of optical purity; and it is shown that all poosible means to do so are indeed used by the organism. As these means can delay but never completely avoid the decay of optical purity, this decay, even if it be slow, i.e.an ageing of optical purity, is a necessity in the course of the life of a living organism. As a loss of optical purity must disturb the normal metabolism, this must, even if no other events occur, limit the individual life.Experimental evidence which would check the prediction of decay of optical purity with age is incomplete; in several instances, however, the experimental technique permits us to detect and to follow the occurrence of finite small amounts of undesired antipodes, e.g. of some amino acids in the proteins of living organisms.

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Vortrag, gehalten vor der Schweizerischen Gerontologischen Gesellschaft in Basel am 11. Dezember 1954.     

Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed <Emphasis Type="Italic">intracellular</Emphasis> proteinase inhibitor clades that protect cells from promiscuous proteolysis

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.Received 24 June 2003; received after revision 16 July 2003; accepted 5 August 2003     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.     

Two mechanisms in the biological clock ofPieris brassicae L.: an oscillator for diapause induction; an hour-glass for diapause termination

Summary Resonance experiments for photoperiodic termination of pupal diapause demonstrated thatPieris brassicae uses a night-measuring hour-glass mechanism. In previous work the same resonance technique for diapause induction revealed that photoperiodic time-measurement is a function of the circadian system. For the first time in a living organism it has been shown that the biological clock operates by means of an oscillator for photoperiodic onset of a phenomenon and according to an hour-glass system for photoperiodic termination.