The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. M?ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.     

Prox1 is differentially localized during lens development

Prox1, the vertebrate cognate of Drosophila Prospero, is a homeodomain protein essential for the development of the lens, liver and lymphatic system. While it is well established that the subcellular distribution of Prospero changes during development, this had not been demonstrated for Prox1. Here, high-resolution confocal microscopy demonstrated that Prox1 protein is predominately cytoplasmic in the lens placode as well as the lens epithelium and germinative zone throughout development. However during fiber cell differentiation, Prox1 protein redistributes to cell nuclei. Finally, as lens fiber cells condense their chromatin in response to lens denucleation, Prox1 remains in the nucleus but does not appear to interact with DNA. Thus, it appears that the function of Prox1, like that of its Drosophila cognate Prospero, is at least partially controlled by changes in its subcellular distribution during development.     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

Presynaptic BDNF required for a presynaptic but not postsynaptic component of LTP at hippocampal CA1-CA3 synapses

Brain-derived neurotrophic factor (BDNF) has been implicated in several forms of long-term potentiation (LTP) at different hippocampal synapses. Using two-photon imaging of FM 1-43, a fluorescent marker of synaptic vesicle cycling, we find that BDNF is selectively required for those forms of LTP at Schaffer collateral synapses that recruit a presynaptic component of expression. BDNF-dependent forms of LTP also require activation of L-type voltage-gated calcium channels. One form of LTP with presynaptic expression, theta burst LTP, is thought to be of particular behavioral importance. Using restricted genetic deletion to selectively disrupt BDNF production in either the entire forebrain (CA3 and CA1) or in only the postsynaptic CA1 neuron, we localize the source of BDNF required for LTP to presynaptic neurons. These results suggest that long-term synaptic plasticity has distinct presynaptic and postsynaptic modules. Release of BDNF from CA3 neurons is required to recruit the presynaptic, but not postsynaptic, module of plasticity.     

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.     

The importance of the prolonged postnatal period in determination of heart atrial muscarinic receptor and beta-adrenoceptor densities

Our previous work indicated that hyperstimulation of muscarinic receptors brought about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors both in vivo and in vitro. Now we try to identify the changes in receptor densities when the neuronal input from the autonomic nerves is disrupted, i.e. when neonatal rats were injected with 6-hydroxydopamine (6-OHDA; able to destroy sympathetic nerves). Although the interruption of neuronal input was successful as indicated by norepinephrine uptake, there was no change in receptor densities in the rat heart atria when 6-OHDA was applied in the first postnatal week only. When we repeated the 6-OHDA treatment consequently (on 14th, 21st and 28th postnatal day) there were clear increases of both receptor types (muscarinic receptors rise to 139% of control, beta-adrenoceptors to 134% of control). The atrial muscarinic receptor number was increased (to 146% of control) also when we combined the first week 6-OHDA application with another factor (when the pups abide with mother till the 56th day). The simple non-weaning of animals has no effect on the receptor densities. We can therefore conclude that the first week application of 6-OHDA was insufficient in changing the receptor number and that only repeated 6-OHDA administration was able to change them. Similarly, combination with other factor: non-weaning led to the muscarinic receptors increase. Our results can indicate the importance of the prolonged postnatal period for the heart muscarinic and beta-adrenergic receptor number determination.     

Cross-linked cell aggregates of Trigonopsis variabilis: D-amino acid oxidase catalyst for oxidation of cephalosporin C

Trigonopsis variabilis CBS 4095 was treated with alkali (pH 11, 30 min), heated (65°C, 60 s) and immobilized. Glutaraldehyde, polyethyleneimine and a cross-linking reagent formed by reaction of polyethyleneimine with glutaraldehyde were used for stabilization of d-amino acid oxidase in the cells, as well as for aggregation and binding of the cells. A specific activity of 82–98 U of d-amino acid oxidase per g dry mass was produced with a yield of about 20%. The half-life time of 142 repeated conversion cycles corresponds to a productivity of 130 kg cephalosporin C oxidized per kg catalyst dry mass.     

A novel lipopeptide, an inhibitor of bacterial adhesion, from the thermophilic and halotolerant subsurface Bacillus licheniformis strain 603

A new Bacillus licheniformis strain, 603, isolated from a mixture of drilling fluid and subsurface thermal water, has been found to produce a cyclic lipopeptide which is released into cultural medium as well as present in cells as the major lipid constituent (57% of the total cell lipids extractable with 2:1 chloroform-methanol). The quantitative ratio of the extracellular and intracellular lipopeptide has been estimated as 23:10. The metabolite represents a heptapeptide, L-Asp-->L-Leu-->L-Leu-->L-Val-->L-Val-->L-Glu-->L-Leu, N-acylated to the N-terminal amino acid, L-Asp, by a 3-hydroxy fatty acid (from 13:0 to 17:0 with n-, iso-, and anteiso-chains), the 3-OH group of which is esterified by the C-terminal amino acid, L-Leu. The chemical structure of the lipopeptide has been established by means of infrared (IR), 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionisation (ESI) mass spectrometry (MS), including secondary ion mass spectrometry, along with chemical and enzymatic degradation. Although a diversity of similar metabolites synthesised by various B. licheniformis strains are presently known, such a structure has not been reported thus far. Added to the growth medium of strain 603 at the concentration of 1.6 microg/ml, the lipopeptide prevents adhesion of cells to a glass surface. Also, it exhibits a considerable growth-inhibiting activity against Corynebacterium variabilis and a much lower activity against Acinetobacter sp.     

Anatomical correlates of venom production in Conus californicus

Like all members of the genus, Conus californicus has a specialized venom apparatus, including a modified radular tooth, with which it injects paralyzing venom into its prey. In this paper the venom duct and its connection to the pharynx, along with the radular sac and teeth, were examined using light and transmission electron microscopy. The general anatomy of the venom apparatus resembles that in other members of the genus, but several features are described that have not been previously reported for other species. The proximal (posterior) quarter of the venom duct is composed of a complex epithelium that may be specialized for active transport rather than secretion. The distal portion of the duct is composed of a different type of epithelium, suggestive of holocrine secretion, and the cells display prominent intracellular granules of at least two types. Similar granules fill the lumen of the duct. The passageway between the lumen of the venom duct and pharynx is a flattened branching channel that narrows to a width of 10 micro m and is lined by a unique cell type of unknown function. Granular material similar to that in the venom duct was also found in the lumen of individual teeth within the radular sac. Mass spectrometry (MALDI-TOF) demonstrated the presence of putative peptides in material derived from the tooth lumen, and all of the more prominent species were also evident in the anterior venom duct. Radular teeth thus appear to be loaded with peptide toxins while they are still in the radular sac.