Intra-protocol repeatability and inter-protocol agreement for the analysis of scapulo-humeral coordination.

Multi-center clinical trials incorporating shoulder kinematics are currently uncommon. The absence of repeatability and limits of agreement (LoA) studies between different centers employing different motion analysis protocols has led to a lack dataset compatibility. Therefore, the aim of this work was to determine the repeatability and LoA between two shoulder kinematic protocols. The first one uses a scapula tracker (ST), the International Society of Biomechanics anatomical frames and an optoelectronic measurement system, and the second uses a spine tracker, the INAIL Shoulder and Elbow Outpatient protocol (ISEO) and an inertial and magnetic measurement system. First within-protocol repeatability for each approach was assessed on a group of 23 healthy subjects and compared with the literature. Then, the between-protocol agreement was evaluated. The within-protocol repeatability was similar for the ST ([Formula: see text] = 2.35°, [Formula: see text] = 0.97°, SEM = 2.5°) and ISEO ([Formula: see text] = 2.24°, [Formula: see text] = 0.97°, SEM = 2.3°) protocols and comparable with data from published literature. The between-protocol agreement analysis showed comparable scapula medio-lateral rotation measurements for up to 120° of flexion-extension and up to 100° of scapula plane ab-adduction. Scapula protraction-retraction measurements were in agreement for a smaller range of humeral elevation. The results of this study suggest comparable repeatability for the ST and ISEO protocols and between-protocol agreement for two scapula rotations. Different thresholds for repeatability and LoA may be adapted to suit different clinical hypotheses.

Microwave techniques in agriculture.

Microwave techniques, methods and instrumentation can be utilized in agriculture to improve the efficiency of crop production, handling and processing, and improve the quality of products. Pertinent microwave theory relative to such applications is discussed, and a review of microwave research on agricultural problems is presented. A wide range of applications is treated briefly, including examples of successful applications of microwaves in agriculture, and some future prospects are discussed.

Broadband microwave wheat permittivity measurements in free space.

Use of broadband horn/lens antennas with a collimated beam in a free-space measurement set-up with a network analyzer operating between 3 and 13 GHz and applying time-domain gating improved the accuracy of permittivity (dielectric properties) measurements on grain. Measurements were taken in the transmission mode on samples from five different classes of wheat over ranges of moisture content, temperature, and bulk density. Dielectric properties were divided by bulk density of the grain for comparison of different wheat lots. No differences in permittivity data were noted among the different wheat classes, so data were combined for presentation. The new data agree well with those reported previously. For further research, to provide dielectric properties over a very broad frequency range for a given grain sample, use of time-domain spectroscopy is suggested.

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.

Nucleoside phosphate analogues of biological interest, and their synthesis via aryl nucleoside H-phosphonates as intermediates.

This review presents a brief account of the chemistry and mechanistic aspects of aryl H-phosphonates, and selected applications of this class of compounds as intermediates in the synthesis of a wide range of biologically important analogues of nucleoside phosphates, and oligonucleotides, in which the phosphate moieties are replaced by other structurally related groups. The aryl nucleoside H-phosphonates, compounds of controlled reactivity, have proven to be more versatile and superior to various mixed anhydrides as synthetic intermediates, particularly for preparation of nucleotide analogues bearing P-N or P-S bonds in various configurational arrangements at the phosphate moiety.

Aryl H-phosphonates. 12. Synthetic and (31)P NMR studies on the preparation of nucleoside H-phosphonothioate and nucleoside H-phosphonodithioate monoesters.

Transformation of nucleoside H-phosphonate monoesters into the corresponding H-phosphonothioate and H-phosphonodithioate derivatives and possible side-reactions that may accompany this process were studied using (31)P NMR spectroscopy. These provided new insight into a possible mechanism involved in this transformation and constituted the basis for development of efficient methods for the preparation of nucleoside H-phosphonothioate and nucleoside H-phosphonodithioate monoesters using readily available H-phosphonate monoesters as starting materials.

Studies on reactions of nucleoside H-phosphonates with bifunctional reagents. Part VI. Reaction with diols.

Reactions of nucleoside H-phosphonates with various diols using different types of condensing agents have been studied. Depending on the coupling procedure and the length of a polymethylene chain of the diol, acyclic H-phosphonate diesters or cyclic phosphite triesters were formed. The course of oxidation with iodine to produce cyclic nucleoside alkyl phosphotriesters or hydroxyalkyl nucleoside phosphodiesters can be controlled by the amount of water present in the reaction medium.

The reactions of H-phosphonates with bifunctional reagents. Part V. Functionalization of support-bound oligonucleotides and synthesis of non-radioactive hybridization probes.

Three methods for the functionalization of oligonucleotides with aminoalkyl moieties have been developed and their efficiencies were evaluated in the preparation of non-radioactive hybridization probes.

Apolipoprotein E genotypes in sporadic early and late-onset Alzheimer's disease.

Recent data have demonstrated that presence of apolipoprotein E (APOE) epsilon *4 allele is a major risk factor of Alzheimer's disease (AD). We determined the APOE genotypes in 64 patients with sporadic probable AD and 43 non-demented aged controls selected from Poznan region and the western part of Poland using the polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) analysis. We confirmed a strong correlation of the APOE epsilon *4 allele with sporadic late-onset AD. In contrast to many previous reports we did not found an association between the APOE epsilon *4 allele and sporadic early-onset AD.

Some aspects of oligoribonucleotides synthesis via the H-phosphonate approach.

This review gives a short account of selected aspects of oligoribonucleotide synthesis via the H-phosphonate method. It includes: (i) recent methods for the preparation of suitably protected ribonucleoside 3'-H-phosphonates (the phosphonylation step), (ii) some chemical and stereochemical features of the formation of H-phosphonate internucleosidic linkages, and (iii) stereoselective synthesis of oligoribonucleoside phosphorothioates using chemo-enzymatic approach.

Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry.

The study of modified nucleoside contributions to RNA chemistry, structure and function has been thwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies. A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta- and octadecamer RNAs has been achieved with automated phosphoramidite chemistry. The 5'-O-(4,4'-dimethoxytrityl-2'-O-tert-butyldimethylsilyl-ribonucleoside- 3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite syntheses of m5C, D, psi, riboT, s2U, mnm5U, m1G and m2A were designed for compatibility with the commercially available major and 2'OH methylated ribonucleoside phosphoramidites. The synthesis of the m5C phosphoramidite was uniquely designed, and the first syntheses and incorporation of the two modified purine ribonucleosides are reported in detail along with that of psi, s2U, and mnm5U. Cleavage of RNA product from the synthesis support column, deprotection of the RNA, its purification by HPLC and nucleoside composition analysis are described. Modified nucleoside-containing tRNA domains were synthesized and purified in mumol quantities required for biophysical, as well as biochemical, studies. The anticodon domain of yeast tRNA(Phe) was synthesized with modified nucleosides introduced at the native positions: Cm32, Gm34, m1G37 (precursor to Y), psi 39 and m5C40. The T loop and stem was synthesized with riboT54 and the D loop and stem with D16 and D17. The E coli tRNA(Glu2) anti-codon codon domain was synthesized with mnm5U at wobble position 34, but an attempt at incorporating s2U at the same position failed. The unprotected thio group was labile to the oxidation step of the cyclical process. Chemically synthesized anticodon and T domains have been used in assays of tRNA structure and function (Guenther et al (1994) Biochimie 76, 1143-1151).

Long-term cadmium exposure accelerates oxidant injury: significance of bound/free water states during long-term metal stress.

Lepidium sativum (cress) and Lycopersicon esculentum (tomato) plants were grown in peatlite in controlled environments with or without long-term (4 weeks) cadmium stress (Cd) (100 micrograms/ml every fourth day) and with a single exposure (6 hr at 35 parts per hundred million (pphm)) or no exposure to the oxidant ozone (O3). Cress plants which received Cd wilted faster during O3 exposure and became a gray-green color by the end of a 6-hr O3 exposure. Those receiving O3 alone also wilted but were normal in color during wilting. Leaf water content (percentage) significantly declined in both O3 + Cd- and O3-treated plants. However, leaves after Cd + O3 exposure were severely dessicated and necrotic, whereas O3-treated plants recovered their water content completely but had some injury. Increased stomatal aperture in cress but not tomato before O3 exposure and significantly lower water content at 1 and 24 hr after the end of O3 exposure were associated with the higher Cd content of leaves before and subsequent to O3 exposure. These factors contributed to a greater injury and cell death observed in the leaves of combined cadmium-oxidant stress. Dielectric properties of Thlaspi arvense (field penny cress) leaves grown at continuous exposure to Cd and/or nickel (Ni) indicated that there were measurable differences between metal-containing vs control leaves with regard to bound/unbound water status. This indicated that there was more free water under metal stress, and that the bound water content significantly declined in the leaves of these plants.

Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon".

The efficiency of translation depends on correct tRNA-ribosome interactions. The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications. Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop. Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding. To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops. The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe). However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding. Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding.

Prevalence of beta allele of the insulin gene in type II diabetes mellitus.

Fifty-two patients and 36 controls were compared in a search for insulin gene variants among type II diabetic patients with fasting hyperinsulinemia (above 90 microU/ml) and a fasting C-peptide to insulin molar ratio between 1.11 and 1.50. Alpha and beta alleles of the insulin gene were characterized by restriction analysis of polymerase chain reaction (PCR) products and direct sequencing. The more frequent occurrence of the alpha allele of the insulin gene within the control population as compared with a prevalence of the beta allele in the diabetic patients (P, 0.05) was observed. The beta allele, usually described as the rare allele, seems to be associated with the disease.

Aminoacyl-tRNA synthetase and U54 methyltransferase recognize conformations of the yeast tRNA(Phe) anticodon and T stem/loop domain.

The enzyme-catalyzed posttranscriptional modification of tRNA and the contributions of modified nucleosides to tRNA structure and function can be investigated with chemically synthesized domains of the tRNA molecule. Heptadecamer RNAs with and without modified nucleosides and DNAs designed as analogs to the anticodon and T stem/loop domains of yeast tRNA(Phe) were produced by automated chemical synthesis. The unmodified T stem/loop domain of yeast tRNA(Phe) was a substrate for the E coli m5U54-tRNA methyltransferase activity, RUMT. Surprisingly, the DNA analog of the T stem/loop domain composed of d(A,U,G,C) was also a substrate. In addition, the DNA analog inhibited the methylation of unfractionated, undermodified E coli tRNA lacking the U54 methylation. RNA anticodon domains and DNA analogs differentially and specifically affected aminoacylation of the wild type yeast tRNA(Phe). Three differentially modified tRNA(Phe) anticodon domains with psi 39 alone, m1G37 and m5C40, or psi 39 with m1G37 and m5C40,stimulated phenylalanyl-tRNA synthetase (FRS) activity. However, one anticodon domain, with m5C40 as the only modified nucleoside and a closed loop conformation, inhibited FRS activity. Modified and unmodified DNA analogs of the anticodon, tDNA(PheAC), inhibited FRS activity. Analysis of the enzyme activity in the presence of the DNA analog characterized the DNA/enzyme interaction as either partial or allosteric inhibition. The disparity of action between the DNA and RNA hairpins provides new insight into the potential allosteric relationship of anticodon binding and open loop conformational requirements for active site function of FRS and other aaRSs. The comparison of the stimulatory and inhibitory properties of variously modified RNA domains and DNA analogs demonstrates that conformation, in addition to primary sequence, is important for tRNA-protein interaction. The enzyme recognition of various DNA analogs as substrate and/or inhibitors of activity demonstrates that conformational determinants are not restricted to ribose and the standard A-form RNA structure.

The inhibitory effect of oligodeoxynucleotides complementary to different fragments of tRNA structure on the enzymatic binding of Phe-tRNA to poly-U-programmed eukaryotic ribosomes.

The effect of several oligodeoxynucleotides complementary to the fragments of yellow lupin tRNA(Phe) was tested in the aminoacylation of tRNA(Phe) and in the binding of Phe-tRNA(Phe) to poly-U-programmed eukaryotic ribosomes. Oligonucleotides tested in the aminoacylation test did not give any inhibition. Monomers and dimers did not have any significant influence on the binding assay, either. A different percentage of inhibition of the binding of Phe-tRNA to ribosomes has been observed for oligonucleotides. Heptamer complementary to the anticodon loop gave 100% inhibition of the binding reaction. However, the oligonucleotides complementary to both the anticodon loop and stem and longer than the heptamer were much less effective inhibitors. A high inhibitory effect was also observed for trimers and for the decamer complementary to the D-loop and CCA-end.

NMR and CD studies on an oligonucleotide containing N4-methylcytosine.

The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.

Simple and rapid procedure for synthesis of deoxynucleoside 3'-2-cyanoethyl-N,N-diisopropyl-amino phosphites.

2-Cyanoethyl-bis/N,N-diisopropyl/phosphoamidite can be prepared from PCl3 by a very fast and efficient procedure. Without purification it was used for the phosphitilation of suitably protected deoxynucleosides which after simple purification are obtained with a high yield, in a stable and easy to manage form.