Pfold: RNA secondary structure prediction using stochastic context-free grammars

RNA secondary structures are important in many biological processes and efficient structure prediction can give vital directions for experimental investigations. Many available programs for RNA secondary structure prediction only use a single sequence at a time. This may be sufficient in some applications, but often it is possible to obtain related RNA sequences with conserved secondary structure. These should be included in structural analyses to give improved results. This work presents a practical way of predicting RNA secondary structure that is especially useful when related sequences can be obtained. The method improves a previous algorithm based on an explicit evolutionary model and a probabilistic model of structures. Predictions can be done on a web server at http://www.daimi.au.dk/~compbio/pfold.     

The alpha-L-iduronidase mutations R89Q and R89W result in an attenuated mucopolysaccharidosis type I clinical presentation

Mucopolysaccharidosis type I (MPS I; McKusick 25280; Hurler syndrome, Hurler-Scheie syndrome and Scheie syndrome) is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase (EC 3.2.1.76). MPS I patients present within a clinical spectrum bounded by the extremes of Hurler and Scheie syndromes. The alpha-L-iduronidase missense mutations R89Q and R89W were investigated and altered an important arginine residue proposed to be a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase. The R89Q alpha-L-iduronidase mutation was shown to result in a reduced level of alpha-L-iduronidase protein (< or =10% of normal control) compared to a normal control level of alpha-L-iduronidase protein that was detected for the R89W alpha-L-iduronidase mutation. When taking into account alpha-L-iduronidase specific activity, the R89W mutation had a greater effect on alpha-L-iduronidase activity than the R89Q mutation. However, overall the R89W mutation produced more residual alpha-L-iduronidase activity than the R89Q mutation. This was consistent with MPS I patients, with an R89W allele, having a less severe clinical presentation compared to MPS I patients with either a double or single allelic R89Q mutation. The effects of the R89Q and R89W mutations on enzyme activity supported the proposed role of R89 as a nucleophile activator in the catalytic mechanism of alpha-L-iduronidase.     

Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Development of an automation system for a tablet coater

An instrumentation and automation system for a side-vented pan coater with a novel air-flow rate measurement system for monitoring the film-coating process of tablets was designed and tested. The instrumented coating system was tested and validated by film-coating over 20 pilot-scale batches of tablets with aqueous-based hydroxypropyl methylcellulose (HPMC). Thirteen different process parameters were continuously measured and monitored, and the most significant ones were logged for analysis. Laser profilometry was used to measure the surface roughness of the coated tablets. The instrumentation system provided comprehensive and quantitative information on the process parameters monitored. The measured process parameters and the responses of the film-coated tablet batches showed that the coating process is reproducible. The inlet air-flow rate influenced the coating process and the subsequent quality of the coated tablets. Increasing the inlet flow rate accelerated the drying of the tablet surface. At high inlet flow rate, obvious film-coating defects (ie, unacceptable surface roughness of the coated tablets) were observed and the loss of coating material increased. The instrumented and automated pancoating system described, including historical data storage capability and a novel air-flow measurement system, is a useful tool for controlling and characterizing the tablet film-coating process. Monitoring of critical process parameters increases the overall coating process efficiency and predictability.     

Shear stress levels in paralyzed legs of spinal cord-injured individuals with and without nerve degeneration.

The purpose of this study was to assess the relationship between inactivity and shear stress, the frictional force of blood against the endothelium, in spinal cord injury (SCI) subjects. SCI group offers a unique "model of nature" to study the effects of inactivity. Nine SCI subjects with upper (SCI-U) and 5 with a lower (SCI-L) motoneuron lesion and 10 able-bodied controls (C) were included. A venous blood sample was withdrawn to determine blood viscosity. Red blood cell velocities and arterial diameters of the common carotid artery (CCA) and common femoral artery (CFA) were measured by using echo-Doppler ultrasound in a supine position. No differences were observed in wall shear stress in the CCA between groups. In the CFA, peak and mean wall shear stress were significantly increased in SCI (14.1 and 1.2 Pa, respectively) compared with C (10.2 and 0.9 Pa, respectively). Because SCI-U and SCI-L showed no differences in shear stress levels, inactivity and not nerve degeneration seems to cause the elevated shear stress levels in the CFA in SCI. However, the lack of central neural control as a causal factor cannot be ruled out.     

Functional genomics of C190T single nucleotide polymorphism in human N-acetyltransferase 2

N-acetyltransferase 2 (NAT2) catalyzes N-acetylation and O-acetylation of many drugs and environmental carcinogens. Genetic polymorphisms in the NAT2 gene have been associated with differential susceptibility to cancers and drug toxicity from these compounds. Single nucleotide polymorphisms (SNPs) have been identified in the human NAT2 coding region. A new allele, NAT2*19, possessing the C190T (R64W) exchange, was recently identified. In order to understand the effect of this new SNP, recombinant NAT2*4 (reference) and NAT2*19 were expressed in yeast (Schizosaccharomyces pombe). The C190T (R64W) SNP in NAT2*19 caused substantial reduction in the NAT2 protein level and stability, but did not cause significant reduction in transformation efficiency or mRNA level. The enzymatic activities for N-acetylation of two arylamine carcinogens (2-aminofluorene, 4-aminobiphenyl), and a sulfonamide drug (sulfamethazine) were over 100-fold lower for NAT2 19 compared to reference NAT2 4. Kinetic studies showed a reduction in Vmax but no significant change in substrate Km. In addition, the SNP caused significant reduction in the O-acetylation of the N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine. These results show that NAT2*19 possessing the C190T (R64W) SNP encodes a slow acetylator phenotype for both N- and O-acetylation, due to a reduction in the amount and stability of the NAT2 19 allozyme.