NMR structure and backbone dynamics of the extended second transmembrane domain of the human neuronal glycine receptor alpha1 subunit

The structure and backbone dynamics of an extended second transmembrane segment (TM2e) of the human neuronal glycine receptor alpha(1) subunit in sodium dodecyl sulfate micelles were studied by (1)H and (15)N solution-state NMR. The 28-amino acid segment contained the consensus TM2 domain plus part of the linker between the second and third transmembrane domains. The presence of a well-structured helical region of at least 13 amino acids long and an unstructured region near the linker was evident from the proton chemical shifts and the pattern of midrange nuclear Overhauser effects (NOE). (15)N relaxation rate constants, R(1) and R(2), and (15)N-[(1)H] NOE indicated restricted internal motions in the helical region with NOE values between 0.6 and 0.8. The squared order parameter (S(2)), the effective correlation time for fast internal motions (tau(e)), and the global rotational correlation time (tau(m)) were calculated for all TM2e backbone N-H bonds using the model-free approach. The S(2) values ranged about 0.75-0.86, and the tau(e) values were below 100 ps for most of the residues in the helical region. The tau(m) value, calculated from the dynamics of the helical region, was 5.1 ns. The S(2) values decreased to 0.1, and the tau(e) values sharply increased up to 1.2 ns at the linker near the C-terminus, indicating that the motion of this region is unrestricted. The results suggest a relatively high degree of motional freedom of TM2e in micelles and different propensities of the N- and C-terminal moieties of the transmembrane domain to assume stable helical structures.     

Statistical analysis of noninferiority trials with a rate ratio in small-sample matched-pair designs

Testing of noninferiority has become increasingly important in modern medicine as a means of comparing a new test procedure to a currently available test procedure. Asymptotic methods have recently been developed for analyzing noninferiority trials using rate ratios under the matched-pair design. In small samples, however, the performance of these asymptotic methods may not be reliable, and they are not recommended. In this article, we investigate alternative methods that are desirable for assessing noninferiority trials, using the rate ratio measure under small-sample matched-pair designs. In particular, we propose an exact and an approximate exact unconditional test, along with the corresponding confidence intervals based on the score statistic. The exact unconditional method guarantees the type I error rate will not exceed the nominal level. It is recommended for when strict control of type I error (protection against any inflated risk of accepting inferior treatments) is required. However, the exact method tends to be overly conservative (thus, less powerful) and computationally demanding. Via empirical studies, we demonstrate that the approximate exact score method, which is computationally simple to implement, controls the type I error rate reasonably well and has high power for hypothesis testing. On balance, the approximate exact method offers a very good alternative for analyzing correlated binary data from matched-pair designs with small sample sizes. We illustrate these methods using two real examples taken from a crossover study of soft lenses and a Pneumocystis carinii pneumonia study. We contrast the methods with a hypothetical example.     

Structure-activity relationships in the hydrophobic interactions of polyphenols with cellulose and collagen

Polyphenol interactions with both cellulose and collagen in the solid state have been studied by using chromatography on cellulose and by evaluating the hydrothermal stability of the polyphenol treated sheepskin collagen. Twenty-four polyphenolic compounds were studied, including seven glucose-based gallotannins, five polyalcohol-based gallotannins, and twelve ellagitannins. In the cellulose-polyphenols systems, the polyphenol's affinity to cellulose is positively correlated with their molecular masses, the number of galloyl groups, and their hydrophobicity (logP). The polyphenol treatment increased the hydrothermal stability of collagen samples, and such effects are also positively correlated with the molecular masses, total number of galloyl groups and the hydrophobicity of polyphenols. Ellagitannins showed much weaker interactions with both biopolymers than gallotannins having similar molecular mass, the same number of galloyl groups, and the same number of phenolic hydroxyl groups. It is concluded that, for the polyphenol interactions with both cellulose and collagen, (1) the galloyl group of polyphenols is the functional group; (2) the strength of interactions are positively correlated with molecular size, the number of galloyl groups and the hydrophobicity of polyphenols; (3) the hydrophobic interactions are of great significance; and (4) the interactions are strongly dependent on the flexibility of galloyl groups.     

Inhibition of an activated Ras protein with genetically selected peptide aptamers

Mutant alleles of Ras maintain an activated, GTP-bound conformation and relay mitogenic signals that cannot be turned off. A genetic selection in Saccharomyces cerevisiae was used to identify peptide aptamers that suppress the growth arrest phenotype of an activated Ras allele. Peptide aptamers were expressed as C-terminal fusions to glutathione-S-transferase. Modifications that alter the coding capacity of the peptide aptamer indicate it is necessary for Ras2-Val19 suppression. Aptamer expression also reduces the elevated levels of cAMP and suppresses the heat shock sensitivity characteristic of Ras-activated yeast cells. The peptide aptamer retains suppressor activity when fused to thioredoxin. The peptide aptamer expression strategy described here indicates that aptamers presented as unconstrained peptides have functional capacity in vivo.     

The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Stability of reconstructed paralyzed shoulders using a reflected long head biceps technique

A new tendon transfer technique is proposed for the reconstruction of the paralyzed shoulders secondary to Brachial Plexus Injury (BPI). In this tendon transfer, the long head of the biceps tendons is utilized as a bridging tendon graft. It is reflected at the exit of the bicipital groove, passed through the deltoid and directed to the trapezius. The technique is referred to here as the Reflected Long Head Bicepts (RLHB) technique. This study evaluated the effect of this tendon transfer on the anterior, posterior, and inferior stability of the reconstructed should using cadaveric specimens. It was shown that loading of the RLHB contributed significantly to anterior stability of the reconstructed shoulder for 90 deg elevation in the scapula plane. The mean displacement was reduced by 56 percent with RLHB loaded (p<0.01), by 56 percent with the rotator cuff loaded (p <0.005), and by 67 percent with both the RLHB and the rotator cuff loaded (p<0.004). For the post-operation conditions, variation of the directions of RLHB had no significant effect on joint displacement in response to anterior loading. The RLHB tendon also contributed to the posterior and inferior stability for the low and middle elevations in the plane of scapula. Two variations of the RLHB tendon transfer procedures, namely the "Sub-Deltoid" and the "Through-Deltoid" techniques, were introduced and studied. These two techniques did not seem to have significantly different effects on the displacement of the humeral head in response to both posterior and inferior loading. The results of this study seemed to support the clinical feasibility of this tendon transfer approach as far as the biomedical stability of the reconstruction is concerned.