Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.     

Enhancing encoding capacity of combinational test stimulus decompressors

While scan-based compression is widely utilized in order to alleviate the test time and data volume problems,the overall compression level is dictated not only by the chain to channel ratio but also the ratio of encodable patterns.Aggressively increasing the number of scan chains in an effort to raise the compression levels may reduce the ratio of encodable patterns,degrading the overall compression level.In this paper,we present various methods to improve the ratio of encodable patterns.These methods are b...     

哈锅670 t/h锅炉炉内冷态流场数值模拟

对 HG-670 /1 40 -9煤粉炉燃烧器改造后的炉内冷态流场进行数值模拟 ,分析了炉内燃烧区烟气的速度分布及压力分布。结果表明该型号锅炉改造后在额定工况下运行时炉内可形成良好的切圆 ,无一次风贴壁现象 ,不会发生壁面结渣 ,同时避免了壁面处高温腐蚀现象的发生     

大鼠放射性皮肤溃疡组织中c-Fos、Rb蛋白表达研究

为研究大鼠放射性溃疡组织中ｃ－Ｆｏｓ、Ｒｂ表达。采用１４０只Ｗｉｓｔａｒ大鼠进行局部照射（γ射线）制备放射性皮肤溃疡动物模型,观察病变１年,然后采用ｃ－Ｆｏｓ、Ｒｂ多克隆抗体及免疫组化方法检测溃疡组织中ｃ－Ｆｏｓ、Ｒｂ的表达情况。结果表明：ｃ－Ｆｏｓ、Ｒｂ的表达阳性率分别为４５．８％（３３／７２）、６３．９％（４６／７２）,两者阳性部位主要见于增生肥大的鳞状上皮细胞胞核、增生的间质成纤维细胞胞质及     

Modification of haloperidol-induced pattern of c-fos expression by serotonin agonists

Acute challenge with clozapine and haloperidol produce different anatomical patterns of c-fos expression in the forebrain. The pharmacological profile of atypical antipsychotics suggests that serotonin might contribute to the unique therapeutic benefits of these drugs. In order to test this possibility, we examined the abilities of 5-HT1A and 5-HT2A/2c agonists to modify the pattern of c-fos expression induced by haloperidol and clozapine. Various groups of rats were pretreated with either saline, DOI, 8-OH-DPAT, and 8-OH-DPAT + DOI 30 min prior to haloperidol or clozapine administration. Rats were killed 90 min after antipsychotic administration. In saline-pretreated rats, haloperidol produced intense Fos-LI in all four striatal quadrants while the effect of clozapine was restricted to the medial part of the striatum. Prior administration of 8-OH-DPAT significantly reduced haloperidol-induced Fos-LI in all four striatal quadrants while DOI and 8-OHDPAT + DOI significantly reduced Fos-LI only in dorso- and ventrolateral quadrants. In the nucleus accumbens, haloperidol induced intense Fos-LI in the core and the shell regions whereas clozapine induced c-fos expression only in the shell. Pretreatment with 8-OHDPAT in haloperidol treated rats reduced Fos-LI in the core region yielding to a c-fos pattern similar to that induced by clozapine. In the prefrontal cortex of saline-pretreated rats, haloperidol produced a moderate c-fos expression compared with the intense expression produced by clozapine. Pretreatment with serotonin agonists before haloperidol brought the number of FOS-positive neurons to the same level as in clozapine treated rats. These results show the ability of 5-HT agonists to transform the typical pattern of c-fos expression induced by haloperidol into a pattern resembling that of clozapine.     

Dental embryology, anatomy, development, and aging

Equine practitioners should be knowledgeable of dental anatomy and development to enhance their skill of age estimation of horses. The permanent teeth of horses are continually undergoing changes in shape and appearance. These changes may be used to suggest a reasonable age range for a horse.     

Direct inhibition of the pancreatic beta-cell ATP-regulated potassium channel by alpha-ketoisocaproate

The ATP-regulated potassium (KATP) channel plays an essential role in the control of insulin release from the pancreatic beta-cell. In the present study we have used the patch-clamp technique to study the direct effects of alpha-ketoisocaproate on the KATP channel in isolated patches and intact pancreatic beta-cells. In excised inside-out patches, the activity of the KATP channel was dose-dependently inhibited by alpha-ketoisocaproate, half-maximal concentration being approximately 8 mM. The blocking effect of alpha-ketoisocaproate was fully reversible. Stimulation of channel activity by the addition of ATP/ADP (ratio 1) did not counteract the inhibitory effect of alpha-ketoisocaproate. In the presence of the metabolic inhibitor sodium azide, alpha-ketoisocaproate was still able to inhibit single channel activity in excised patches and to block whole cell KATP currents in intact cells. No effect of alpha-ketoisocaproate could be obtained on either the large or the small conductance Ca2+-regulated K+ channel. Enzymatic treatment of the patches with trypsin prevented the inhibitory effect of alpha-ketoisocaproate. Based on these observations, it is unlikely that the blocking effect of alpha-ketoisocaproate is due to an unspecific effect on K+ channel pores. Leucine, the precursor of alpha-ketoisocaproate, did not affect KATP channel activity in excised patches. Our findings are compatible with the view that alpha-ketoisocaproate not only affects the beta-cell stimulus secretion coupling by generation of ATP but also by direct inhibition of the KATP channel.     

A new method of readout in radiochromic film dosimetry

Radiochromic film as a dosimetry medium offers several advantages in high-resolution radiography. A new technique of readout was developed to measure the optical density distributions of the film in purely directed light. This technique implements radiochromic film dosimetry near the film's absorption maximum by using a single-mode top-surface emitting laser diode (675.2 nm). The effective sensitivity of the film, compared with a helium-neon laser densitometer (632.8 nm), is increased approximately threefold. Good accuracy, high spatial resolution and simple assembly of the readout system is achieved. Beam profiles of the four final collimator helmets of a Leksell Gamma Knife (Elekta Inc., Sweden) were experimentally determined. Measured profiles and full-widths at half maximum are consistent with the computer generated data of the dose planning system (Kula 4.4, Elekta Inc., Sweden). The output factor of the 4 mm collimator (the smallest collimator with the steepest dose gradient), essential for the application of well defined doses, was checked. The measurements established an output factor of 826 +/- 9 that lies 9 +/- 1% lower than the adjusted one.