Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Activity-dependent remodeling of presynaptic inputs by postsynaptic expression of activated CaMKII

Competitive synaptic remodeling is an important feature of developmental plasticity, but the molecular mechanisms remain largely unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can induce postsynaptic changes in synaptic strength. We show that postsynaptic CaMKII also generates structural synaptic rearrangements between cultured cortical neurons. Postsynaptic expression of activated CaMKII (T286D) increased the strength of transmission between pairs of pyramidal neuron by a factor of 4, through a modest increase in quantal amplitude and a larger increase in the number of synaptic contacts. Concurrently, T286D reduced overall excitatory synaptic density and increased the proportion of unconnected pairs. This suggests that connectivity from some synaptic partners was increased while other partners were eliminated. The enhancement of connectivity required activity and NMDA receptor activation, while the elimination did not. These data suggest that postsynaptic activation of CaMKII induces a structural remodeling of presynaptic inputs that favors the retention of active presynaptic partners.     

T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang

Mammalian telomeres contain a duplex TTAGGG-repeat tract terminating in a 3' single-stranded overhang. TRF2 protein has been implicated in remodeling telomeres into duplex lariats, termed t-loops, in vitro and t-loops have been isolated from cells in vivo. To examine the features of the telomeric DNA essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overhangs were constructed. As assayed by electron microscopy, looped molecules containing most of the telomeric tract are observed with TRF2 at the loop junction. A TTAGGG-3' overhang of at least six nucleotides is required for loop formation. Termini with 5' overhangs, blunt ends or 3' termini with non-telomeric sequences at the junction are deficient in loop formation. Addition of non-telomeric sequences to the distal portion of a 3' overhang beginning with TTAGGG repeats only modestly diminishes looping. TRF2 preferentially localizes to the junction between the duplex repeats and the single-stranded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.     

Effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal flora of swine

The effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal tract of swine were studied in animals from a farm that had not been supplementing antimicrobials in feed for the past 10 years. In one study, 10 finisher hogs were heat stressed (34 degrees C) for 24 h. Antimicrobial resistance levels after stress were significantly higher (P < 0.05) when compared with pre-stress levels for amikacin, ampicillin, cephalothin, neomycin and tetracycline from faecal samples. This high level of resistance persisted to slaughter that occurred at 10 days post-stress for most of the antimicrobials mentioned. In a second study, samples of different sections of the gastrointestinal tract were collected after heat stress and compared with control, non-stressed animals. Results indicated that E. coli which colonized the ileum and caecum had a higher level of resistance to ampicillin and tetracycline than the E. coli which colonized the colon and rectum. When animals were exposed to heat stress, resistance to ampicillin and tetracycline of E. coli in the lower digestive tract increased (P < 0.05) to a level similar to that observed in the ileum and caecum. Based on these findings, an investigation was made to test the hypothesis that (a) an increase in intestinal motility increases shedding of resistant E. coli and (b) heat stress induces a reduction in intestinal transit time in swine. For each study, two groups of three, randomly selected finisher hogs each were formed (treated and control groups). In study (a), induction of increased motility and peristalsis was obtained using an intramuscular injection of the cholinergic drug neostigmine methylsulphate. Escherichia coli isolates were obtained from the ileum, caecum, colon and rectum after animals were slaughtered. A higher level of ampicillin-resistant E. coli was found in the caecum (40%) than in other segments of the intestinal tract. In treated animals, level of resistance increased for organisms from the colon and rectum. Similar results were obtained for tetracycline resistance. In study (b), intestinal transit time was measured using chromium-EDTA as a marker. Swine were euthanized and samples were collected throughout the intestinal tract (duodenum to rectum) 8 h after administration of the marker to control and heat-stressed animals. Results indicated a reduced transit time for the stressed group. These findings corroborate the initial hypothesis that an outflow of resistant organisms moves from the upper tract (ileum and caecum) to the lower tract (colon and rectum).     

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.     

The nitrogen handling characteristics of white clover (Trifolium repens L.) cultivars and a perennial ryegrass (Lolium perenne L.) cultivar

Ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) have contrasting responses to soil mineral N availability and clover has the ability to fix atmospheric N(2) symbiotically. It has been hypothesized that these differences are the key to understanding grass-clover coexistence and vegetative dynamics in pastures. However, the whole plant response of clover and ryegrass to mineral N availability has not been fully characterized and inter-cultivar variability in the N-handling dynamics of clover has not been assessed. A detailed experimental study to address these issues was undertaken. For all clover cultivars and ryegrass, mass specific mineral N uptake rates (of whole plants) were similar saturating functions of mineral N availability. For all clover cultivars total N assimilation rates, whole plant C : N ratios and root : shoot ratios were independent of mineral N availability. Clover growth rates were also independent of mineral N availability except for a slight (<10%) reduction at very low N availability levels. Specific N(2) fixation rate (whole plant) was precisely controlled to ensure fixation balanced the deficit between mineral N uptake and the total N assimilation required to maintain constant whole plant C : N ratio. There was always a deficit between N uptake and the total N assimilation required to maintain C : N ratio. Consequently, some N(2) fixation remained engaged even at high mineral N availability levels. All inter-cultivar variation in N(2) fixation dynamics could be attributed to variations in growth rate. Clover mass specific growth rate declined as plant size increased. Ryegrass specific growth rate, whole plant C : N ratio and root : shoot ratio were dependent on N availability. Increased N availability led to increased growth rate and decreased C : N and root : shoot ratios. Specific growth rate was also dependent on plant size, growth rate declining as plant size increased. It is concluded that clover inter-cultivar variation in field performance is unlikely to be a consequence of variation in N-handling characteristics. Inter-cultivar differences in growth rate are likely to be a much more important source of variation.     

Ligand design for alpha1 adrenoceptor subtype selective antagonists

Alpha1 adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring D analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human alpha1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (alpha1A, alpha1B, alpha1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the alpha1D adrenoceptor subtype is also reported.     

Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.     

High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the urokinase receptor in human glioma cells

The distribution of the urokinase-type plasminogen activator receptor (uPAR) on human glioma cells was examined as a function of culture conditions, using immunofluorescence and immunophotoelectron microscopy. Both uPAR colocalization with focal adhesion proteins and glioma cell motility were maximal in medium containing whole serum or a serum fraction retained by a 500,000 mol wt cutoff centrifugal concentration filter. High motility also took place in medium containing a serum fraction passed by the 500,000 cutoff filter but retained by a 100,000 cutoff filter and in minimal medium containing added vitronectin; however, under these conditions only a small percentage of the otherwise abundant focal adhesions contained colocalized uPAR. Glioma cells in minimal medium with added laminin migrated with a highly elongated morphology but without either classical focal adhesions or well-defined uPAR labeling. In contrast, glioma cells in minimal medium with no additions did not migrate, nor did they adhere well or display defined labeling patterns for focal adhesion proteins or uPAR. The results indicate that high-molecular-weight serum protein complexes promote both uPAR-focal adhesion colocalization and cell migration in glioma cells. However, conditions can be selected in which migration takes place with minimal uPAR-focal adhesion localization, as well as in the absence of apparent focal adhesions.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.