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To evaluate spray-freeze drying and spray drying processes for fabricating micron-sized particles of darbepoetin alfa (NESP, Aranesp) with uniform size distribution and retention of protein integrity, requirements for encapsulation. |
Fluid-bed spray-coating process is widely used to prepare non-protein pharmaceutical solid dosage forms using macro-size seed particles (200-1000 microm) at kilogram batch sizes. In this study we developed a small-scale fluid-bed spray-coating process (20 g) to produce micro-sized vaccine powder formulations (40-60 microm) for epidermal powder immunization (EPI) METHODS: A bench-top spray coater was used to spray two vaccines, diphtheria toxoid (dT) and alum-adsorbed hepatitis-B surface antigen (Alum-HBsAg), onto crystalline lactose particles of 40-60 microm in diameter. Particle properties such as particle size, surface morphology, and degree of particle agglomeration were determined. Protein stability was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenicity of the vaccine was evaluated in vivo by needle injection and epidermal powder immunization (EPI) of mice or guinea pigs. |
A number of neurohormones and cytokines are activated in heart failure and their activity is related to the progression of heart failure. Inhibiting the deleterious effects of the activated sympathetic and the renin-angiotensin-aldosterone systems with b-blockers, angiotensin-converting enzyme inhibitors, and aldosterone blockers has been remarkably successful in improving the survival of patients with heart failure. However, inhibition of the other neurohormones and cytokines has not provided the expected incremental benefit, and in some cases has been deleterious, suggesting a ceiling effect of further neurohormonal blockade. This article reviews some of these studies and analyzes the possible causes of these observations. |
Left ventricular (LV) remodeling describes dynamic changes in ventricular size and shape that result from hemodynamic and metabolic insults to the failing heart. The remodeling hypothesis in heart failure asserts that LV remodeling is the central pathophysiologic lesion whereby alterations in cardiac structure are followed by impairment in function, with a wide range of genetic-environment interactions that determine the ultimate phenotype. Several therapeutic targets of LV remodeling have already been exploited (such as neurohormonal and cytokine activation). On the other hand, great efforts are still being made to understand the complex array of genetic and metabolic derangements. Nevertheless, we have realized that there is no single phenotypic change, protein expression, or signal-transduction pathway that is dominant in the process of cardiac remodeling. This implies that better characterization of this heterogeneous heart failure phenotype is desperately needed. |
Results of prospective trials of effects of various antihypertensive drugs on left ventricular hypertrophy in patients with hypertensive disease are reviewed. According to 2 meta-analyses angiotensin converting enzyme inhibitors are most effective inducers of regression of left ventricular hypertrophy. However in comparative randomized trials in patients with hypertension ability of diuretics, lipophilic beta-adrenoblockers, long-acting calcium antagonists, and angiotensin receptor blockers to cause regression of left ventricular hypertrophy was not inferior to that of angiotensin converting enzyme inhibitors. |
About 60% of patients with mild and moderate hypertension have insulin resistance and half of them have clinically manifest metabolic syndrome which comprises abdominal obesity, hyperlipidemia, impaired glucose tolerance, hypertension and insulin resistance. In a framework of metabolic syndrome hypertension is characterized by disturbed circadian profile without nocturnal blood pressure lowering and concentric left ventricular hypertrophy. There exist 2 mechanisms of linkage between hypertension and metabolic syndrome: impaired ion transport and neurohormonal and humoral activation. Antihypertensive drugs for correction of hypertension in metabolic syndrome should be long acting, provide protection of target organs, and induce positive or neutral metabolic effect. Together with normalization of blood pressure these actions can cause lowering of risk of atherosclerosis development. Representatives of the following classes of antihypertensive agents can be used as drugs of choice: angiotensin converting enzyme inhibitors, long-acting calcium antagonists, selective beta1-adrenoblockers, and thiazide diuretics. |
A library of fluorous, (1H,1H,2H,2H-perfluoroalkyl)silyl-substituted derivatives of triphenylphosphine, Ph(3-a)P[C(6)H(5-y)[SiMe(3-b)(CH(2)CH(2)C(x)F(2x+1))(b)](y)-pos](a) [a = 1-3; b = 1-3; x = 4, 6, 8, or 10; pos = 3, 4 (y = 1) or 3,5 (y = 2)], was prepared using parallel synthetic techniques. Upon variation of these four parameters, a total of 108 different fluorous phosphines can be synthesized. Using factorial design, 37 phosphines were selected and their partition coefficients in the typical fluorous biphasic solvent system PFMCH/toluene (PFMCH = perfluoromethylcyclohexane) determined. By fitting of the partition coefficient data to linear functions of the parameters a, b, and x, the partition coefficients of the remaining 71 fluorous phosphines, which were not prepared, could be predicted. Using this approach, some unexpected trends in the dependence of the partition coefficient on variations of the four parameters became clear, resulting in a better understanding of the optimum fluorous substitution pattern for obtaining the highest partition coefficient (P). In this way, the partition coefficient was increased by 2 orders of magnitude, i.e., from the initial value P = 7.8 for 1(3, 2, 6, C4) to P > 238 for 1(2, 3, 6, C3C5). Para- and 3,5-substituted phosphines showed irregular behavior in the sense that elongation or increase of the number of perfluoroalkyl tails did not necessarily lead to higher partition coefficients. Particularly high values were found for phosphines containing a total of 72 fluorinated carbon atoms on the meta position(s) of the aryl rings. Linear relationships were found between the predicted log P of 1(a, b, x, C4) and the experimentally determined log P values of fluorous diphosphines [CH(2)P[C(6)H(4)(SiMe(3-b)(CH(2)CH(2)C(6)F(13))(b))-4](2)](2) and monophosphines Ph(3-a)P(C(6)H(4)(CH(2)CH(2)C(6)F(13))-4)(a). One of the most fluorophilic phosphines, i.e., 1(3, 1, 8, C3C5), was applied and efficiently recycled in rhodium-catalyzed, fluorous hydrosilylation of 1-hexene by HSiMe(2)Ph using PFMCH as the fluorous phase and the substrates as the organic phase. It was demonstrated that a higher partition coefficient of the ligand in PFMCH/toluene at 0 degrees C indeed resulted in less leaching of both the catalyst and the free ligand during phase separation. |
To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. |
To investigate the killing effect on SKOV3 cells by fusion protein HER2-specific antibody-reversed caspase-3 in the secreted form. |
To prepare and characterize mAb to Fc fragment of Ig fusion protein, and to establish sandwich ELISA for detecting Ig fusion proteins and affinity chromatography method for Ig fusion protein purification. |
To investigate the effect of momordica anti-HIV protein of M(r ) being 30 000 (MAP30) on HBV expression by laser scanning confocal microscopy. |
To study the expression and localization of p27(KIP1)-GFP fusion protein in hepatocellular carcinoma cell line HCC-9204. |
To express fusion protein of GST and vWf binding domain(GP302) of platelet GPIbalpha in E.coli and its preparation of rabbit anti-serum. |
To construct, express and characterize the eukaryotic expression vector of encoding human CD226 (PTA1) extracellular region Ig fusion protein gene containing 3C protease-restricted site. |
To investigate the role of P38 mitogen-activated protein kinase (P38MAPK) in expression of P-selectin on human umbilical vein endothelial cells and diabetic atherosclerosis. |
To assess in a randomized open study effect of 12-15 week use of angiotensin converting enzyme inhibitors (ACEI) with and without trimetazidine on myocardial perfusion reserve in patients with ischemic heart disease (IHD) and/or hypertension associated with type II diabetes. |
To study the activation effect of BCR/ABL antigen on T cells from CML patients mediated by protein transduction domain (PTD). |
To express the fusion protein TGF-betaR II/Fc in large amounts by using recombinant Bac-TR II baculovirus expression system constructed by our laboratory and to purify and characterize it. Then, to verify whether the fusion protein TGF-betaR II/Fc can be able to block the biological activity of cytokine TGF-beta1. |
The C1423T polymorphism in von Willebrand factor-cleaving protease (vWF-cp) gene affects its enzyme activity. The present study was to investigate the polymorphism frequency among Chinese Han population and its relevance to arterial thrombotic disorders. |
To investigate the expression of VEGF mRNA and secretion of VEGF protein in NB4 and HL-60 cells affected by all-trans retinoic acid (ATRA) and daunorubincin (DNR) respectively. |
During last 10 years the potential interaction between aspirin and angiotensin converting enzyme inhibitors (ACEI) has received considerable attention. Retrospective analysis of randomized trials and their meta-analyses gave conflicting results concerning existence and effects of this interaction. Taking into consideration proven benefit of both aspirin and ACEI, restricting their combined use is premature. Further studies of this issue are warranted. |
E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells. |
In the present study we describe the evaluation of the effectiveness of gonad protection in diagnostic radiology based on the measurement of organ and the effective doses with and without lead clothing to gonads. We devised in-phantom dosimetry system and measured organ and effective doses in x-ray radiography and CT examinations with the new dosimetry system. From the data of organ and the effective doses we assessed the effectiveness of radiological protection by the use of lead clothing to gonads. Although in chest radiography and chest CT examinations, the effectiveness of radiological protection was not found, in the case of hip joint radiography (AP), gonad doses decreased remarkably by using lead clothing. The effectiveness of radiological protection, i.e. the ratio of the decreased dose to the dose value without protection, in testis and ovary were found to be 91.4% and 68.0%, respectively. It was also found that gonad doses observed with and without gonad protection were extremely lower than those of threshold for sterility recommended by the International Commission on Radiological Protection 60 (ICRP Publ. 60). |
SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the Institute of Physical and Chemical Research (RIKEN). The beamline consists of two experimental stations for protein crystallography and small-angle X-ray scattering. Both types of experiments can be carried out simultaneously, with dichromatic synchrotron radiation emitted from two coaxial undulators with vertical polarization. The branched beams are generated by a transparent diamond crystal. With synchrotron radiation, the multiple-wavelength anomalous-dispersion (MAD) method, which gives phases from a single anomalous scatterer, has been developed. Anomalous scattering contributes a small proportion of the diffraction intensity so that the accuracy of intensity data is important. The protein crystallography branch of RIKEN beamline I has been designed based on a 'trichromatic concept' to optimize MAD data collection. This concept requires the quasi-simultaneous collection, by use of a 'trichromator', of three intensity data sets at three different wavelengths from a single protein crystal without changing any settings. The main feature of the concept is the minimization of systematic errors in the measurement of anomalous diffraction for the MAD method. Initial commissioning of the beamline has provided three different monochromated undulator beams, which were successfully observed on the phosphor screen located at the near end of the trichromator. |
A two-dimensional microstrip gas chamber (MSGC) has been developed with a 10 cm-square detection area and an ultrafast read-out system. The MSGC was made using multi-chip module (MCM) technology, and has a very thin substrate of 17 micro m and many anodes and back strips, both with 200 micro m pitches. The new read-out system, in which the hit addresses of the electrodes were sequentially encoded to the hit positions by a synchronous clock, handles data rates of up to 10(7) events s(-1) from MSGCs. This enables the acquisition of fast and sequential digital images. Furthermore, since the MSGC is a real photon-counting detector, the timing of the photons, to an accuracy of a few tens of nanoseconds, and energy can be recorded. Here, the performance of the MSGC system as a real-time area detector is reported, and the abilities of this system are discussed. |
A multilayer monochromator was installed on a bending-magnet beamline at the Cornell High Energy Synchrotron Source (CHESS) and was used to provide an unfocused pseudo-monochromatic X-ray beam for protein crystallography experiments. Datasets were collected from lysozyme at room temperature and human methylthioadenosine phosphorylase at 100 K. The wide energy bandpass of the multilayer allowed short exposure times, typically only a few times longer than on a focused multipole wiggler beamline. The diffraction images were processed using unmodified monochromatic data-processing software to yield datasets of good quality. These first measurements demonstrate that multilayer monochromators can be readily applied to the rapid structure determination of many typical-sized macromolecules. |
A new multipole wiggler device has been designed for the 2.0 GeV Synchrotron Radiation Source at Daresbury Laboratory in the UK. The nine-pole 2.0 T device will provide radiation for two beamlines dedicated to protein crystallography, one of which will be of high intensity. This article provides details of the design of the two stations and outlines methods being developed to combine dealing with the high heat load from the radiation while allowing both stations to be built as close to the centre of the fan as possible. |
A novel X-ray beam-position detection device that we call a position-sensitive photoconductive detector (PSPCD) is designed to have synthetic diamond as its substrate material. We proved that it is feasible to use synthetic diamond to make a hard X-ray position-sensitive detector based on the photoconductivity principle and that it acts as a solid-state ion chamber. Experiments on different PSPCD samples using synthetic diamond with a high-heat-flux white undulator beam, as well as with monochromatic hard X-ray beams, have been performed at the Advanced Photon Source. Recent test results with the PSPCD in the quadrant configuration as an X-ray beam-position monitor and in a multipixel array as an X-ray beam profiler are presented in this paper. |
The first insertion-device beamline at the Pohang Light Source is designed for high-resolution spectroscopy and spectromicroscopy. The beamline will contain a variable-included-angle plane-grating monochromator (VIA-PGM) using a grating substrate which has seven different grooves with different depths. The advantages of this scheme will be the fixed exit-slit position and the mechanical stability of the grating scan mechanism while changing the photon energy range. The beamline is designed to cover the photon energy range 20-2000 eV. The estimated spectral resolution, E/DeltaE, is above 8000 in the photon energy range below 500 eV, and above 4000 for the remaining photon energy range. The estimated flux at the end-station is of the order of 10(12) photons s(-1) (0.1% bandwidth)(-1). |
A semitransparent Mo/Si multilayer beam splitter with a completely self-standing active area (10 x 10 mm) and a flatness of 1.1 nm (r.m.s.) was fabricated. The influence of the roughness of the membrane substrate on the reflectivity of a beam splitter was investigated for different materials and deposition schemes. Precise control of multilayer stress to give a slightly tensile state not only enables the fabrication of a large and flat reflection surface, but also makes it possible to etch away the supporting membrane and obtain a completely self-standing structure. The performance evaluation using synchrotron radiation revealed that the fabricated beam splitter works as a one-to-one beam splitter whose reflectivity and transmittance are both 27% (s-polarization, 45 degrees, lambda = 13.4 nm). |
The optical performance of platinum-carbon multilayers deposited onto different substrates has been examined. Specular reflectivity and non-specular diffuse scattering were measured to study the replication of substrate roughness into the multilayer structure. Surface topography was measured before and after deposition using a scanning probe microscope and a mechanical profiler. |
A UHV surface X-ray scattering system has been constructed at the SRRC, providing users with a state-of-the-art system for performing X-ray scattering studies of two-dimensional crystallography, in situ growth mechanisms as well as phase transitions of surfaces and interfaces. A study of the phase transition of the Si(001) reconstructed surface was conducted to commission both the scattering system and the SRRC X-ray beamline. The detailed design and performance of the SRRC surface X-ray scattering system together with the results of the Si(001) study are presented. |
As a basic layered structure for giant magnetoresistive (GMR) heads, NiFe/Cu/NiFe/Ta/Si substrate was measured by X-ray reflectometry at Cu Kalpha, Cu Kbeta and Cu K-absorption-edge energies. The accuracy of both the Cu thickness and the interface width between the upper NiFe and the Cu layers was found to improve in the order Cu Kalpha < Cu Kbeta < Cu K-edge. The final thickness and interface width values obtained from Cu Kbeta reflectivity are in good agreement with those from the Cu K-edge. The anomalous-dispersion effect is useful in the more accurate analysis of the layered structure of transition metal multilayers because it causes a large difference in the refractive indices of specific elements near the absorption edge. The Kbeta X-rays, which can be produced from conventional X-ray sources, are also available for the accurate analysis of reflectivity measurements. |
Accurate diffraction intensity data have been collected from a twinned P6(3) crystal of the 24-haem protein hydroxylamine oxidoreductase, from a nitrifying chemoautotrophic bacterium Nitrosomonas europaea, using synchrotron radiation at station BL6A of the Photon Factory. Estimation of the twinning fraction and deconvoluted intensity data, including native and heavy-atom derivative data, gave an improved Patterson function. Four diffraction data sets were collected from one crystal and an estimation of the twinning fraction to confirm the phenomena was undertaken. The successfully detwinned data sets were utilized in the structure analysis of the present enzyme. The mechanism of twinned-crystal formation is also discussed. |
To evaluate the application of the conversion-electron-yield (CEY) method in catalyst analysis, the intensities of the CEY and X-ray fluorescence (XRF) as a function of glancing angle were measured simultaneously. The probing depth of the CEY method is shallower than that of the XRF method. The CEY method also shows potential application for the analysis of even a powder specimen of a low-concentration zeolite catalyst. |
Angle-resolved UV photoelectron spectra (ARUPS) were measured for thin films of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) deposited on cleaved MoS(2) surfaces. The take-off angle (theta) dependence of the photoelectron intensity of the highest pi band showed a sharp maximum at theta = 32-34 degrees. A spectral feature of the binding energy at approximately 8.9 eV, which is believed to originate from a pi state, showed a remarkably different theta dependence from that of the pi band. A quantitative analysis of the observed theta dependencies clearly indicates that (a) the feature at approximately 8.9 eV originates from the oxygen 2p non-bonding states and (b) the molecules lie flat on the substrate surface. |
To investigate the changes in the pulmonary surface tension and the tissue content of surfactant substance protein B (SP-B) in rabbits during early post-injury stage after smoke inhalation injury. |
This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome. |
We have determined the Rubisco components and activity, whole leaf protein and amino acid components of thermo-sensitive mutant line 1103s of rice (Oryza sativa ssp. indica) leaves during induces green and yellow banding in this study. The results are as follows: The structure and components of Rubisco in the mutant are the same as in the wild form and relatively stable, but the activity of the mutant Rubisco greatly changes as a special protein of molecular weight 56.2 kD (PI=4.5) appears and disappears. When the green-yellow bands appear, the special protein disappears, and the activity of the mutant-rice Rubisco decreases, whereas when the green-yellow bands in the same part of the leaves disappear, the special protein appears and the activity of Rubisco is increase. The above shows the changes of the activity of the mutant-rice Rubisco during photosynthesis are closely related to the special protein in the leaves and its structure and components, and possibly to the regulating protein of Rubisco. The protein particularly regulates metabolic processes of amino acids preventing regulation of the preceding amino acids, such that the formation of the structural material in chloroplast is prevented, and finally the chloroplast thylakoid structure degenerates. |
The present study has determined the cellular distribution of cytochrome P450scc in human early placenta by immunohistochemistry and assessed the abundances of cytochrome P450scc protein in the villous tissue at 6-9 weeks of human pregnancy by Western blotting. The results showed that immunoreactive P450scc was mainly localized in the villous syncytiotrophoblast cells but not in the cytotrophoblast cells and the villous core, and that the expression of protein for cytochrome P450scc in human early placental villous tissues increased gradually with advancing weeks of pregnancy. Taken together, these findings suggest that the syncytiotrophoblast cells are the major site expressing P450scc in human early placenta, and that increasing expression of cytochrome P450scc in placental villi might establish a foundation, in terms of enzymology, for site-shift of progesterone biosynthesis from the corpus luteum to the placenta during human early pregnancy. |
According to results of multiple studies depression and anxiety are found in more than 50% of patients with hypertension. Presence of affective disorders elevates risk of progression of hypertension. That is why complex therapy comprising antihypertensive drugs from various groups and antidepressants takes on higher and higher significance. Modern antidepressants produce no substantial cardiotoxic effects. Combination of an antihypertensive drug (angiotensin converting enzyme inhibitor captopril or beta-adrenoblocker metoprolol) with an antidepressant affects favorably clinical course of hypertension, 24-hour blood pressure profile, characteristics of intracardiac hemodynamics, structural and geometric left ventricular parameters, allows to achieve sufficient antihypertensive effect with acceptable tolerability. |
To elucidate effect of beta-blocker bisoprolol on hibernating myocardium in patients with congestive heart failure (CHF) of ischaemic etiology without concomitant use of angiotensin converting enzyme inhibitors. |
In order to shed light on the mechanisms of TGF-beta action in the testis,we examined the expression and function of Smad4 protein, the common-mediator Smads, which is one of intracellular signaling molecules of TGF-beta superfamily members, in rat testis during postnatal development. Whole testes were collected from SD rats aged 3 days, 7 days, 14 days and 28 days, and adult. In this study, we examined, by means of western blots, the protein expression of Smad4 during rat testicular development and its cellular localization by immunohistochemical ABC method with glucose oxidase-DAB-nickel enhancement technique. The results showed that the protein of Smad4 was present in rats from 3 days of age to adulthood, and the immunoreactivity for Smad4 was exclusively localized to the cytoplasm of Leydig cells with negative nuclei in the interstitial tissue at any time point. No expression was detected in germ cells. Therefore, our data provide evidence for the molecular mechanism of TGF-beta action in rat testes during postnatal development and spermatogenesis of rats. |
The localization of the new estrogen receptor, ER-beta, in the rat brain was studied by immunocytochemical technique, and the results revealed that ER-beta immunoactive material was predominantly localized in the neuronal nucleus, but it was also detectable in the cytoplasm and neuronal processes. High levels of ER-beta immunopositive signals were detected in the cerebral cortex, vertical limb of the diagonal band, Purkinje cells, locus ceruleus, and motor trigeminal nucleus. Moderate levels were found in the medial septum, lateral amygdaloid nucleus, substantia nigra, and central gray. Weak signals were localized in some subregions of the hypothalamus and amygdaloid complex. Some differences of the expression of ER-beta immunoreactivity between male and female rats were also noticed. The above results provide the first detailed evidence that ER-beta protein is widely distributed in the rat brain, and ER-beta may be involved in some important brain function such as learning and memory. |
The centrosomal protein, Cenexin, is a molecular marker of mature centriole. To elucidate the variability and function of mature centriole in spermatogenesis, the high titer polyclonal antibody against rat cenexin was obtained by immunizing mice with recombinant cenexin which was made up in this study. The expression of cenexin in rat spermatogenesis was carried out by semi-quantitative RT-PCR, immunofluorescence and Western Blot. The results demonstrated that the level of Cenexin mRNA was higher in spermatogonia and spermatocytes, then decreased in following stages, while Cenexin protein was located on one centriole from spermatogonia to spermatids, showing mature centriole existed in these stages. Cenexin protein was localized to the basal body of the flagellum in elongated spermatids and the stained signal disappeared in the most of epidydimal sperms. These results suggested that the expression pattern of cenexin in rat spermatogenesis might be related to the initiation of the flagella formation. |
Differentially expressed genes between normal liver and hepatocellular carcinomas were investigated using differential display. In previous study, human F-LANa was identified as a differentially expressed gene, up-regulated in hepatocellular carcinoma. In this study, we developed an in silico cloning approach to rapidly and accurately characterize the mouse ortholog of the human F-LANa. Mouse F-LANa encodes a 239 aa protein exhibiting 97.9% similarity to the human ortholog gene. Homology analysis was carried out in various species and showed that F-LANa was evolutionarily conserved from yeast to human. Based on the alignment results, phylogenetic tree was established here. |
The most commonly observed change that has been linked to resistance development is the increase in activity of carboxylesterases. The putative mechanism involves an overproduction of this enzyme for the sequestration and the hydroxylation of various organophosphate and carbamate insecticides. Carboxylesterases A2 cDNA was amplified from Culex quinquefasciatus by RT-PCR and sequenced consequently. Target gene was inserted into pET-28a to create prokaryotic expression plasmid pET-EstA2. When pET-EstA2 was transformed into E. coli BL21, the recombinant was induced by IPTG. A pure recombinant protein was obtained by affinity purification. Compared with carboxylesterase A2 purified from Culex quinquefasciatus, carboxylesterase A2, purified from the product of the transgenic of E. coli, has the same Km, but the Vm was higher than that of it, which shows that carboxylesterase A2, purified from the product of E. coli by affinity, is purer than that from Culex quinquefasciatus. The study on the expression and characterization of carboxylesterase A2 in E. coli is more useful for its future application. |
In order to explore the role of MNSFbeta in the process of implantaion, MNSFbeta and its antibodies are required. The expression plasmid pBV220/MNSFbeta-hCGbeta was constructed, and then transferred into E. coli to express the fusion protein MNSFbeta-hCGbeta. The anti-hCGbeta antibody was used to identify the fusion protein. The result demonstrated that MNSFbeta-hCGbeta was expressed correctly and its molecular weight was consistent with the anticipated one. Finally the expression product MNSFbeta-hCGbeta was preliminarily purified and used to immunize Balb/C mouse to generate the antibodies. In the meantime, the expression plasmid pGEX-4T-2/MNSFbeta was also constructed and transferred into E. coli to express the fusion protein GST-MNSFbeta. GST-MNSFbeta was purified and used to stimulate the immunized mouse before the preparation of hybridomas cells. The prepared polyclonal and monoclonal antibodies against MNSFbeta were checked and measured by fusion protein GST-MNSFbeta. The prepared polyclonal antibody was then used to perform the immunohistochemistry analysis. The result suggested that the level of MNSFbeta in interimplantation sites was significantly higher as compared with implantation sites in the mouse uterine on Day 4.5 of pregnancy. |
In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed. |
Bt-CpTI fusion protein gene was transferred to the explants of hypocotyl, cotyledon with petiole and shoot apex of cabbage (Brassica oleracea L. var. capitata) variety "Zhonggan No 8" via Agrobacterium tumefaciens and particle bombardment, and 13 kanamycin-resistant plants were obtained. PCR and Southern blotting hybridization verified that all these plants of the kanr plants of type I regenerated from hypocotyl and cotyledon with petiole mediated by A. tumefaciens were transgenic plants, 2 karr plants of type II stemed from shoot apex mediated by A. tumefaciens were "false-positive"plants and one of 2 kanr plants of type III regenerated from shoot apex via particle bombardment was non-transformed plants. It was showed that part of transgenic plants had high activity of trypsin inhibitor and strong resistance to resist common cabbage worm through the analysis of CpTI relative capacity and insect-resistant test. |
The term RNA editing is generally used to describe those molecular processes in which the information content is altered in an RNA molecule. This process is not limited to mRNA since alterations of non-informational RNA have also been found. RNA editing exists extensively in the higher plant mitochondria, and is the necessary step for forming functional proteins. In this paper, the research materials are the gametopthyte male sterility line (A), maintainer line (B) and F1 hybrid (F1) of HL-type CMS. 15 editing sites are found in the transcripts of coxII by comparing cDNAs and DNAs sequences. A,B and F1 have same Editing sites. When editing occurs at the first or second position of codons, the encoded amino acid is likely to be altered. As a result, the conservation of the predicted protein is improved as compared with other organisms. |
The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12. |
Fluo-3/AM, a calcium indicator, was introduced by low temprature loading method into callus protoplasts of Stevia rebaudiana Bertoni. The microscope observation results showed that NaCl in different concentrations (30-200mmol/L) can elevate the intracellular calcium concentration in protoplasts. The elevation was related to the amount of salinity. The effect of salinity stress was inhibited by LiCl pretreatment but restored by inositol pretreatment. This suggested that salinity stress promoted the cytoplasmic calcium activity, perhaps by activating the phosphoinoditide system. As a result of salinity stress signals, elevated calcium activity may trigger corresponding metabolic changes, such as synthesis of enzyme via activating other members of calcium signal system, fit the cells for the change of the environment. |
The bcl-2 protein, which widely expressed in the developing central and peripheral nervous systems, has the functional role of blocking apoptosis. The purpose of this study was to map bcl-2 expression in the human enteric nervous system and investigate the value of bcl-2 immunohistochemical method in the diagnosis of Hirschsprung's disease (HD), as this has not previously been done. Rectal specimens were obtained from definitive operation of 20 patients with HD. Specimens were analyzed with immunohistochemical methods, using antibodies against bcl-2. The bcl-2 protein was expressed in myenteric and submucous neurons in normal adult and HD expand segment, but no bcl-2 immunoreactive enteric neurons was revealed in the narrow segment. And nerve fibers of the enteric plexuses that were bcl-2 immunoreactive were few in all examined specimens. From the conclusion, expression of bcl-2 is displayed in enteric neurons and immunohistochemical analysis of bcl-2 may also be valuable for identification of the enteric neurons in HD. |
The study on proteome of human cancer is helpful to explain its pathogenesis and make good effect on its prevention and cure. We compared the 2-DE maps of whole proteins of human lung cancer line A549 at 37 degrees C, 42 degrees C and 45 degrees C for the purpose of studying the expression of its heat shock proteins. Three temperature-sensitive differential spots were obtained and named as P1, P2, P3, respectively. Analyzed by MALDI-TOF-MS and Peptident software searched in the SWISS-PROT database, the three differential proteins were elementarily identified. P1 matches with two proteins belong to the Aldo-keto reductase family, P2 may be a new protein and P3 is Zinc finger protein 11A. |
The phosphorylation of the fibroblast growth factor receptor (FGFR) kinase substrate SNT1 (also called FGFR substrate 2, FRS2) by FGFR tyrosine kinases is both host cell- and receptor isotype-specific. To study the determinants of the host cell-specific phosphorylation of SNT1 by FGFR1 tyrosine kinase, we constructed a chimeric receptor FGFR2IIIb/R1 that consisted of an FGFR2IIIb ligand-binding ectodomain and an FGFR1 tyrosine kinase domain. The chimeric FGFR2IIIb/R1 kinase mediated robust phosphorylation of SNT1 immediately after transfection in mouse 3T3 cells where the FGFR1 kinase was residential, and in proliferative aged prostate tumor epithelial cells (DTE-R1/100) that ectopically expressed FGFR1 kinase. This is in contrast to the fact that the robust phosphorylation of SNT1 by ectopic FGFR1 kinase is an acquisition property in DTE premalignant prostate epithelial cells, which normally do not express FGFR1. The data suggest that the microenvironment of intracellular, rather than components in the extracellular compartment, of host cells is important in permitting FGFR1 kinase to strongly phosphorylate SNT1. Together with our previous data that the acquisition of SNT1 phosphorylation activity is concurrent with the acquisition of mitogenic activity of FGFR1 in prostate epithelial cells, the results here further demonstrate that the phosphorylation of SNT1 is host cell specific and that alterations induced by chronic exposure to ectopic FGFR1 kinase are involved in acquisition of SNT1 phosphorylation activity to the ectopic FGFR1 in prostate epithelial cells. |
Pot experiment was used to study the responses of membrane protection enzyme system of tobacco leaves on Hg, Cd and Pb stresses in soil. The results showed that POD activity gradually increased with increasing concetrations of Hg, Cd and Pb. CAT and SOD activity gradually decreased under three heavy metals common existing and SOD variation curve showed unimodal curve under single or two elements existing with increase of concentration of Hg, Cd and Pb. The effects of Hg, Cd and Pb in soil: three elemets together > two elements together > single element only. The effects resulted in an imbalance--activated oxygen produce and scavenge and physiological biochemical process disorder. There was a synergistic action for the effect of Hg, Cd and Pb in soil on membrane protection enzyme system in tobacco leaves. |
Chalcone synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. The production of flower pigment is specifically regulated by the activity of CHS. We cloned the cDNA sequence of CHS-A gene from Narcissus by PCR and analyzed the coding sequence of gene. The result demonstrated that the sequence of the coding region was 1167bp, encoding a protein of 389 amino acid which was more than 80% homology with CHS of the other 8 plants, such as Nicotine abacus and Solana tuberosum. |
Increases of functional T-type calcium channel (T-channel) expression have been associated with cellular proliferation although evidence for this remains controversial. In the present study, we have used a variety of cellular, molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation. The results showed that stable over-expression of alpha1G T-channel subunit in HEK-293 cells conferred a significant growth advantage. Thus, cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants, compared to control cultures (22.1 +/- 1.1 h) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/G1 phases of the cell cycle compared to controls. The selective T-type calcium channel blocker, mibefradil, induced a dose-dependent inhibition of proliferation in alpha1G tansfectants. Furthermore, the Western blotting results proved that the level of protein expression of CDK2, cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures. Our results demonstrate that the T-type calcium channel provides a significant growth advantage to HEK-293 cells that might occur via effects on the G1/S cell cycle mechanism. |
Abscisic acid (ABA) was critical in plant seed development and response to environmental factors such as stress situations. To study the possible ABA related signaling transduction pathways, we tried to isolate the ABA-regulated genes through fluorescent differential display PCR (FDD-PCR) technology using rice seedling as materials (treated with ABA for 2, 4, 8 and 12h). In the 17 fragments isolated, 14 and 3 clones were up-and down-regulated respectively. Sequence analyses revealed that the encoded proteins were involved in photosynthesis (7 fragments), signal transduction (1 fragments), transcription (2 fragments), metabolism and resistance (6 fragments), and unknown protein (1 fragments). 3 clones, encoding putative alpha/beta hydrolase fold, putative vacuolar H+ -ATPase B subunit, putative tyrosine phosphatase, were confirmed to be regulated under ABA treatment by RT-PCR and northern blot analysis. FDD-PCR and possible functional mechanisms of ABA were discussed. |
Silver crucian carp (Carassius auratus gibelio) has been known for its gynogenetic reproduction. In this paper, eggs from clone F of gynogenetic silver crucian carp were inseminated respectively by sperms of clone D, clone A and common carp, and polymorphic patterns of transferrin and isozymes of the produced three offspring FD, FA and FL were studied comparatively to explore the diversity in reproductive modes of silver crucian carp. As control, zymograms of FL progenies exhibited a maternally clonal inheritance, and gynogenesis was reconfirmed. However, differentiation of morphs and electrophoretic patterns was both observed among the FD progenies, and detection of bands specific for the clone F or clone D in some FD individuals urged the occurrence of recombination. Furthermore, extreme linkage disequilibrium for different protein loci suggested that linkage groups composed of different genes might function as the fundamental unit in the recombination. With respect to FA group, phenotypes of parental clones were both detected in the F2 generation (FA x FA progenies) while only maternal phenotypes were detected in the F1 generation. It appeared that both the parental chromosome sets could be transmitted to the offspring but expression of paternal genes were fundamentally upset in the F1 generation because of incompatibility of the parental genes. Generally, genetic analysis of FD and FA offspring primarily exhibited a particular syngamy for silver crucian carp. Besides the clonal reproduction of gynogenesis, syngamy could provide opportunities of recombination for silver crucian carp, which might remove some genetic loads from the genome and introduce new genotypes. Diversity in reproductive modes might play an important role in ecological adaptation of silver crucian carp. |
The ultrastructures of storage protein accumulation in cotyledons and aleurone layer in buckwheat (Fagopyrum esculentum Monch) were investigated by electron microscopy. (1) On 15 days after anthesis (DAA), there are many dictyosomes and vacuoles accumulating protein in cytoplasm of the outer layer endosperm cells. These cells containing abundant aleurone grains (1-2 microm in diameter) from vacuoles accumulated storage protein form aleurone layer where division of aleurone grain is also observed on 25 DAA. (2) On 20 DAA, protein begins to accumulate in vacuoles of cotyledon cells. At the early stage of protein deposition, vacuoles are becoming smaller ones by ingrowth of tonoplast or by pinch-off. A multitude of ribosomes, many dictyosomes and electron-dense vesicles (0.1-0.7 microm in diameter) coated with membrane are observed in cytoplasm of cotyledon cells at every stage of protein accumulation. Originated from the separation of saccules containing protein at the fringe of dictyosome, these vesicles (0.1-0.2 microm in diameter) probably increase their volume by fusion each other. They play the leading role in transporting storage protein into vacuoles that become PSVs in cotyledon cells of buckwheat. On 25 DAA, there are many PSVs (1-3 microm in diameter) and some vesicles (0.1-0.7 microm) filled with protein in mature cotyledon cells. These vesicles may be also play a part in accumulating storage protein in mature buckwheat seed. |
Congenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive inherited disorder, characterized by deficiency of adrenal and gonadal steroid hormones. Recent studies have shown that mutations in the gene for steroidogenic acute regulatory protein (StAR) cause this most severe genetic disorder in steroid hormone biosynthesis. StAR is a mitochondrial protein promotes cholesterol transfer from outer mitochondrial membrane to the inner mitochondrial membrane, where the cholesterol serves as a substrate for P450scc and initiates steroidogenesis. So far, more than 30 different mutations in the StAR gene have been found in the patients with CLAH from various ethnic groups. None of CLAH patients in the Chinese population has been previously reported. In the present study we analyzed the StAR gene in a Chinese patient with CLAH. |
To investigate the relationship of Fas mRNA and protein expression and apoptosis in human oral squamous cell carcinoma. |
While studies of schistosome antigens have proceeded rapidly over the past ten years, studies of schistosome enzymes have also been increasing apace. Now the two `lines' of research are coming together. Parasites such as schistosomes hardly present antigens merely to stimulate a host immune response, so it is not surprising that many antigens turn out to have functions, for example, as enzymes. One type of antigenic schistosome enzyme - glutathione S-transferase - already shows promise as a vaccine candidate. Here, Jim McKerrow and Mike Doenhoff review another important class of enzyme, many of which are clearly antigenic, the schistosome proteases. |
Neonatal septicemia is a critical disease in neonatal period. Its incidence among live births is between 1 per thousand and 8 per thousand. Mortality of neonatal septicemia may be as high as 50% for infants who are not treated. The early signs of septicemia in the newborn are generally nonspecific. Blood culture and the other clinical diagnostic measures are not sufficiently sensitive. The present study aimed at evaluating potential use of soluble intercellular adhesion molecule-1 (sICAM-1), procalcitonin (PCT) and C-reactive protein (CRP) in diagnosis of septicemia. |
To enhance the immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3, the secreted 6his-hCGbeta-C3d3 fusion protein and 6his-hCGbeta were expressed in CHO cells and purified with Ni(2+)-chelating chromatography and Sephadex G-150 column. Then we investigated the potential of three copies of C3d as the molecular adjuvant of hCGbeta protein antigen. The antibody response to hCGbeta-C3d3 conjugates was compared with those resulting from immunization with hCGbeta alone and hCGbeta plus CFA/IFA in BALB/c mice. Our results showed that the antibody titer of hCGbeta-C3d3 was 1995-fold higher than that of hCGbeta alone and the anti-serum was capable effectively neutralizing the bioactivity of hCG. The immunity-enhancing action of C3d3 was 10-fold (primer) and 32-fold (booster) greater than CFA/IFA. These results indicated that C3d3 conjugates might be a better way to overcome the poor immunogenicity of hCGbeta contraceptive vaccine. |
A full length cDNA clone encoding invertase inhibitor was isolated by RT-PCR combined with 5' RACE from potato (S. tuberosum) tubers of cv. JH and designated as St-inh. The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids. The DNA fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E. coli. Co-incubation of the proteins produced by St-inh in E. coli and the invertase extracts from potato tubers of cv. E1, JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively. These results indicated that products of St-INH protein had a function of invertase inhibitors. The analysises of the nucleotide and amino acid sequences using BLAST and T-COFFEE demonstrated that St-inh cDNA was of over 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein [L, I, V, M]-X-D-X-[E, D, N, T, Y-[D, G]-[R, K, H, D, E, N, Q]-X-[L, I, V, M]-X(5)-Y-X-[L, I, V, M. Therefore, it was conjectured that St-inh could be a member of Kunitz-type gene family. |
To study the pathogenesis of dysosphresia in patients with chronic sinusitis, to discuss the expression and significance of olfaction marker protein in olfactory mucosa. |
Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 resulted in a 6-fold reduction of promoter activity. The promoter activity is not affected if these sites are mutated and cannot be methylated. Reactivation of MAT1A correlated with demethylation of +10 and +88. DNase I footprinting analysis using the probe containing nucleotides -346 to +160 and human liver nuclear extract or recombinant TATA binding protein (TBP) showed that HpaII methylation at +10 and +88 prevented TBP binding to the TATA box, but not if the sites were mutated. ChIP analysis confirmed TBP binding to MAT1A only in MAT1A expressing cells. Collectively our data support the novel finding that methylation of the MAT1A coding region can influence TBP binding to the TATA box and shut down gene transcription. |
To elucidate relationships between blood levels of matric metalloproteinases (MMP), C-reactive protein (CRP), markers of thrombinemia, and development of restenosis after percutaneous coronary interventions (PCI) in patients with stable angina. |
To investigate the expression and distribution of extracellular-regulated protein kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) under the mechanical pressure in rabbit mandibular condylar chondrocytes (MCC). |
The surface of oral mucous is highly specialized stratified squamous epithelium that plays an important role in protecting the tissue beneath it from physical, chemical or biological damages. However, the change of the epithelium structure as well as the related deficiency in functions due to some genetic or immunological factors have already become the obstacle of patients' life quality and clinical treatment. Recently, with the development of molecular biology and genetic technology, more and more researches have been done on the epithelia microstructure, and a better understanding has been made not only of the epithelia constructive protein which takes an important part in maintaining the integration and functions of the epithelium tissue, but also of the pathology of some human hereditary and acquired diseases related to the abnormality of epithelium constructive protein. In this way, it provides a new direction for the diagnosis and therapy of this kind of diseases. |
In order to illustrate the possible roles of PML-RARalpha protein in arsenic trioxide (AsO3)-induced NB4 cell apoptosis. |
To detect the expression of glycosyl-phosphatidyl-inositol (GPI) anchored protein on the blood cell membrane and its implication in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). |
To investigate the Factor V Leiden mutation associated with activated protein C resistance (APCR) in Chinese. |
To explore the regulatory mechanism of iron responsive element binding protein (IRE-BP) in iron metabolism. |
Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function. |
To study the oncogene transcriptor c-myc, stimulatory protein 1 (SP1) expression in ameloblastoma (AB) and their relation with telomerase reverse transcripase (hTERT), and to investigate the clinical biological characteristics of AB. |
Dilated cardiomyopathy (DCM) is characterized by enlargement and dilation of all heart compartments associated with serious decrease of its contractile function. DCM hallmark is the combination of dystrophic and hypertrophic alterations of cardiomyocytes. Since the power output of cardiac cells is directly related to remodeling of their contractile machinery we investigated expression of selected contractile and cytoskeletal proteins in the left ventricle of DCM patients using immunoblotting. The content of the recognized protein markers of cardiomyocyte hypertrophy such as tubulin, desmin and slow skeletal myosin heavy chain isoform, MHCbeta, was significantly elevated in DCM compared to normal myocardium. In addition, marked increase in the content of several smooth muscle proteins (smooth muscle alpha-actin, Myosin Light Chain Kinase, Kinase Related protein SM22) that are normally expressed in embryonic myocardium, was observed in DCM hearts. Thus, cardiomyocyte hypertrophy in DCM is associated with activation of embryonic protein expression program and smooth muscle proteins could serve as markers of this process. Understanding their involvement in sarcomere assembly and pathways of their expression activation during cardiac hypertrophy may bring new insights in treatment of various forms of cardiomyopathy. |
In part V of a series of papers on epidemiology and drug prevention of stroke and other thromboembolic complications of atrial fibrillation the authors analyze data of randomized trials exhibiting ability of long term use of beta-adrenoblockers, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists and statins to prevent atrial fibrillation. They also discuss results of short term studies demonstrating improved efficacy of electrical and pharmacological cardioversion in patients with atrial fibrillation after pretreatment with verapamil, beta-adrenoblockers, and angiotensin receptor antagonists, and present data indicating that monotherapy with verapamil, beta-adrenoblockers, angiotensin receptor antagonists and statins can facilitate maintenance of sinus rhythm after cardioversion in patients with persistent atrial fibrillation. |
A series of hyperbranched poly(amine-ester)s were synthesized using one-step and pseudo-step procedure respectively, and their molecular structures were characterized by IR, hydroxyl value and viscosity. These polymers were coated on the inner surface of the fused-silica capillaries in a chemical bonding process. The electroosmotic flow and the separation capability for basic proteins of the coated capillaries were investigated. The results demonstrated that the coating in the capillaries could greatly reduce the electroosmotic flow and effectively suppress protein adsorption in the pH range of 3-7. The separation efficiency of the coated capillary of hyperbranched poly(amine-ester)s with trimethylolpropane and pentaerythritol as the core was 10(5)/m and 10(7)/m, respectively. The separation efficiency and the stability of basic proteins on these coated capillaries were found excellent. |
Hyperphosphorylated microtubule-associated protein tau is the major protein component of neurofibrillary tangles in the brain of patients with Alzheimer's disease (AD). Until now, there is no effective cure to arrest this hyperphosphorylation. The present study was designed to explore the in vivo preventive effect of melatonin on Alzheimer-like tau hyperphosphorylation. Isoproterenol, a beta-receptor agonist, was used to induce tau hyperphosphorylation, and for preventive effect of melatonin, the rats were injected intraperitoneally with melatonin for 5 d before hippocampi infusion of isoproterenol. The level of tau phosphorylation was detected by Western blot and immunohistochemistry using sites specific antibodies (PHF-1 and Tau-1), and it was normalized by non-phosphorylation dependent total tau antibody (111e). The results by Western blot showed that the immunoreaction of tau at PHF-1 epitope was enhanced, and the reaction at Tau-1 epitope was weakened significantly at 48 h after injection of isoproterenol, suggesting hyperphosphorylation of tau at Ser 396/Ser 404 (PHF-1) and Ser199/Ser 202 (Tau-1) sites. Similar results were observed by immunohistochemistry staining, in which hyperphosphorylated tau was mainly detected in mossy fibers of hippocampal CA3 region. Pre-injection of rats with melatonin intraperitoneally arrested effectively the isoproterenol-induced tau hyperphosphorylation at both Tau-1 and PHF-1 sites, implying the preventive effect of melatonin in Alzheimer-like tau hyperphosphorylation. |
Increased vascular endothelial growth factor (VEGF) biosynthesis in vascular endothelial cells has been reported to play an obligatory role in promoting angiogenesis. Nevertheless, the intracellular signaling mechanisms of hypoxia-induced VEGF release remain largely unknown. Human umbilical vein endothelial cell lines (ECV304) were cultured in normoxic or hypoxic conditions for 12 approximately 24 h and harvested for determination of VEGF mRNA expression and phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase (p38 MAPK) by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Secreted VEGF protein was measured by enzyme-linked immunosorbent assay (ELISA). It has reported that PD98059, an ERK inhibitor, was able to blunt the hypoxia-induced activation of the expression of VEGF gene. In accordance with this report, an increase in ERK1/2 phosphorylation and VEGF biosynthesis was observed in ECV304 cells cultured in hypoxia, and this increase was blocked by PD98059. The novel finding of the present study is that an activation of p38 MAPK is involved in hypoxia-induced increase in VEGF biosynthesis. SB202190, an inhibitor of p38 MAPK was able to blunt the hypoxia-induced increase in VEGF biosynthesis. These dada provide the first direct evidence for a role of p38 MAPK in mediating hypoxia-induced increase in VEGF biosynthesis in human endothelial cells. |
A reversed-phase HPLC assay has been developed for measuring cefotaxime (CTX) in human serum. A solid-phase extraction column (Bond Elute C18) was used for the pre-purification of sample. Serum (200 microL) was mixed with 100 microL of internal standard (acetylamino phenol) solution and 200 microL of sodium acetate buffer (0.01 mol/L, pH 5.8), loaded onto the conditioned column and washed with 2 mL of acetate buffer, the compounds were eluted with 0.5 mL x 2 of methanol-acetate buffer (40:60, V/V ). The eluate (20 microL) was injected directly into an Apex ODS column (250 mm x 4.6 mm i.d., 5 microm) with a mobile phase of acetate buffer (0.01 mol/L, pH 5.8)-methanol (80:20, V/V ), detection was achieved at 254 nm. The average extraction recoveries were 96.7% and 97.7% for CTX and internal standard respectively, which were higher than that of CTX (91.0%) obtained by protein precipitation with methanol. The solid-phase extraction method also provided much clearer samples for HPLC analysis. The concern of acid degradation of CTX when an acid is used as a protein precipitation agent was avoided. The calibration curve of CTX was linear between 10 and 150 mg/L of serum concentration with a correlation coefficient of 0.9992, and the minimum detection limit was 2 mg/L. The precision was tested with three sample concentrations, and within-day RSD below 3.0% and day-to-day RSD below 4.1% were achieved. Maximum serum concentrations of 25.95 and 22.89 mg/L were measured at 45 min after intramuscular injection of 1 g CTX to two healthy volunteers respectively. The chromatographic behaviour of CTX was also studied. |
A rapid high performance liquid chromatography for determination of demethylvancomycin in neonate serum has been developed. The procedure involved a simple protein precipitation by acetonitrile-isopropanol (1:1) and then the sample was chromatographed on a reversed phase C18 column with UV detection at 236 nm. The mobile phase was CH3CN: 0.05 mol/L KH2PO4 = 8:92 (V/V). The calibration curve was Y = 35,721.89X - 13,031.54, r = 0.9998 and he detection limit was 0.3 mg/L. The average recovery was 94.7% +/- 1.2%. Intra-day and inter-day RSD were 2.23% and 2.62% respectively. It can be concluded that this method meets the requirement for routine clinical application. This method has been used to determine serum concentration of demethylvancomycin in neonates. The data obtained showed that the method was simple, rapid, sensitive and precise. |
Based on inorganic matrix controlled pore glass (CPG) and macro-pore silica sphere, by using polyethylene glycol (PEG 1000) as a ligand, a preparation method of hydrophobic interaction chromatographic (HIC) packing material was improved by adding a proper catalyst during the bonding process. The packing material can be synthesized in a scale-up batch, for example 150g for each batch, both for analytical and preparative columns. The retention of proteins, such as cytochrome C (Cyt-C), chymotrypsingen-A (Chy-A), lysozyme (Lys) and ribonuclease(Rnase), is increased with the increasing of (NH4)2SO4 concentration in the eluant 2.5 mol/L of salt concentration for the mobile phase was chosen by considering the separation efficiency and equipment life. After comparing the effect of pH for the retention of proteins it is found that the proteins are well separated at pH 7. The time of linear gradient elution program was optimized in considering the separation efficiency and speed. It is better to take 30 minutes of the gradient program for the separation. Six standard proteins can be well separated with the high-performance HIC column in the linear gradient elution program from 2.5 to 0 mol/L of (NH4)2SO4 in 50 mmol/L of phosphate buffer solution within 30 minutes. Cyt-C, Rnase, Lys and Chy-A can be separated by the HIC column based on CPG matrix. Six proteins, Cyt-C, Rnase, Lys, Chy-A, insulin(Ins) and lipase (Lip) can be well separated on the column based on silica matrix with gradient elution program. The recovery of trypsin detected with BAEE method is over 95% after purification with the HIC column. |
Hydrophobic interaction chromatography (HIC) is an attractive method for protein purification. The paper gives a review on the characteristics of HIC packings, including matrices and hydrophobic ligands. The efficiencies for protein separations were compared for different HIC packings. The present situation and the developing trends of the preparative HIC packings were described. |
L-4-oxalysinyl-norvalinyl-N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (I-677-Nva-FMDP) is a new tripeptide synthesized in our group. This peptide exhibits potent anticandida Albicans activity. In the presence of serum, the antifun gal activity of I-677-Nva-FMDP decreases after incubating over 1h. In order to investigate the relationship between the degradation and antifungal activity of I-677-Nva-FMDP, the concentrations of I-677-Nva-FMDP and N3-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) were determined at different incubating time in mice serum. The incubating time range was from 0 to 300 min and the incubating samples were measured at intervals of 30 min. For this measurement reversed-phase HPLC was used and the mobile phase was composed of 8% methanol and 0.1% trifluoroacitic acid-triethyl amine buffer (pH 3). In this condition the retention time of FMDP and I-677-Nva-FMDP were 3.9 min and 16.5 min respectively. Methanol was superior to other reagents for the removal of protein from the incubating medium and there was not any interference peak before the retention time of I-677-Nva-FMDP. A decrease in concentration of I-677-Nva-FMDP was observed from 0 min to 180 min and no I-677-Nva-FMDP could be detected at 210 min whereas the concentration of FMDP incresed from 30 min to 2h and reached a maximum at 120 min. The results showed that the half life (t1/2) of I-677-Nva-FMDP was 70 min. This result coincided with the antifungal test in vitro. |
From the seeds of Pachyrrhizus errosus, three protein constituents, namel PE1, PE2 and PE3, have been isolated and purified by extraction with 5mmol/L phosphate saline (0.9% NaCl) buffer (PB) at pH 7.2, and S-Sepharose Fast Flow Column (2.6cm x 15cm) chromatography which eluted with 5mmol/L phosphate buffer (pH 7.0) containing 1mmol/L NaCl. Three proteins were burther separated on two connected Protein-Pak 60+Protein-Pak 125 [7.5mm x 39cm, 10microm] columns with mobile phase of 0.2mol/L phosphate buffer (pH 6.5). The flow rate was kept constant at 0.8mL/min by YSB-2 type high press pump. The effluent was monitored at a wavelength of 280nm on photodiode array detector. These three proteins are proved to be homogeneous by SDS-PAGE, IEF and HPGFC experiments, and all present the typical absorption spectra in ultraviolet region. The moleculer weights of the three proteins are approxiamtely 33000D, 14500D and 14000D respectively by SDS-PAGE. But as using HPGFC analysis, the MW value of PE2 is 28000D. This indicates PE2 may be composed of two chains joined by disulfide bond, which is further proved from the latter amino acid composition analysis. The isoelectric points of three proteins are 4.5, 6.5 and 7.5 respectively by using IEF. The amion acids compositions of the three proteins were determined with OPA post-column derivatization/fluorescence detection. |
A new type of SnO2/SnCl2 semiconductor as detector in gas chromatography has been developed. The sensing crystalloid with SnO2 as main block has an outstanding sensitivity to the reducing gases in a certain oxidative circumstance. The reducing gases can be reversibly adsorbed on the surface of crystal and make the resistance of the sensing element changed. Therefore, this type of sensing element may be used as detector in GC. We studied and prepared such sensing element with SnCl2 as catalyst to improve the sensitivity. An optimum procedurefor preparation has been received. We also discussed the possibilily as the gas-sensing element to replace the flame ionization detector in GC. The results showed that at the condition of 300 degrees C, the sensitivity, stability, detectability and linear range were satisfactory. This new type of detector can meet the needs of practical sample analysis. |
A capillary isoelectric focusing (CIEF) method was developed for the separation of proteins with different pI. Uncoated capillary (57 cm x 50 microm i.d.) was used instead of commercial coated column because of its ease in use and a longer lifetime. Polymeric additive, methylcellulose (MC-1500), acts as dynamic coating of the silica wall to suppress electroendosmosis. TEMED was used to extend the pH range of ampholine. Taurine was added to the ampholyte mixture to eliminate the aggregation of proteins. The uniform design, a new experimental design method suited for experiments of a large number of factors and levels, was used for the optimization of experimental conditions, such as concentrations of ampholine, MC-1500, TEMED and Taurine. U6* (6(4)) table was chosen to arrange experiments. Regression equations of maximum current, mean migration time and criterion of resolution power were obtained after the treatment of experimental results by SAS software. Optimized experimental conditions were obtained according to multivariate regression equations. Carrier ampholyte which consists of 3.2%-4.8% ampholine 3-10, 0.06%-0.18% MC-1500, 0.8% TEMED and 1%-2% taurine will produce good resolution. Cathode and anode solutions were 20 mmol/L NaOH and 20 mmol/L HsPO4, respectively. Low pressure (34.5kPa) was applied at the inlet of the capillary simultaneously after 31 minutes of isoelectric focusing at 25 kV to mobilize the focused acidic protein zone to pass through the detector. Human serum albumin (HSA) and human epidermal growth factor (hEGF) were separated by this method using an ampholyte mixture of 4% ampholine 3-10, 0.8% TEMED, 1% taurine and 0.12% MC-1500. Data analysis indicated that the coefficients of variation of pressure and the method are all 1.2%. The results demonstrated that the method established is applicable to basic, neutral and acidic proteins, and can be used for the determination of pI by internal calibration. |
High-performance ion exchange chromatography (HPIEC) is extensively used in the separation of peptides and proteins, especially in the biotechnology process. The principle of separation of proteins is based on the changes of pH and salt concentration in the mobile phase for the chromatographic model. A new synthetic method with the help of a catalyst for the bounding of diethylaminoethyl group on a home-made macro-pore silica sphere (the trade mark is Sinopak-s, with sphere size of 5 microm and pore diameter of 100 nm) was developed in our laboratory for the application of the scale-up separation of biotechnological target products in China. The Sinopak-s-DEAE weak anion ion exchange matrix for HPLC was prepared and characterized with various proteins. The pH value and reaction time were discussed for the reaction efficiency of ligand to the silica sphere. The coverage of the DEAE ligand on the silica surface were among 1.6 to 2.1 micromol/m2 for six batches of packings. The influences of the pH value and the salt concentration in mobile phase upon the retention of proteins on the DEAE column were also discussed. A bio-activity recovery up to 98% for trypsin was arrived after purification with the DEAE column under the chosen chromatographic conditions. The capacity of matrix for BSA was 80 mg/g. The column was successfully applied to separate a mixture of several standard proteins in a linear gradient elution condition from 0 to 0.4 mol/L of NaCl in a 50 mmol/L of Tris/HCl buffer (pH 7.0) at 1.0 mL/min flow rate and detected at 280 nm wavelength. |
A high performance liquid chromatographic system is described for the purification to homogeneity of recombinant human gamma-interferon (gamma-IFN) from the inclusion bodies produced in genetically transformed Escherichia Coli cells. Crude products of gamma-IFN obtained from typical preparative liquid chromatographic separation on Sephacryl S200 column was applied to analytical HPLC with RP-C18 column and eluted with convex gradient of 0-82% acetonitrile in 30 min followed by linear gradient of 82%-100% in 30 min. A constant level of 0.1% trifluoroacetic acid was maintained throughout the gradient process. The retention of gamma-IFN was investigated as a function of pH, temperature as well as concentration of salt in the mobile phase. For denatured and renatured gamma-IFN the chromatographic profiles were analyzed under various conditions. The retention time of renatured gamma-IFN was compared with that of denatured gamma-IFN and a significant difference in the retention time of profiles was observed. Both biological activities were reduced to different degree followed with incorrect disulfide bond gamma-IFN or oligomer gamma-IFN. The purified gamma-IFN has a specific activity about 6 x 10(6) units/mL. The eluats were assayed for identification of gamma-IFN using Enzyme-Linked Immunosorbent Assays (ELISA). Its molecular weight was 17.5 kD, as determined by SDS-PAGE. The results support that renatured sample with tR = 24.8 min and denatured one with tR = 30.1 min were identical protein with different conformation. The chromatographic behavior is explained by the change in net charge and polarity of gamma-IFN when the mobile phase condition and the conformation vary. The oligomer or incorrect disulfide bonds were formed during gamma-IFN renaturation and also the biological activity was found to be native conformation dependent. |
Analysis of butyraldehyde obtained by the interaction of butanol with cytochrome P450 II E1 in rat liver microsomes prepared by centrifugation and with the NADPH added is described in this paper. The biotransformation rate of butanol into butyraldehyde can be used as an index for the assessment of the enzyme activity of cytochrome P450 II E1. A headspace gas chromatograghic method to determine the butyraldehyde has been developed. The detection limit and CV of this method for butyraldehyde in microsomes were 0.7 micromol/L and 8.1%-9.3% respectively. The recovery was 85.3%. The results show that this is a rapid and sensitive method with less interferences and fairly good precision. The method developed has made a reliable analytical methodological foundation for the assessment of the enzyme activity of cytochrome P450 II E1. |
A series of unexpected products were obtained when 1,4-dichloro-2-butyne reacted with allyl bromide with the catalyst PdCl2 (PhCN)2. It is difficult to separate these products from each other and to identify the structure of each one due to their similar properties. In order to solve this problem, gas chromatography-mass spectrometry (GC-MS) was chosen to analyse the above products because of its good sensitivity and selectivity. The analytical results showed that (A) the 8 compounds were 5,6-dichloro-4-chloromethyl-1, (E) 4-hexadiene (III), 5,6-dichloro-4-chloromethyl-1, (Z) 4-hexadiene (IV), 5-bromo-6-chloro-4-chloromethyl-1, (E) 4-hexadiene (V), 5-bromo-6-chloro-4-chloromethyl-1, (Z) 4-hexadiene (VI), 5-bromo-6-chloro-4-bromomethyl-1, (E) 4-hexadiene (VII), 5-bromo-6-chloro-4-bromomethyl-1, (Z) 4-hexadiene (VIII), 5,6-dibromo-4-bromomethyl-1, (E) 4-hexadiene (IX) and 5,6-dibromo-4-bromomethyl-1, (E) 4-hexadiene (X). Most compounds, except (V) and (VI), were the results of exchange reaction between chloride and bromide anion; each pair of the products are geometrical isomers. (B) in the four pairs of geometrical isomers, the ratio of Z-isomer/E-isomer decreased from 95:5 to 53:47 as molecular weight increased from compounds (III) and (IV) to compounds (IX) and (X). |
Catalytic hydrogenation of nitrobenzene with supported palladium catalyst is a new method to produce p-aminophenol. p-Aminophenol, aniline and 4,4'-diaminodiphenyl ether obtained from this method were determined by reversed phase high performance liquid chromatography. The factors, e.g., concentration of methanol, pH and ionic strength which could affect separation efficiency were studied. UV spectra of p-aminophenol, aniline and 4,4'-diaminodiphenyl ether were recorded. Good separation was performed by using a 100 mm x 4.6 mm column with 5 microm Hypersil ODS, a mixture of 60% aqueous 8.0 mmol/L KH2PO4 buffered to 6.5 with 4.0 mmol/L Na2HPO4 and 40% methanol as mobile phase at a flow rate of 1.0 mL/min, and UV spectrophotometric detector at 232 nm wavelength. The calibration curves of p-aminophenol, aniline and 4,4'-diaminodiphenyl ether have good linearity over concentration range of 5-250, 5-150 and 0.2-120 mg/L, respectively. Minimum detectable limits at a signal-to-noise ratio of 2 were 0.1, 0.6 and 0.6 ng. This method has been applied to analysis of the reaction products of ultrasonic catalytic hydrogenation and industrial samples with good results and reproducibility. |
An RP-HPLC method is described for the assay of propofol (2,6-diisopropylphenol), a new anesthesia drug, in human plasma. Protein in sample was precipitated with methanol, followed by extraction of propofol with cyclohexane. Both propofol and the internal standard thymol were derivatized with Gibbs' reagent and then chromatographed on an ODS column (Ultrasphere, 250 mm x 4.6 mm i.d.) with a mobile phase of acetonitrile-water-trifluoroacetic acid (80:20:0.1) and UV detection at 276 nm. The detectable limit of propofol was 24.8 microg/L (S/N>2) and calibration curves were linear between 50 to 1500 microg/L (r=0.9991). The average coefficient of variation was 6.1%. |
The hydrolytic solution of proteins mainly contains free amino acids and peptides. The separation of amino acids and di- and tri-peptides is very significant and also a complicated work. This report presents a chelated metal ion affinity chromatographic (CMAC) method for the separation of alpha-amino acids and peptides. Sephadex G10 was used as the solid matrix. It was epoxy-activated by epichlorophydrin; then coupled with iminodiacetate (IDA) and chelated with copper ion to produce immobilized copper-ion affinity chromatographic packing. Some examples are given for the chromatography of model mixtures of L-Val, L-His, L-Tyr, L-Try, Tyr-Try dipeptides and protein hydrolyzing solution of fish. The separation was based on the different stabilities of copper complexes of alpha-amino acids, peptides and IDA-Sephadex G10. The components which form weak complexes with copper apparently move along with the solvent front. Alpha-amino acids-copper complexes with a stability comparable to that of copper-IDA-Sephadex G10 are retained on the matrix. Peptides form strong complexes and catch copper from the matrix. They are only slightly retained. The results showed that alpha-amino acids and peptides were completely separated under the experimental conditions. |
The levels of R- and S-mephenytoin in human urine were determined by chiral capillary gas chromatography with nitrogen-phosphorus detector. The conditions of the chromatography and detection included a chiral capillary column (Chirasil-Val, 25 m x 0.25 mm i.d., Alltech), column temperature (T) 190 degrees C, injector T 220 degrees C, detector T 240 degrees C, and the flow-rates of 0.85, 3.5 and 120 mL/min for nitrogen, hydrogen and air respectively. Based on the above conditions, the satisfactory separation of R- and S-mephenytoin was gained, and other interference from urine sample was not found. The linear curves for both tested compounds ranged between 12.5 and 2500 microg/L, with a minimum detectable concentration of about 6 microg/L. Because of its simplicity, rapidity, sensitivity and accuracy, this method has been extensively used for testing metabolic ability of S-mephenytoin polymorphic oxidation in the Chinese populations and for the determination of the activity of hepatic drug-metabolizing enzyme CYP2C19 using mephenytoin S/R ratio of the urine samples of subjects as an indicator. |