Do metalloproteinases destabilize vulnerable atherosclerotic plaques?

PURPOSE OF REVIEW: Atherosclerotic plaque rupture and thrombosis underlie most myocardial infarctions. Matrix metalloproteinases are a family of enzymes that remodel the extracellular matrix. Metalloproteinases could stabilize rupture-prone plaques by promoting smooth muscle cell migration and proliferation. Alternatively, metalloproteinases could destabilize vulnerable plaques by promoting matrix destruction, angiogenesis, leucocyte infiltration, and apoptosis. Evidence is reviewed from genetically modified mice and human biomarker and genetic studies that sheds light on this dual role of metalloproteinases. RECENT FINDINGS: Inhibition of metalloproteinases in mice using tissue inhibitors of metalloproteinases increases plaque stability; however, double knockouts of apolipoprotein E with matrix metalloproteinase 2, 3, 7, 9, 12, and 13 have more or less stable plaques, consistent with harmful or protective effects of individual metalloproteinases. Overexpression studies in mice or rabbits show that high activities of matrix metalloproteinase 9 and 12 decrease stability. Biomarker and human genetic studies demonstrate that increased metalloproteinase activity is associated with vascular repair or myocardial infarction. SUMMARY: Recent studies reinforce evidence for a dual role of matrix metalloproteinases in plaque stabilization and rupture, which probably depends on the stage, site, and severity of disease. Dysregulated metalloproteinase activity in end-stage coronary artery disease appears a valid target for therapy.     

Multiplex genotyping of novel tetranucleotide microsatellites from a marine foodfish species crimson red snapper (Lutjanus erythropterus)

Crimson red snapper (Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA‐microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy–Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

Phylogeographic analysis of nuclear and mtDNA supports subspecies designations in the ostrich (Struthio camelus)

We investigated the phylogeography and subspecies classification of the ostrich (Struthio camelus) by assessing patterns of variation in mitochondrial DNA control region (mtDNA-CR) sequence and across fourteen nuclear microsatellite loci. The current consensus taxonomy of S. camelus names five subspecies based on morphology, geographic range, mtDNA restriction fragment length polymorphism and mtDNA-CR sequence analysis: S. c. camelus, S. c. syriacus, S. c. molybdephanes, S. c. massaicus and S. c. australis. We expanded a previous mtDNA dataset from 18 individual mtDNA-CR sequences to 123 sequences, including sequences from all five subspecies. Importantly, these additional sequences included 43 novel sequences of the red-necked ostrich, S. c. camelus, obtained from birds from Niger. Phylogeographic reconstruction of these sequences matches previous results, with three well-supported clades containing S. c. camelus/syriacus, S. c. molybdophanes, and S. c. massaicus/australis, respectively. The 14 microsatellite loci assessed for 119 individuals of four subspecies (all but S. c. syriacus) showed considerable variation, with an average of 13.4 (±2.0) alleles per locus and a mean observed heterozygosity of 55.7 (±5.3)%. These data revealed high levels of variation within most subspecies, and a structure analysis revealed strong separation between each of the four subspecies. The level of divergence across both marker types suggests the consideration of separate species status for S. c. molybdophanes, and perhaps also for S. c. camelus/syriacus. Both the mtDNA-CR and microsatellite analyzes also suggest that there has been no recent hybridization between the subspecies. These findings are of importance for management of the highly endangered red-necked subspecies (S. c. camelus) and may warrant its placement onto the IUCN red list of threatened animals.     

Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 5'-exonuclease,and hybridization probe assays

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.     

Self-assembling of polymer-enzyme conjugates at oil/water interfaces

Interface-binding enzymes are desirable for biphasic reactions in that they offer simultaneous access to substrates dissolved in both phases across the interface. It has been shown that conjugating water-soluble enzymes with hydrophobic polymers facilitated the assembling of enzymes at oil/water interfaces. In this work, the interfacial assembling of alpha-chymotrypsin conjugated with polystyrene, poly(methyl methacrylate), and poly(l-lactic acid) was examined using the pendant drop method. The interface-assembling process of the conjugates from the organic phase followed a similar pattern of that of native alpha-chymotrypsin from the aqueous buffer phase, i.e., the interfacial tension decreased gradually with time. However, when the conjugates were dispersed in the form of particulates in the aqueous phase, in which the conjugate was insoluble, the assembling occurred faster and the interfacial tension quickly approached zero. It was suspected that the assembling in this case involved two steps, i.e., the adsorption of the particulates and the subsequent rearrangement, dissociation, and redispersion of the conjugates at the interface. The effect of other factors, including the polarity of organic solvent and pH and ionic strength of the aqueous phase, was evaluated. It was found that the polar solvent slightly facilitated the assembling, whereas pH and ionic strength showed minimal effects.     

Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.     

云南省林地薇甘菊防控研究进展

薇甘菊是世界十大有害杂草之一,引起广泛关注,因其入侵能力强,给入侵地的生态系统造成了极大威胁。本文系统总结云南省林地薇甘菊防治研究进展:开发出"林地薇甘菊监测预警信息系统"和"薇甘菊风险评估管理信息系统",提高了薇甘菊在云南省潜在分布区域预测的可靠性;筛选及复配出林地防效好的森草净+2,4-D钠盐,对土壤相对较为安全的2,4-D+敌草快复合药剂;选用旱冬瓜、千果榄仁、柱花草等替代控制薇甘菊,防控的同时还能创造更大的经济效益;发现薇甘菊颈盲蝽是控制薇甘菊的专化性强且取食量大的重要天敌昆虫,实现了对薇甘菊种子扩散和无性传播的有效控制。对薇甘菊的防治,集成了监测预警、应急除治、生物防治、生态修复技术,形成林地薇甘菊绿色防控技术体系,但其防控仍是局部的,未来还需要不断突破,使对薇甘菊的局部应急防控逐步转为大面积的持续生态控制。     

华山松不同部位挥发性单萜烯的含量及其对华山松木蠹象行为的影响

通过GC—MS分析,发现健康的与遭受华山松木蠹象Pissodes punctatus Langor et Zhang危害的华山松Pinusarmandii Franch树干以及华山松枝梢释放的挥发性单萜烯在含量上存在较大差别;利用Y-型双向选择嗅觉仪进行的室内趋向反应实验发现,处于补充营养期和交尾产卵期的华山松木蠹象对单萜烯表现出不同的趋向反应。在华山松枝梢挥发物中含量较高的（-）-β-蒎烯、月桂烯和柠檬烯引起补充营养期华山松木蠹象较大的趋向反应;在华山松树干韧皮部挥发物中含量较高的3-（＋）-蒈烯则引起交尾产卵期的华山松木蠹象较大的趋向反应。根据试验结果,对华山松和华山松木蠹象互作的机制进行了初步探讨。