Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228

Metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of β-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine β-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of β-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.     

NO在胆红素神经毒性中的作用

目的:探讨NO的神经毒性是否能促使高胆红素血症新生兔脑损伤的形成.方法:采用新生兔制作高胆红素血症动物模型,检测脑中EAAs及NO的含量.结果:模型组脑组织中EAAs含量显著低于对照组(P＜0.01),而NO浓度显著高于对照组(P＜0.01).结论:NO可能通过介导EAAs兴奋性神经毒性及其本身对神经细胞的直接毒性作用而加重胆红素所诱导的脑损伤.     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Soil Diversity and Land Use in the United States

Soils are dynamic components of terrestrial ecosystems that historically have been viewed as economic resources by government and private interests. The large-scale conversion of many sections of the United States to agriculture and urban land uses, combined with the growing awareness of the role of soils in global biogeochemistry and ecology, ultimately requires an assessment of the remaining distribution of undisturbed soils in the country. Here we conduct the first quantitative analysis of disturbed and undisturbed soil distribution in the USA using a GIS-based approach. We find that a sizable fraction (4.5%) of the nation's soils are in danger of substantial loss, or complete extinction, due to agriculture and urbanization. In the agricultural belt of the country, up to 80% of the soils that were naturally of low abundance are now severely impacted (greater than 50% conversion to agricultural/urban uses). Undisturbed soils provide ecosystem services that warrant their preservation, including a somewhat complex relationship with rare or endangered plants. The known and unknown attributes of undisturbed soils suggests the need for an integrated biogeodiversity perspective in landscape preservation efforts.     

果蝇原生殖细胞特化的分子机制

原生殖细胞在许多有性生殖动物的胚胎发育早期就已特化出来,并进一步分化为生殖细胞以产生新的子代。动物原生殖细胞的特化主要有生殖质决定和诱导两种模式,果蝇原生殖细胞的特化模式属于前者。研究表明,果蝇原生殖细胞特化过程中生殖质组装的关键基因是osk,其调控下游基因转录产物的定位和翻译,如vas和tud。此外,基因转录沉默是原生殖细胞特化过程的一个重要特征,其与生殖质中的成分如基因nos、gcl、pgc的表达产物密切相关。现对果蝇原生殖细胞特化分子机制进行综述。     

Successive chromosome walking by compatible ends ligation inverse PCR

Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR) for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out.