Inhibition of MDR1 expression with altritol-modified siRNAs

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3′ end of the sense or antisense strands, while modification at the 5′ end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5′-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.     

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828)

Campylobacter jejuni is a major human enteric pathogen that displays genetic variability via genomic reorganization and phase variation. This variability can adversely affect the outcomes and reproducibility of experiments. C. jejuni strain 81116 (NCTC11828) has been suggested to be a genetically stable strain (G. Manning, B. Duim, T. Wassenaar, J. A. Wagenaar, A. Ridley, and D. G. Newell, Appl. Environ. Microbiol. 67:1185-1189, 2001), is amenable to genetic manipulation, and is infective for chickens. Here we report the finished annotated genome sequence of C. jejuni strain 81116.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.     

Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation

Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K(+) or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K(+) initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant lambda: 371 +/- 42 mum). After N(omega)-nitro-l-arginine (l-NNA), lambda increased to 507 +/- 30 mum, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced lambda after l-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, lambda was 530 +/- 80 mum and thus similar to that in wild-type mice after l-NNA. Exogenous NO likewise reduced lambda in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 mum and were not altered by l-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.     

Identifying variables responsible for clustering in discriminant analysis of data from infrared microspectroscopy of a biological sample.

In the biomedical field, infrared (IR) spectroscopic studies can involve the processing of data derived from many samples, divided into classes such as category of tissue (e.g., normal or cancerous) or patient identity. We require reliable methods to identify the class-specific information on which of the wavenumbers, representing various molecular groups, are responsible for observed class groupings. Employing a prostate tissue sample divided into three regions (transition zone, peripheral zone, and adjacent adenocarcinoma), and interrogated using synchrotron Fourier-transform IR microspectroscopy, we compared two statistical methods: (a) a new "cluster vector" version of principal component analysis (PCA) in which the dimensions of the dataset are reduced, followed by linear discriminant analysis (LDA) to reveal clusters, through each of which a vector is constructed that identifies the contributory wavenumbers; and (b) stepwise LDA, which exploits the fact that spectral peaks which identify certain chemical bonds extend over several wavenumbers, and which following classification via either one or two wavenumbers, checks whether the resulting predictions are stable across a range of nearby wavenumbers. Stepwise LDA is the simpler of the two methods; the cluster vector approach can indicate which of the different classes of spectra exhibit the significant differences in signal seen at the "prominent" wavenumbers identified. In situations where IR spectra are found to separate into classes, the excellent agreement between the two quite different methods points to what will prove to be a new and reliable approach to establishing which molecular groups are responsible for such separation.     

Milk composition of three free-ranging African elephant (Loxodonta africana africana) cows during mid lactation

Data are presented that indicate the dynamic changes of nutrients in milk from three free ranging African elephant (Loxodonta africana africana) cows during lactation. At the respective collection times of 12, 14 and 18 months of lactation the nutrient content was 47.3, 52.0 and 68.6 g protein; 60.7, 87.4 and 170.8 g fat; 1.6, 2.1 0.5 g lactose and 20.9, 21.5 and 8.6 g oligosaccharides per kg milk. The protein fraction respectively consisted of 18.0, 31.7 and 45.9 g caseins/kg milk and of 29.3, 20.3 and 22.7 g whey proteins/kg milk. Electrophoresis and identification of protein bands showed that polymorphs of one whey protein may be present in elephant's milk similar to polymorphs of alpha-lactalbumin found in cow's milk. From the middle of the lactation time lactose was replaced by oligosaccharides as major carbohydrate, and the major compound of these was identified as isoglobotriose by 1H NMR spectroscopy. The lipid fraction contains a high content, of capric and lauric acids, approximately 70% of the total fatty acids, and low content of myristic, palmitic and oleic acids. During these lactation times the content of short chain fatty acids, capric and caprylic acids increased, while fatty acids lauric acid and longer decreased.     

Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity

Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.     

Metabolism of mono- and dihalogenated C1 and C2 compounds by <Emphasis Type="Italic">Xanthobacter autotrophicus</Emphasis> growing on 1,2-dichloroethane

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.