Light-driven amino acid uptake in Streptococcus cremoris or Clostridium acetobutylicum membrane vesicles fused with liposomes containing bacterial reaction centers.

Reaction centers of the phototrophic bacterium Rhodopseudomonas palustris were introduced as proton motive force-generating systems in membrane vesicles of two anaerobic bacteria. Liposomes containing reaction center-light-harvesting complex I pigment protein complexes were fused with membrane vesicles of Streptococcus cremoris or Clostridium acetobutylicum by freeze-thawing and sonication. Illumination of these fused membranes resulted in the generation of a proton motive force of approximately -110 mV. The magnitude of the proton motive force in these membranes could be varied by changing the light intensity. As a result of this proton motive force, amino acid transport into the fused membranes could be observed. The initial rate of leucine transport by membrane vesicles of S. cremoris increased exponentially with the proton motive force. An H+/leucine stoichiometry of 0.8 was determined from the steady-state level of leucine accumulation and the proton motive force, and this stoichiometry was found to be independent of the magnitude of the proton motive force. These results indicate that the introduction of bacterial reaction centers in membrane vesicles by the fusion procedure yields very attractive model systems for the study of proton motive force-consuming processes in membrane vesicles of (strict) anaerobic bacteria.     

Structure of the bovine eye lens gamma s-crystallin gene (formerly beta s)

The organization of a number of crystallin genes has already been resolved. One of the remaining genes of which the structure was hitherto unknown is the gamma s gene (formerly beta s). We determined the complete sequence of the bovine gamma s-crystallin-coding gene, apart from the middle region of the first intron. Since it contains three exons and two introns, we conclude that the former beta s, also at the gene level is gamma-crystallin-like. However, it is located on chromosome 3, in contrast to other gamma genes which occur in tandem on the human chromosome 2.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

The innervation of the lymphoid tissue at the ileocolonic transition: an enzyme and immunohistochemical study.

Using enzyme and immunohistochemical methods on whole-mount preparations and cryostat sections, a morphologic and semiquantitative study was performed of the nervous tissue in the appendix and the ileum (areas with and without Peyer's patches) of the rabbit. The plexus submucous externus (Meissner) consists of a network of small ganglia, vaguely associated with the vascular submucosal plexus. From the nerve cell bodies, cell processes occasionally penetrate the lymphoid follicles at the junction between the mucosa and the submucosa while other extensions form a dense plexus in the lamina propria of the mucosa. No nerve fibers are present in the dome of the follicles. The plexus submucous internus (Henle), consisting of large cell bodies and large processes, closely follows the blood vessels. The numeration of the nerve fibers of the submucosal plexus endorses the histological finding that the appendix is a richly innervated lymphoid organ. In addition, the plexus myentericus (Auerbach) of the appendix is a network of small meshes, while in the ileum, in the area of Peyer's patches, the same plexus is composed of a network with large meshes. These differences point to a higher density of innervation in the appendix. Yet a specialized anatomic distribution of the innervation of lymphoepithelial structures cannot be demonstrated.     

Autoradiographic model experiments with 67Ga and 99mTc

Summary In order to evaluate the feasibility to localize correctly ⁶⁷Ga citrate and ^99mTc pertechnetate in tissues, the resolution of these radioactive compounds were measured in a model system using four different autoradiographic techniques, wet as well as dry.Wet autoradiographic techniques gave an almost complete loss of ⁶⁷Ga. In deparaffinized sections of fixed and paraffin-embedded tissue the remaining ⁶⁷Ga, which was probably bound to proteins, gave a resolution of 2.6 . ^99mTc was either completely lost in wet techniques or the procedure could not be performed at all because of the very short half life of ^99mTc.The resolution of ⁶⁷Ga in a dry autoradiographic technique (according to Stumpf) was 6.9 and the resolution of ^99mTc 22.6 .The technique in which frozen sections are thawed on dry film and consequently dryed, gave slightly better resolutions than the dry technique (according to Stumpf) with ⁶⁷Ga as well as ^99mTc.It is concluded that for the histological localization of ⁶⁷Ga and ^99mTc a dry technique is required. However, the use of a wet technique can be considered, provided a loss of the radioisotope is acceptable and the procedure is controlled by a dry technique.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

The reliability of product-specific eco-labels as an agrobiodiversity management instrument

This paper seeks to understand why multinationals prefer to launch a label specific to their own product and examines how reliable these product-specific eco-labels are. A new methodology is applied to assess the extent to which eco-labels live up to claims about their contribution to conservation and the sustainable use of agricultural biodiversity. Product-specific eco-labels are considered as industry self-regulation and all three regulatory stages are studied: the planning, implementation and outcome stage. There are major differences between the product specific eco-labels in the degree in which agrobiodiversity management is part of the normative labeling schemes. Although there are some problems of reliability, such as transparency in the implementation stage and the monitoring in the outcome stage, the degree of reliability of product-specific labels is comparable with eco-labels of international labeling families. The conclusion is that only one of the product-specific eco-labels examined here is reliable when examined in the light of all three stages. The main reason why multinationals establish a product-specific eco-label instead of adopting one from an existing labeling family is that they want to profile themselves as distinct from other companies. The unique character of a product-specific label creates a market opportunity for them.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Can the excretion of metabolites by bacteria be manipulated?

Bacteria can release metabolites into the environment by various mechanisms. Excretion may occur by passive diffusion or by the reversal of the uptake process when the internal concentration of the metabolite exceeds the thermodynamic equilibrium level. In other cases, solutes are excreted against the concentration gradient by special extrusion systems. Their mode of energy coupling is different to that of the well-studied group of uptake systems. A thorough understanding of the transport processes will help to improve the excretion of metabolites of commercial interest, allow a more efficient production of metabolites in bulk quantities, and permit their exploitation to establish new markets.