BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry

Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to influence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a specific receptor whose activation is highly controversial. As a member of the cytokine receptor superfamily, it has been predicted to be activated by ligand-induced dimerization. However, recent evidence has indicated that this receptor exists as a dimer in both ligand-free and ligand-bound states. Here, the BiaCore has been used to measure the kinetics and stoichiometry of the interaction between the leptin and its receptor. Human or mouse receptor chimeras comprising two receptor extracellular domains fused to the Fc region of IgG(1) were captured on to the sensor via protein G. Kinetic data fitted to the simplest 1/1 model. The observed stoichiometry at ligand saturation was 1:1. Analyzing the binding mode and the reaction stoichiometry allowed us to conclude that the leptin receptor dimerization is not induced by ligand binding. This contradicts the common paradigm of cytokine receptor activation. Furthermore, data demonstrated a high-affinity interaction. The KD was 0.23+/-0.08 nM, with ka = (1.9 +/- 0.4) x 10(6) M(-1)s(-1) and kd = (4.4 +/- 0.6) x 10(-4) s(-1) for human leptin with its cognate receptor. Similar results were observed for the affinity of different species of leptin binding to mouse leptin receptor.     

High-field (9.4 T) 1H magnetic resonance microscopy of mouse brain

The FLASH and STEAM pulse sequences were used to perform the microimaging and localized spectroscopy of brain of living and dead mice, respectively. The phase-shift presaturation approach was used to sup-press water NMR signal. The experimental results show that the differences in localized spectra and MR images of brain between live and dead mice can be observed by means of magnetic resonance microscopy.     

Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer agents

In our pursuit to develop novel non-carbohydrate small molecule Galectin-1 Inhibitors, we have designed a series of 1-benzyl-1H-benzimidazole derivatives and demonstrated their anticancer activity. The compound 6g, 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-hydroxyphenyl) benzamide was found to be most potent with an IC₅₀ of 7.01 ± 0.20 µM and arresting MCF-7 cell growth at G2/M phase and S phase. Induction of apoptosis was confirmed by morphological changes like cell shrinkage, blebbing and cell wall deformation, dose dependent increase in the mitochondrial membrane potential (ΔΨm) and ROS levels. Further, dose dependent decrease in Gal-1 protein levels proves Gal-1 mediated apoptosis by 6g. Molecular docking studies were performed to understand the Gal-1 interaction with compound 6g. In addition, RP-HPLC studies showed 85.44% of 6g binding to Gal-1. Binding affinity studies by fluorescence spectroscopy and Surface Plasmon Resonance (SPR) showed that 6g binds to Gal-1 with binding constant (K_a) of 1.2 × 10⁴ M⁻¹ and equilibrium constant K_D value of 5.76 × 10⁻⁴ M respectively.     

Sequence-specific 1H, 13C, and 15N resonance assignments of GRP1 PH domain

The carboxy-terminal pleckstrin homology (PH) domain recruits GRP1 to the plasma membrane through the specific binding to phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. Here, we describe backbone and side chain assignments of the GRP1 PH domain determined by triple resonance experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved 1HN-detected triple resonance TROSY-based experiments

A pulse scheme resulting in improved sensitivity in TROSY-based 1HN-detected triple resonance experiments is presented. The approach minimizes relaxation losses which occur during the transfer of transverse magnetization from 15N to 1HN immediately prior to detection. The utility of the method is demonstrated on a complex of methyl protonated, highly deuterated maltose binding protein (MBP, 370 residues) and - cyclodextrin. Sensitivity gains relative to previous TROSY schemes of approximately 10 and 20% are noted in HNCO spectra of MBP recorded at 25 and 5°C, respectively, corresponding to molecular correlation times of 23 and 46 ns.     

1H n.m.r. study of geletin gels

Proton spin-lattice and spin-spin relaxation times have been measured in the gelatin-water system during gelatin at various concentrations and quenching temperatures. The spin-spin relaxation time is shown to be a good parameter for following the kinetics of gelation and can be related to the amount of helices measured by polarimetry. The results are analysed within the framework of the Zimmerman-Brittin theory.     

Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1

Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.     

Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments

In experiments with proteins of molecular weights around 100 kDa the implementation of [¹⁵N,¹H]-TROSY-elements in [¹⁵N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of sensitivity enhancement elements. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the ¹³ C ¹⁵N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the ¹⁵N multiplet components.     

3.0T 1H-MRS对脑胶质瘤及脑转移瘤的鉴别诊断

目的：利用3.0T氢质子磁共振波谱对胶质瘤和转移瘤的肿瘤组织区、瘤周水肿区进行细胞代谢物水平的检测,试图找出胶质瘤和脑转移瘤的鉴别诊断的依据,以及胶质瘤高、低级别组间的差别。方法：对经病理证实的20例高级别胶质瘤组、16例低级别胶质瘤组和19例脑转移瘤组患者,先行MRI平扫及增强扫描,波谱均在增强扫描的基础上获得,使用MR点分辨波谱序列,检测肿瘤组织区、瘤周水肿组织区NAA/Cr、Cho/Cr、NAA/Cho、NAA、Cho、Cr、Lip/Lac等值,进行比较。结果：（1）高级别胶质瘤与转移瘤在肿瘤组织区NAA/Cr代谢物浓度的比值有统计学意义。（2）高、低级别胶质瘤肿瘤组织内Cho/Cr比值有统计学意义。（3）转移瘤与高、低级别胶质瘤在瘤周水肿区NAA/Cr,以及低级别胶质瘤与转移瘤Cho/Cr代谢物浓度的比值有统计学意义;高级别胶质瘤与转移瘤瘤周区NAA代谢物浓度有明显差异。（4）胶质瘤高、低级别组间在肿瘤周围区NAA峰、Cho峰及NAA/Cho Cho/Cr代谢物浓度比值有统计学意义。（5）高级别胶质瘤和转移瘤分别与低级别胶质瘤在肿瘤组织区及瘤周水肿区Lip/Lac有显著性差异（P〈0.01）。结论：利用氢质子波谱可对胶质瘤和转移瘤进行鉴别诊断;Cho/Cr及NAA/Cho比值可对胶质瘤进行分级;Lip/Lac峰的出现与肿瘤的恶性度呈正相关,但不特异。     

Effects of PRE and POST therapy drug-pressure selected mutations on HIV-1 protease conformational sampling

Conformational sampling of pre- and post-therapy subtype B HIV-1 protease sequences derived from a pediatric subject infected via maternal transmission with HIV-1 were characterized by double electron–electron resonance spectroscopy. The conformational ensemble of the PRE construct resembles native-like inhibitor bound states. In contrast, the POST construct, which contains accumulated drug-pressure selected mutations, has a predominantly semi-open conformational ensemble, with increased populations of open-like states. The single point mutant L63P, which is contained in PRE and POST, has decreased dynamics, particularly in the flap region, and also displays a closed-like conformation of inhibitor-bound states. These findings support our hypothesis that secondary mutations accumulate in HIV-1 protease to shift conformational sampling to stabilize open-like conformations, while maintaining the predominant semi-open conformation for activity.     

In vivo phenotyping of the ob/ob mouse by magnetic resonance imaging and 1H-magnetic resonance spectroscopy

Objective: We studied ob/ob and wild‐type (WT) mice to characterize the adipose tissues depots and other visceral organs and to establish an experimental paradigm for in vivo phenotyping. Research Methods and Procedures: An in vivo evaluation was conducted using magnetic resonance imaging and ¹H‐magnetic resonance spectroscopy (¹H‐MRS). We used T1‐weighted images and three‐dimensional spin echo T1‐weighted images for the morphological analysis and ¹H‐MRS spectra on all body mass, as well as ¹H‐MRS spectra focalized on specific lipid depots [triglyceride (TG) depots] for a molecular analysis. Results: In ob/ob mice, three‐dimensional evaluation of the trunk revealed that ～64% of the volume consists of white adipose tissue, which is 72% subcutaneous and 28% visceral. In vivo ¹H‐MRS showed that 20.00 ± 6.92% in the WT group and 58.67 ± 6.65% in the ob/ob group of the total proton content is composed of TG protons. In in vivo‐localized spectra of ob/ob mice, we found a polyunsaturation degree of 0.5247 in subcutaneous depots. In the liver, we observed that 48.7% of the proton signal is due to water, whereas in the WT group, the water signal amounted to 82.8% of the total proton signal. With the sequences used, the TG amount was not detectable in the brain or kidneys. Discussion: The present study shows that several parameters can be obtained by in vivo examination of ob/ob mice by magnetic resonance imaging and ¹H‐MRS and that the accumulated white adipose tissue displays low polyunsaturation degree and low hydrolipidic ratio. Relevant anatomical alterations observed in urinary and digestive apparatuses should be considered when ob/ob mice are used in experimental paradigms.