Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.     

Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.     

The role of mammalian NEIL1 protein in the repair of 8-oxo-7,8-dihydroadenine in DNA

8-oxo-7,8-dihydroadenine (8-oxoAde) is a major product of adenine modification by reactive oxygen species. So far, only one mammalian DNA glycosylase, 8-oxoguanine-DNA-glycosylase 1 (OGG1), has been shown to excise 8-oxoAde, exclusively from pairs with Cyt. We have found that endonuclease VIII-like protein 1 (NEIL1), a mammalian homolog of bacterial endonuclease VIII, can efficiently remove 8-oxoAde from 8-oxoAde:Cyt pairs but not from other contexts. In an in vitro reconstituted system, reactions containing OGG1 produced a fully repaired product, whereas NEIL1 caused an abortive initiation of repair, stopping after 8-oxoAde removal and DNA strand cleavage. This block was partially relieved by polynucleotide kinase/3′-phosphatase. Thus, two alternative routes of 8-oxoAde repair may exist in mammals.     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.     

Distinct correlations of vasopressin release within the lateral septum and the bed nucleus of the stria terminalis with the display of intermale aggression

Arginine vasopressin (AVP) has been implicated in a wide variety of social behaviors ranging from affiliation to aggression. However, the precise functional involvement of AVP in intermale aggression is still a matter of debate. In fact, very little is known about AVP release patterns within distinct brain regions during the display of intermale aggression and, in turn, the behavioral consequences of such release. We used intracerebral microdialysis to monitor local AVP release within the lateral septum (LS) and the bed nucleus of the stria terminalis (BST) of adult male Wistar rats during the resident-intruder (RI) test. Resident males were cohabitated with a female prior to the RI test to stimulate intermale aggression toward the intruder male. AVP release within the LS correlated positively with intermale aggression. The specific AVP V1a receptor antagonist d(CH₂)₅Tyr(Me)AVP (10 μg/ml) administered via retrodialysis (3.3 µl/min, 30 min) into the LS of high-aggressive rats prior to the second RI test, prevented an increase in aggression in the second compared with the first RI test as seen in vehicle-treated high-aggressive rats. In contrast to the LS, AVP release within the BST correlated negatively with intermale aggression. Moreover, retrodialysis of synthetic AVP (1 µg/ml) administered into the BST of high-aggressive rats significantly reduced the display of aggression during the second RI test. These data reveal that AVP can both promote and inhibit intermale aggression, depending upon the brain region in which AVP is released. Although challenging the general view that central AVP release enhances intermale aggression in rodents, our data support a model in which AVP coordinates a range of social behaviors by eliciting region-specific effects.     

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

What Noseleaves Do for FM Bats Depends on Their Degree of Sensorial Specialization

Background

Many bats vocalizing through their nose carry a prominent noseleaf that is involved in shaping the emission beam of these animals. To our knowledge, the exact role of these appendages has not been thoroughly investigated as for no single species both the hearing and the emission spatial sensitivities have been obtained. In this paper, we set out to evaluate the complete spatial sensitivity of two species of New World leaf-nosed bats: Micronycteris microtis and Phyllostomus discolor. From an ecological point of view, these species are interesting as they belong to the same family (Phyllostomidae) and their noseleaves are morphologically similar. They differ vastly in the niche they occupy. Comparing these species allows us to relate differences in function of the noseleaf to the ecological background of bat species.

Methodology/Principal Findings

We simulate the spatial sensitivity of both the hearing and the emission subsystems of two species, M. microtis and P. discolor. This technique allows us to evaluate the respective roles played by the noseleaf in the echolocation system of these species. We find that the noseleaf of M. microtis focuses the radiated energy better and yields better control over the emission beam.

Conclusions

From the evidence presented we conclude that the noseleaves serve quantitatively different functions for different bats. The main function of the noseleaf is to serve as an energy focusing mechanism that increases the difference between the reflected energy from objects in the focal area and objects in the periphery. However, despite the gross morphological similarities between the noseleaves of the two Phyllostomid species they focus the energy to a different extent, a capability that can be linked to the different ecological niches occupied by the two species.     

A protocol for TILLING and Ecotilling in plants and animals

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.