In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

哺乳动物精子体外发生

精子的发生是一个高度复杂而有序的过程 ,涉及到细胞的增殖和分化。体外培养生精细胞在近年来取得了较大进展 ,建立了睾丸组织培养、曲精细管小段培养、支持细胞 生精细胞共培养及藻酸钙胶囊包裹培养等方法。建立生精细胞体外培养模型有助于 :(1)研究精子发生的调控机制 ;(2 )直接对雄性生殖细胞进行遗传修饰 ;(3)用于辅助生育技术 ,治疗精子发生阻滞的患者。如何改善培养条件 ,进一步提高生殖细胞的存活、分化、增殖效率 ,是使哺乳动物体外精子发生发展成为一项适用性较强的技术所必须解决的问题。     

登革病毒包膜蛋白的原核表达及免疫原性研究

登革病毒感染引起的登革热和登革出血热/登革休克综合征是目前流行最为广泛的虫媒病毒病,但其发病机制不明,也无疫苗和特异性抗病毒药物用于防治。登革病毒包膜蛋白(E蛋白)在病毒致病和免疫过程中发挥重要作用。本研究构建了登革病毒2型E蛋白基因的重组质粒E/pGEX-6P-1,并优化表达条件,获得登革病毒E蛋白的高效可溶性表达;用谷胱甘肽琼脂糖凝胶4B亲和层析柱纯化,获得纯度较高的蛋白。该蛋白具有较好的免疫原性,免疫小鼠后可获得高效价抗体。本研究为进一步了解登革病毒E蛋白的功能及其在相关疾病中的应用奠定了基础。     

In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features

The "protein only" hypothesis of prion propagation postulates that the abnormal isoform of the prion protein, PrP(Sc), acts as a causative and transmissible agent of prion disease. In attempt to reconstitute prion infectivity in vitro, we previously developed a cell-free conversion protocol for generating amyloid fibrils from a recombinant prion protein encompassing residues 89-231 (rPrP 89-230) [Baskakov et al. (2002) J. Biol. Chem. 277, 21140]. When inoculated into transgenic mice, these amyloid fibrils induced prion disease, which can be efficiently transmitted to both wild-type and transgenic mice [Legname et al. (2004) Science 305, 673]. Here we show that the polymerization of rPrPs into the fibrils displays a number of distinctive kinetic features that are not typical for polymerization by other amyloidogenic polypeptides. Specifically, the lag phase of polymerization showed only modest dependence on protein concentration, and the conversion reaction displayed a dramatic volume-dependent threshold effect. To explain these unique kinetic features, we proposed that the conversion reaction is regulated by the dynamics between the rates of multiplication and deactivation of self-propagating fibrillar isoforms. Our further studies demonstrated that surface-dependent sorption of fibrillar isoforms is responsible for their deactivation in vitro, while fibril fragmentation seems to account for the multiplication of the active centers of polymerization. Our findings support the hypothesis that development of prion disease is controlled by a fine dynamic balance between self-propagation and clearance/deactivation of PrP(Sc).     

In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS

The major biochemical and thermodynamic features of nucelocapsid protein of SARS coronavirus (SARS_NP) were characterized by use of non-denatured gel electrophoresis, size-exclusion chromatographic and surface plasmon resonance (SPR) techniques. The results showed that SARS_NP existed in vitro as oligomer, more probably dimer, as the basic functional unit. This protein shows its maximum conformational stability near pH 9.0, and it seems that its oligomer dissociation and protein unfolding occur simultaneously. Thermal-induced unfolding for SARS_NP was totally irreversible. Both the thermal and chemical denaturant-induced denaturation analyses showed that oligomeric SARS_NP unfolds and refolds through a two-state model, and the electrostatic interactions among the charge groups of SARS_NP made a significant contribution to its conformational stability.     

In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.     

Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments.

Thin-filament preparations from four smooth muscle types (gizzard, stomach, trachea, aorta) all activate myosin MgATPase activity, are regulated by Ca2+, and contain actin, tropomyosin and a 120000-140000-Mr protein in the molar proportions 1:1/7:1/26. The 120000-140000-Mr protein from all sources is a potent inhibitor of actomyosin ATPase activity. Peptide-mapping and immunological evidence is presented showing that it is identical with caldesmon. Quantitative immunological data suggest that caldesmon is a component of all the thin filaments and that the thin-filament-bound caldesmon accounts for all the caldesmon in intact tissue. The myosin light-chain kinase content of thin-filament preparations was found to be negligible. We propose that caldesmon-based thin-filament Ca2+ regulation is a physiological mechanism in all smooth muscles.     

In vitro incorporation of tritium into native DNA

An exchange method is described for producing tritium-labeled native DNA in vitro with minimal physical damage to the DNA. Tritium-labeled calf thymus DNA prepared in this way has a specific activity of about 100 μCi/mmole of nucleotide (i.e., about 2 × 10⁸ dpm/mmole). Sedimentation velocity at neutral and alkaline pH indicate that the product has an average of two single strand breaks per duplex molecule of molecular weight 6 × 10⁶ daltons. The optical and thermal denaturation properties of the product are those of native DNA. The method should be particularly useful for labeling DNA from organisms that cannot be labeled conveniently in vivo.     

In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein.

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.     

In vitro palmitoylation of native bovine brain Goα

The native Goα was purified from bovine brain cortex and palmitoylated in vitro. The in vitro palmitoylation site was the same as that in vivo. The internal palmitoylation of purified native Goα was found to be largely maintained. The apparant palmitoylation ratio was significantly increased after the Goa was treated with DTT. The GTPg S binding characteristic of Goα was not influenced by palmitoylation, however, the affinity for LUVs was increased dramatically. The in vitro palmitoylation model of Goα provides a better basis for studying the functional role of G protein palmitoylation in signal transduction.     

In vitro characterization of protein kinase CKII beta mutants defective in beta-beta dimerization

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The beta subunit mediates tetramer formation through beta-beta homodimerization and alpha-beta heterodimerization. In a previous study R26 and R75, point mutants of CKIIb defective in beta-beta dimerization, were isolated. In the present work we characterized these CKIIbeta mutants in vitro. Purified R26 and R75 bound to CKIIalpha but were defective in binding to CKIIbeta. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with CKIIbeta and to activate CKIIalpha.     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.     

In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance

Mammalian chromosomes terminate with a 3' tail which consists of reiterations of the G-rich repeat, d(TTAGGG). The telomeric tail is the primer for replication by telomerase, and it may also invade telomeric duplex DNA to form terminal lariat structures, or T loops. Here we show that the ubiquitous and highly conserved mammalian protein hnRNP D interacts specifically with the G-rich strand of the telomeric repeat. A single gene encodes multiple isoforms of hnRNP D. All isoforms bind comparably to the G-rich strand, and certain isoforms can also bind tightly and specifically to the C-rich telomeric strand. G-rich telomeric sequences readily form structures stabilized by G-G pairing, which can interfere with telomere replication by telomerase. We show that hnRNP D binding to the G-rich strand destabilizes intrastrand G-G pairing and that hnRNP D interacts specifically with telomerase in human cell extracts. This biochemical analysis suggest that hnRNP D could function in vivo to destabilize structures formed by telomeric G-rich tails and facilitate their extension by telomerase.     

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.     

In vitro microtubule assembly on centrioles from mammalian spermatids

In the present study we have shown that the centriolar structures, which form the neck region of the spermatid tail, can act as microtubule-organizing centers.     

In vitro characterization of anti-glucosylceramide rabbit antisera

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.     

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl-D-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil相似文献   

In vitro characterization of a koala retrovirus

Recently, a new endogenous koala gammaretrovirus, designated KoRV, was isolated from koalas. The KoRV genome shares 78% nucleotide identity with another gammaretrovirus, gibbon ape leukemia virus (GALV). KoRV is endogenous in koalas, while GALV is exogenous, suggesting that KoRV predates GALV and that gibbons and koalas acquired the virus at different times from a common source. We have determined that subtle adaptive differences between the KoRV and GALV envelope genes account for differences in their receptor utilization properties. KoRV represents a unique example of a gammaretrovirus whose envelope has evolved to allow for its expanded host range and zoonotic potential.