花椒属植物生物碱研究进展

花椒属植物全世界约有250种,我国约有39种14变种。近年来研究发现花椒属植物生物碱具有多种药理作用与临床功能,现就近20年来花椒属生物碱和花椒生物碱的提取、分离、测定方法以及其药理作用等研究作一综述。     

Plant community complexity in the arid valley of Minjiang River, southwestern China

Biocomplexity theory is becoming increasingly important in understanding natural vegetation dynamics and interrelation among all components of the ecosystem. In this study, based on the field investigation of plant species and environmental factors (altitude, microtopography, soil water content, and soil nutrients) in an arid valley of the upper reaches of Minjiang River, Sichuan Province, southwestern China, plant community complexity and its relationship with environmental factors, community diversity, species evenness and richness were studied. Both total and structural complexities of the communities showed a “high- low-high” tendency with the increase in altitude of the area, which meant that the complexity of communities was the highest at the sites of low and high altitude, whereas it was the lowest at the sites of intermediate altitude. It was found that the total community complexity had significant quadratic correlations with soil organic matter (SOM) content, total nitrogen (N), hydrolyzable N, soil water content, and available potassium (K), whereas it had no significant correlations with soil total K, total phosphorus (P), available P, and pH value. The total community complexity positively correlated with community diversity, species evenness and species richness, whereas the structural complexity negatively correlated with the community evenness. Of the two components of the total community complexity, namely, the structural complexity and the structural diversity, the structural complexity was more sensitive than the structural diversity to the changes of species in the community, which was not only related to the community evenness but also to the community richness. The relative contribution of both the structural complexity and the structural diversity to the total complexity would be different for different study areas or ecosystems.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

Litterfall Production Along Successional and Altitudinal Gradients of Subtropical Monsoon Evergreen Broadleaved Forests in Guangdong, China

Evaluation of litterfall production is important for understanding nutrient cycling, forest growth, successional pathways, and interactions with environmental variables in forest ecosystems. Litterfall was intensively studied during the period of 1982–2001 in two subtropical monsoon vegetation gradients in the Dinghushan Biosphere Reserve, Guangdong Province, China. The two gradients include: (1) a successional gradient composed of pine forest (PF), mixed pine and broadleaved forest (MF) and monsoon evergreen broadleaved forest (BF), and (2) an altitudinal gradient composed of Baiyunci ravine rain forest (BRF), Qingyunci ravine rain forest (QRF), BF and mountainous evergreen broadleaved forest (MMF). Mean annual litterfall production was 356, 861 and 849 g m⁻² for PF, MF and BF of the successional gradient, and 1016, 1061, 849 and 489 g m⁻² for BRF, QRF, BF and MMF of the altitudinal gradient, respectively. As expected, mean annual litterfall of the pioneer forest PF was the lowest, but rapidly increased over the observation period while those in other forests were relatively stable, confirming that forest litterfall production is closely related to successional stages and growth patterns. Leaf proportions of total litterfall in PF, MF, BF, BRF, QRF and MMF were 76.4%, 68.4%, 56.8%, 55.7%, 57.6% and 69.2%, respectively, which were consistent with the results from studies in other evergreen broadleaved forests. Our analysis on litterfall monthly distributions indicated that litterfall production was much higher during the period of April to September compared to other months for all studied forest types. Although there were significant impacts of some climate variables (maximum and effective temperatures) on litterfall production in some of the studied forests, the mechanisms of how climate factors (temperature and rainfall) interactively affect litterfall await further study.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant human keratinocyte growth factor-2 (rhKGF-2) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as the inducer. In order to determine whether IPTG could be replaced with the cheap and natural lactose to induce rhKGF-2 expression, we examined the expression of rhKGF-2 in flask culture and 30-l fermentation using lactose as the inducer. The optimized fermentation induced with lactose resulted in 1,382 g of cell mass, corresponding to a 84% enhancement in cell mass compared with IPTG induction. While the expression level of rhKGF-2 induced with lactose was comparable to that induced with IPTG, the solubility of target protein was increased by lactose induction than by IPTG induction. The recombinant protein was further purified by cation exchange and heparin-affinity chromatography. 255 milligrams of pure rhKGF-2 was achieved per liter culture by lactose induction, 52% higher than that obtained by IPTG induction. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that the purified lactose-induced rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of FGFR2-IIIb-transfected mouse BaF3 cells as IPTG-induced rhKGF-2 could do.     

花椒总生物碱镇痛、抗炎、止痒作用研究

目的:研究花椒生物碱镇痛、抗炎及止痒作用。方法:分别采用热板法考察其镇痛作用二甲苯致炎法考察其抗炎作用、低分子右旋糖酐-40诱发小鼠皮肤瘙痒研究其止痒作用。结果:中、高浓度的花椒生物碱对小鼠热刺激引起的疼痛具有较强的镇痛作用;高浓度的花椒生物碱能较强抑制二甲苯所致小鼠耳肿胀,并能缓解分子右旋糖酐-40诱发的小鼠皮肤瘙痒。具有较强的抗炎、止痒作用。结论:花椒生物碱具有较强的镇痛、抗炎、止痒作用。     

A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.     

B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF(-/-) and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.