Structural characterization of LNA and alpha-L-LNA hybridized to RNA

LNA and alpha-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we present a short overview of the structural results we have obtained for LNA:RNA and alpha-L-LNA:RNA hybrids. Specifically, we have shown that LNA acts as an A-type mimic, while alpha-L-LNA acts as a B-type mimic when built into oligonucleotides.     

Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 x 10(6) cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by avirulent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

Enantiopure derivatives of 1,2-alkanediols: substrate requirements of lipase B from Candida antarctica

Efficient methods for kinetic resolution of 1-phenoxy-2-butanol, 1-phenylmethoxy-2-butanol, and 1-phenoxy-2-pentanol were developed using lipase B from Candida antarctica as catalyst. Resolutions were performed in order to investigate the substrate requirements needed to obtain a high E-value. The effect of the substrate structure on E is different for transesterifications in organic media as compared to hydrolysis. The influence of different acyl donors on the E-value was also investigated.     

Single amino acid substitutions in kappa-conotoxin PVIIA disrupt interaction with the shaker K+ channel

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid peptide with three disulfide cross-links, isolated from the venom of Conus purpurascens, is the first conopeptide shown to inhibit the Shaker K(+) channel (Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151). Recently, two groups independently determined the solution structure for kappa-PVIIA using NMR; although the structures reported were similar, two mutually exclusive models for the interaction of the peptide with the Shaker channel were proposed. We carried out a structure/function analysis of kappa-PVIIA, with alanine substitutions for all amino acids postulated to be key residues by both groups. Our data are consistent with the critical dyad model developed by Ménez and co-workers (Dauplais, M., Lecoq, A., Song, J. , Cotton, J., Jamin, N., Gilquin, B., Roumestand, C., Vita, C., de Medeiros, C., Rowan, E. G., Harvey, A. L., and Ménez, A. (1997) J. Biol. Chem. 272, 4802-4809) for polypeptide antagonists of K(+) channels. In the case of kappa-PVIIA, Lys(7) and Phe(9) are essential for activity as predicted by Savarin et al. (Savarin, P., Guenneugues, M., Gilquin, B., Lamthanh, H., Gasparini, S., Zinn-Justin, S., and Ménez, A. (1998) Biochemistry 37, 5407-5416); these workers also correctly predicted an important role for Lys(25). Thus, although kappa-conotoxin PVIIA has no obvious sequence homology to polypeptide toxins from other venomous animals that interact with voltage-gated K(+) channels, there may be convergent functional features in diverse K(+) channel polypeptide antagonists.     

Characterisation of a homogeneous plant aminoaldehyde dehydrogenase

According to our knowledge, this is the first purification method developed, enabling isolation of a homogeneous aminoaldehyde dehydrogenase (AMADH) from etiolated pea seedlings. The procedure involved initial purification with precipitants followed by three low pressure chromatographic steps. Partially purified enzyme was further subjected to fast protein liquid chromatography on a Mono Q column and to affinity-interaction chromatography on 5'-AMP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chromatofocusing. Pea AMADH exists as a tetramer of 230 kDa in the native state, a molecular mass of one subunit was determined as 57 kDa. The enzyme was found to be an acidic protein with pI 5.4. AMADH showed a broad substrate specificity utilising various aminoaldehydes (C3-C6) as substrates. The best substrate of pea AMADH was 3-aminopropionaldehyde, the enzyme also efficiently oxidised 4-aminobutyraldehyde and omega-guanidinoanalogues of the aminoaldehydes. Pea AMADH was inhibited by SH reagents, several elementary aldehydes and metal-binding agents. Although AMADH did not oxidise betaine aldehyde at all, the N-terminal amino acid sequence of the enzyme shows a high degree of homology with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in comparison with BADH from cod liver of known crystal structure.     

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.     

Ease of calving, blood chemistry, insulin and bovine growth hormone of newborn calves derived from embryos produced in vitro in culture systems with serum and co-culture or with PVA

Blood chemistry (pH, pCO2, pO2, glucose, lactate) as well as plasma insulin and growth hormone of calves derived from embryos produced under 2 different in vitro culture systems (modified SOFaa with 20% serum and co-culture with bovine oviduct epithelial cells [IVP serum, n=8] or with 3 mg/mL PVA [IVPdefined, n=6]) were compared with those of calves derived from AI (n=5). Calvings were classified according to the ease (unassisted, light traction, heavy traction). Blood samples were taken from the jugular vein of calves at 5, 15, 30 and 60 min, and at 2, 3, 6, 12, 18 and 24 h after delivery, then daily for 6 d. At the second day of life after 4 feedings and a 4-h fasting period, a glucose tolerance test was performed to evaluate glucose metabolism and insulin secretion. Calves in the IVP serum group had higher birth weights than AI calves (LS mean +/- SEM, IVP serum: 45.2 +/- 1.4 kg vs AI: 40.4 +/- 1.7 kg; P < 0.05), while the birth weights of calves in the IVP defined group were in between (IVPdefined: 41.9 +/- 1.6 kg). More IVP serum calves (75%) needed assistance than IVP defined (33%) or AI (40%) calves. The effect of ease of calving vs the effect of embryo culture was compared in relation to blood parameters at birth. There was an effect of ease of calving but not of embryo culture conditions on blood pH, lactate and PCO2. Calves requiring heavy traction had lower pH during the first 3 h after calving, a higher lactate during the first 60 min after calving and a higher pCO2 the first 2 h after calving than calves born unassisted. Calves requiring heavy traction also had lower pH the first 2 h and higher lactate the first 3 h after calving than calves born by light traction. IVP defined calves had lower lactate than IVP serum calves the first 60 min after calving. At 6 h after delivery, all blood parameters had stabilized. There was no effect of either embryo culture or ease of calving on basal insulin and growth hormone level, or the ability of the calves to handle glucose postnatally and during a glucose tolerance test.     

Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells

Body dimensions, birth and organ weights of calves derived from embryos produced in 2 in vitro culture systems (modified SOFaa with 20% cattle serum and co-cultured with oviduct-epithelium cells [IVPserum, n=8], and modified SOFaa with 3 mg/mL PVA [IVPdefined, n=6]) were compared with calves originating from artificial insemination (AI, n=85). Three additional IVP calves were included which had been vitrified as mature oocytes by the open pulled straw (OPS) method, warmed, fertilized and cultured to the blastocyst stage in modified SOFaa with 5% cattle serum, then again OPS-vitrified and warmed prior to transfer (IVPops, n=3). At birth, gestation length and birth weights were registered for all calves. At 1 wk of age all 17 IVP and 7 of the AI calves were killed, and their body dimensions and organ weights recorded. Birth weight was higher for the IVPserum and IVPops calves than for AI control calves (kg +/- SEM: IVPserum 46.9+/-1.8, IVPops 50.6+/-2.4, AI 41.8+/-0.8; P < 0.002). There was no difference between IVP and AI calves regarding gestation length and no effect of culture conditions on body dimensions or organ weights, except for longer hind legs in IVPdefined calves compared with AI calves (cm +/- SEM: IVPdefined 93+/-2, AI 87+/-2; P < 0.04). The IVPops calves had an increased liver weight compared with AI and the other IVP calves (g +/- SEM: IVPops 1.457+/-59; AI 1,117+/-37; IVPserum 1,159+/-34, IVPdefined 1,073+/-39; P < 0.0003). It is concluded that in vitro culture of bovine embryos in the presence of serum and oviduct epithelium cells increased birth weight but not organ weight and body dimension in 1-wk-old calves. However, vitrification of the ova as oocyte and again as blastocysts increased birth weight and liver size. This possible effect of cryopreservation of oocytes on subsequent fetal development awaits further investigation.