真菌组蛋白赖氨酸甲基转移酶的研究进展

甲基化修饰是蛋白翻译后修饰的主要方式之一。真菌中,多种赖氨酸甲基转移酶能够执行组蛋白特定位点上赖氨酸的甲基化。组蛋白上赖氨酸的甲基化与真菌DNA的复制、转录以及异染色质的形成相关。甲基化参与了多种生物学过程,如真菌发育、昼夜节律调节、次级代谢基因簇表达、水解酶合成、致病真菌毒力形成。本文结合笔者工作,对目前真菌中已经发现的组蛋白赖氨酸甲基转移酶的命名、分类、结构域特征、催化域的三维结构以及它们所执行的甲基化在各种真菌中的作用进行了总结,提出了目前研究的不足并对未来的研究方向和内容进行了展望。     

Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier

Neurotrophins, such as brain derived neurotrophic factor (BDNF), do not cross the blood-brain barrier (BBB). Certain monoclonal antibodies (MAb) to the human insulin receptor (HIR) do cross the BBB via receptor-mediated transport, and can act as a molecular Trojan horse to ferry across the BBB an attached drug. A genetically engineered fusion protein was produced whereby the amino terminus of human BDNF is fused to the carboxyl terminus of the heavy chain of a chimeric HIRMAb. The HIRMAb-BDNF fusion protein reacted equally with antibodies to human IgG and BDNF. The bi-functionality of the fusion protein was retained as the affinity of the fusion protein for the HIR was identical to that of the chimeric HIRMAb, and the affinity of the fusion protein for the trkB receptor was identical to that of BDNF. The fusion protein was equi-potent with BDNF in a neuroprotection assay in human neural cells. The pharmacokinetics (PK) of the fusion protein was examined in the adult Rhesus monkey. The mean residence time (MRT) of the fusion protein in blood was >100-fold longer than the MRT of BDNF. Therapeutic levels of BDNF were produced in primate brain following the intravenous administration of the fusion protein. A fusion protein tandem vector was engineered that allowed for isolation of a CHO cell line that produced the fusion protein at high levels in serum free medium. Neurotrophins, such as BDNF, can be re-formulated to enable these molecules to cross the human BBB, and such fusion proteins represent a new class of human neurotherapeutics.     

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.     

Enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose using GerB (dTDP-4-keto-6-deoxy-D-glucose aminotransferase)

Over-expressed GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from dTDP-4-keto-6-deoxy-D-glucose (1). [Carbohydrate structure: see text]. Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase (DH), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phosphate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270min. After purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by (1)H, (13)C, (1)H-(1)H COSY NMR spectrometry.     

Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.     

Behaviour influences cholesterol plasma levels in a pig model

Little is known about the relationship between feed intake behaviour and cholesterol levels in humans. This can be attributed to the fact that feed intake behaviour in humans is difficult to assess. The relationships between feed intake, feed efficiency and feed intake behaviour, and cholesterol and triglyceride levels were investigated at an average age of 187 days, in a pig model consisting of 202 Duroc barrows. Feed intake and feed intake behaviour were recorded individually and daily by means of an electronic identification system. Animals with high levels of total cholesterol also had high levels of high-density lipoprotein (HDL), low-density lipoprotein (LDL) cholesterol and triglycerides. Animals with high levels of HDL also had high levels of LDL and triglycerides, and animals with high levels of LDL also had high levels of triglycerides. Animals with higher BW, higher backfat thickness, higher BW gain, higher gain of backfat deposition, higher feed intake, higher residual feed intake (RFI) and higher feed intake rate had higher levels of total, HDL and LDL plasma cholesterol. Results indicate that the relationship between feed intake and cholesterol levels is a long-term relationship, while the relationship between RFI and cholesterol levels is more of a short-term nature. The relationship between intake rate and cholesterol plasma levels disappeared after correction for the amount of feed consumed. Results indicate that feed intake independent of metabolic BW, growth and fatness, i.e. 'RFI', was positively correlated with cholesterol plasma levels. This suggests that eating food over and above the maintenance and growth requirements constitutes a health risk independent of the level of fatness.     

Parametric dependence in model epidemics. I: contact-related parameters

One of the interesting properties of nonlinear dynamical systems is that arbitrarily small changes in parameter values can induce qualitative changes in behavior. The changes are called bifurcations, and they are typically visualized by plotting asymptotic dynamics against a parameter. In some cases, the resulting bifurcation diagram is unique: irrespective of initial conditions, the same dynamical sequence obtains. In other cases, initial conditions do matter, and there are coexisting sequences. Here we study an epidemiological model in which multiple bifurcation sequences yield to a single sequence in response to varying a second parameter. We call this simplification the emergence of unique parametric dependence (UPD) and discuss how it relates to the model's overall response to parameters. In so doing, we tie together a number of threads that have been developing since the mid-1980s. These include period-doubling; subharmonic resonance, attractor merging and subduction and the evolution of strange invariant sets. The present paper focuses on contact related parameters. A follow-up paper, to be published in this journal, will consider the effects of non-contact related parameters.     

LoCoH: nonparameteric kernel methods for constructing home ranges and utilization distributions

Parametric kernel methods currently dominate the literature regarding the construction of animal home ranges (HRs) and utilization distributions (UDs). These methods frequently fail to capture the kinds of hard boundaries common to many natural systems. Recently a local convex hull (LoCoH) nonparametric kernel method, which generalizes the minimum convex polygon (MCP) method, was shown to be more appropriate than parametric kernel methods for constructing HRs and UDs, because of its ability to identify hard boundaries (e.g., rivers, cliff edges) and convergence to the true distribution as sample size increases. Here we extend the LoCoH in two ways: "fixed sphere-of-influence," or r-LoCoH (kernels constructed from all points within a fixed radius r of each reference point), and an "adaptive sphere-of-influence," or a-LoCoH (kernels constructed from all points within a radius a such that the distances of all points within the radius to the reference point sum to a value less than or equal to a), and compare them to the original "fixed-number-of-points," or k-LoCoH (all kernels constructed from k-1 nearest neighbors of root points). We also compare these nonparametric LoCoH to parametric kernel methods using manufactured data and data collected from GPS collars on African buffalo in the Kruger National Park, South Africa. Our results demonstrate that LoCoH methods are superior to parametric kernel methods in estimating areas used by animals, excluding unused areas (holes) and, generally, in constructing UDs and HRs arising from the movement of animals influenced by hard boundaries and irregular structures (e.g., rocky outcrops). We also demonstrate that a-LoCoH is generally superior to k- and r-LoCoH (with software for all three methods available at http://locoh.cnr.berkeley.edu).     

A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry

A rapid LC-MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25-500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.