Novel analogs of D-e-MAPP and B13. Part 2: signature effects on bioactive sphingolipids

Novel isosteric analogs of the ceramidase inhibitors (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-1,3 (B13) with modified targeting and physicochemical properties were developed and evaluated for their effects on endogenous bioactive sphingolipids: ceramide, sphingosine, and sphingosine 1-phosphate (Cer, Sph, and S1P) in MCF7 cells as determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Time- and dose-response studies on the effects of these compounds on Cer species and Sph levels, combined with structure-activity relationship (SAR) data, revealed 4 distinct classes of analogs which were predominantly defined by modifications of the N-acyl-hydrophobic interfaces: N-acyl-analogs (class A), urea-analogs (class B), N-alkyl-analogs (class C), and omega-cationic-N-acyl analogs (class D). Signature patterns recognized for two of the classes correspond to the cellular compartment of action of the new analogs, with class D acting as mitochondriotropic agents and class C compounds acting as lysosomotropic agents. The neutral agents, classes A and B, do not have this compartmental preference. Moreover, we observed a close correlation between the selective increase of C(16)-, C(14)-, and C(18)-Cers and inhibitory effects on MCF7 cell growth. The results are discussed in the context of compartmentally targeted regulators of Sph, Cer species, and S1P in cancer cell death, emphasizing the role of C(16)-Cer. These novel analogs should be useful in cell-based studies as specific regulators of Cer-Sph-S1P inter-metabolism, in vitro enzymatic studies, and for therapeutic development.     

NMR evaluation of adipocyte fatty acid binding protein (aP2) with R- and S-ibuprofen

We have examined global chemical shift perturbations for aP2 ligand complexes and compared these with amide temperature coefficients. Hydrogen bond potential was monitored by amide chemical shift's temperature coefficient. Based on this information, we propose that the binding energy contribution can be spread out to multiple distant residues. For aP2, the ability of the receptor protein to change its hydrogen bond interactions in the beta-strands to accommodate different ligand scaffolds seems to make this receptor difficult for structure based drug design. While stabilization energy differential on hydrogen bonds is likely to be small for individual residues, the accumulative effect on multiple hydrogen bonds may have a dramatic impact on ligand affinity.     

Seasonal Dynamics in Resource Partitioning to Growth and Storage in Response to Drought in a Perennial Rhizomatous Grass, <Emphasis Type="Italic">Leymus chinensis</Emphasis>

A natural grassland in northeastern China dominated by Leymus chinensis (Trin.) was subject to drought treatments to determine the seasonal dynamics in resource partitioning to shoot and storage organs in response to drought. The growing season was divided into six stages according to the phenology of L. chinensis. Plant samples of L. chinensis were collected at each stage to determine biomass, gross calorific value, relative water content, and key mineral contents of plant parts, including rhizomes, roots, leaves, and stems. Resource partitioning to shoot and storage organs as measured by biomass, gross calorific value, and N, K⁺, and Na⁺ contents varied significantly among phenologic stages. Drought treatment (natural precipitation, 50–60 % of field capacity) significantly reduced biomass, gross calorific value, relative water content of shoot, and N and K⁺ contents in both shoot and storage organs, but it enhanced rhizome : shoot ratio and Na⁺ content. A negative correlation in biomass, gross calorific value, and K⁺ and Na⁺ contents between shoot and storage organs was found throughout the growing season, which may have been accentuated by drought when soil moisture was limited. Our results indicate that resource partitioning to storage plays an important role in regulating plant growth of L. chinensis, especially under drought conditions.     

Dynamic property is a key determinant for protein-protein interactions

Dynamic property is highly correlated with the biological functions of macromolecules, such as the activity and specificity of enzymes and the allosteric regulation in the signal transduction process. Applications of the dynamic property to protein function researches have been discussed and encouraging progresses have been achieved, for example, in enzyme activity and protein-protein docking studies. However, how the global dynamic property contributes to protein-protein interaction was still unclear. We have studied the dynamic property in protein-protein interactions based on Gaussian Network Model and applied it to classify biological and nonbiological protein-protein complexes in crystal structures. The global motion correlation between residues from the two protomers was found to be remarkably different for biological and nonbiological complexes. This correlation has been used to discriminate biological and nonbiological complexes in crystal and gave a classification rate of 86.9% in the cross-validation test. The innovation of this feature is that it is a global dynamic property which does not rely directly on the interfacial properties of the complex. In addition, the correlation of the global motions was found to be weakly correlated with the dissociation rate constant of protein complexes. We suggest that the dynamic property is a key determinant for protein-protein interaction, which can be used to discriminate native and crystal complexes and potentially be applied in protein-protein dynamic rate constants estimations.     

Cross-species bacterial artificial chromosome-fluorescence in situ hybridization painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species.     

载脂蛋白E3的克隆及原核表达

载脂蛋白Ｅ(ａｐｏｌｉｐｏｐｒｏｔｅｉｎＥ ,ａｐｏＥ)由 2 99个氨基酸组成 ,分子量 34ｋＤ ,是维持人体正常脂质代谢的必需蛋白质 .它是乳糜微粒 (ＣＭ )、极低密度脂蛋白 (ＶＬＤＬ)和高密度脂蛋白 (ＨＤＬ)的组分 ,是极低密度脂蛋白受体的重要配体 ,是脂质进入细胞不可缺少的中介 .ａｐｏＥ有 3种同分异构体 :ａｐｏＥ2、ａｐｏＥ3、ａｐｏＥ4 ,分别具有不同的生理作用 .ａｐｏＥ4与血浆高胆固醇、心血管疾病和老年痴呆等疾病关联[1～ 3 ] .ａｐｏＥ2与Ⅲ型高脂血症有关 ,并对老年痴呆有防治作用[4,5] .ａｐｏＥ3是大多数健康人所具有…     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.