Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by snake venom metalloproteinases

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels.     

Identification of protein fragments as pattern features in MALDI-MS analyses of serum

The use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to acquire spectral profiles has become a common approach to detect proteomic biomarkers of disease. MALDI-MS signals may represent both intact proteins as well as proteolysis products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis can tentatively identify the corresponding proteins Here, we describe the application of a data analysis utility called FragMint, which combines MALDI-MS spectral data with LC-MS/MS based protein identifications to generate candidate protein fragments consistent with both types of data. This approach was used to identify protein fragments corresponding to spectral signals in MALDI-MS analyses of unfractionated human serum. The serum also was analyzed by one-dimensional SDS-PAGE and bands corresponding to the MALDI-MS signal masses were excised and subjected to in-gel digestion and LC-MS/MS analysis. Database searches mapped all of the identified peptides to abundant blood proteins larger than the observed MALDI-MS signals. FragMint identified fragments of these proteins that contained the MS/MS identified sequences and were consistent with the observed MALDI-MS signals. This approach should be generally applicable to identify protein species corresponding to MALDI-MS signals.     

Occurrence of Sanguinicola occidentalis Van Cleave and Mueller, 1932 in Perca flavescens and Campeloma decisum from a Michigan creek

Sanguinicola occidentalis (Trematoda: Sanguinicolidae) infected 58 (48%) of 120 yellow perch collected in 1997 and 40 (50%) of 80 yellow perch collected in 1998 from Silver Creek in lower Michigan. The mean intensity and mean abundance of this blood fluke were higher in 1998 than in 1997. The fluke was found in the bulbous arteriosus of the perch heart, free in the petri dishes when the gill filaments were teased apart, and in the body cavity washings, and 1 individual was associated with an eye. Of the 269 S. occidentalis examined from perch, none had eggs. Most perch examined and infected were only 1+ yr in age. Spearman's correlation coefficients between S. occidentalis intensity and host length in 1997 and 1998 were not significant. An additional 25 yellow perch (0+ yr in age) collected in 1998 were not infected. Thirty-seven (33%) of 113 snails (Campeloma decisum) examined in July and August 1999 from Silver Creek were infected with S. occidentalis.     

从公共数据库中利用数据挖掘的方法开发15条黄金鲈EST-SSR标记

黄金鲈是美国中西部地区重要的淡水经济型鱼类。TypeⅠ型标记对于比较作图和遗传学研究有相当重要的作用, 但是黄金鲈的TypeⅠ标记数量相当少。研究通过数据挖掘的方法从公共数据库中挖掘黄金鲈的TypeⅠ型即EST-SSR标记。实验结果分析了21968条EST序列, 约14.4%的序列包含不同类型的核心重复单元。CA重复是最丰富的二碱基重复单元。从中挑选62条包含SSR的EST序列设计引物, 筛选得到15对多态的微卫星位点。群体遗传学参数显示15对EST-SSR位点的等位基因数范围为4—19(平均数为9), 观测杂合度和期望杂合度的范围分别为0.103—0.929和0.116—0.934, 有4个位点偏离HWE平衡。研究得到的EST-SSR标记适用于黄金鲈的群体遗传学和基因组作图研究。     

Evidence of predatory control of yellow perch (Perca flavescens) recruitment in Lake Erie, U.S.A.

Predation can be a major factor in recruitment success of yellow perch ( Perca flavescens Mitchitl). Trawl catches of age-0 yellow perch in western Lake Erie declined from 870·3 per trawling h in June to 3·3 per trawling h in late July 1988. Coincident with the decline in relative abundance of age-0 yellow perch we found large numbers of age-0 yellow perch in the stomachs of small walleyes ( Stizostedion vitreum Mitchill). From this evidence we hypothesized that predation by walleyes may have caused the demise of the 1988 year-class of yellow perch. We used a population and bioenergetics modelling approach to estimate the impact of walleye predation on the abundance of age-0 yellow perch. Modelling showed that 6·8 × 10⁹ age-0 yellow perch that had attained 18 mm total length ( t.l. ), were eaten by small (age-2 and younger) walleye from June through July 1988. We estimated that walleye ate 28·4–89·7% of the yellow perch reaching 18 mm t.l. during 1988. The majority of this predation (77% of total) was by the abundant age-2 cohort of walleyes. We concluded that, even in a large system such as Lake Erie, predation can play a major role in structuring year-class strength of yellow perch and, thus, management of percid fisheries should be conducted on a fish-community basis.     

Bioaccumulation of mercury in yellow perch (Perca flavescens) and common loons (Gavia immer) in relation to lake chemistry in Atlantic Canada

Mercury biomagnifies in aquatic foodwebs in freshwater lakes, and common loons (Gavia immer) breeding in eastern Canada can be exposed to reproductively toxic concentrations of mercury in their fish prey. We assessed the bioaccumulation and biomagnification of mercury in juvenile and adult common loons, and their preferred prey: yellow perch (Perca flavescens) in Kejimkujik National Park (KNP), Nova Scotia by measuring mercury levels and stable isotope ratios in tissues. Total mercury levels and stable-carbon (δ¹³C) and nitrogen isotope ratios (δ¹⁵N) were determined in composite whole-fish samples from lakes in KNP and blood samples from juvenile and adult loons captured on lakes in KNP and southern New Brunswick. Geometric mean mercury concentrations were 0.15 and 0.38 μg/g (wet wt.) in small (9-cm fork length) and large (17-cm fork length) yellow perch, and were 0.43 and 2.7 μg/g (wet wt.) in blood of juvenile and adult common loons, respectively. Mercury concentrations in perch and loons were positively associated with body mass and δ¹⁵N values. Juvenile loons and large yellow perch had similar mercury levels and δ¹⁵N values, indicating similar trophic status despite their 22-fold difference in body mass. Mercury concentrations were higher in yellow perch and common loons in acidic lakes. Our findings highlight the importance of both chemical and ecological factors in understanding mercury biomagnification in lakes and associated risks to fish-eating wildlife. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Gape limitation and piscine prey size‐selection by yellow perch in the extreme southern area of Lake Michigan, with emphasis on two exotic prey items

The trophic linkage between yellow perch Perca flavescens and two exotic prey items, alewife Alosa pseudoharengus and round goby Neogobius melanostomus , was investigated in the extreme southern area of Lake Michigan during the summer of 2002. Yellow perch ≥100 mm total length, L _T( n  = 1293) exhibited size selective feeding, with 148 fish containing round gobies and 120 fish containing alewives. The mean round goby L _T, preyed on by yellow perch, was 23% of the predator L _T, with a range of 7 to 47%, and mean alewife L _T was 32% of yellow perch L _T, with a range of 18 to 46%. Although the selection of prey size by yellow perch increased proportionally with yellow perch L _T, prey consumed appeared smaller than theoretically possible based on gape size.     

Perch or plankton: top‐down control of Daphnia by yellow perch (Perca flavescens) or Bythotrephes cederstroemi in an inland lake?

1. Seasonal termination of the vernal clear-water phase in Long Lake, Grand Traverse Co., Michigan coincided with severe size-selective predation on juvenile Daphnia pulicaria from 0.8 to 1.8 mm in length. This could be caused by predation by age-0 yellow perch ( Perca flavescens ) or by the exotic predatory zooplankter Bythotrephes cederstroemi .
2. During the initial decline of Daphnia , Ivlev's electivity coefficient for yellow perch from 15.0 to 20.0 mm in length was 0.50 for copepods and −0.75 for D. pulicaria .
3. Bioenergetics modelling of both yellow perch and Bythotrephes demonstrates that, during the initial Daphnia decline, Bythotrephes consumed 1.5–5 times greater total mass than yellow perch. Furthermore, models in which Bythotrephes consumed juvenile Daphnia were more consistent with the timing of the Daphnia decline than those in which yellow perch consumed juvenile Daphnia .
4. The invasion of Bythotrephes into Long Lake seems to be a significant perturbation, introducing effects that propagate throughout the food chain. Bythotrephes created a possible bottleneck for age-0 yellow perch in late June by suppressing Daphnia .     

Molecular characterization and sex-specific tissue expression of estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens)

Yellow perch (Perca flavescens) exhibits an estrogen-stimulated sexual size dimorphism (SSD) wherein females grow faster and larger than males. To aid in the examination of this phenomenon, the cDNA sequences encoding estrogen receptor-alpha (esr1), estrogen receptor-betaa (esr2a) and ovarian aromatase (cyp19a1a) for the teleost yellow perch were obtained. Several tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNAs of yellow perch esr1, esr2a and cyp19a1a consist of 3052 bp, 2462 bp and 1859 bp with open reading frames encoding putative proteins of 576 amino acids, 555 amino acids and 518 amino acids, respectively. Esr1 and esr2a expression was highest in female ovary and liver tissues with low to moderate expression in other tissues. Esr2a showed a more global tissue expression pattern than esr1, particularly in males but also in females. Cyp19a1a expression was highest in both male and female spleen tissue and oocytes with moderate expression in male pituitary and gill tissue. Cyp19a1a expression was moderately high in female liver tissue with undetectable expression in male liver tissue, suggesting its involvement in sexually dimorphic growth. These sequences are valuable molecular tools that can be used in future studies investigating estrogen mechanisms and actions, such as SSD, in yellow perch.     

Chemical and physical factors associated with yellow perch abundance in Great Lakes coastal wetlands: patterns within and among wetland types

Great Lakes coastal wetlands provide important spawning and nursery habitat as well as abundant food resources for yellow perch (Perca flavescens). We examined multiple years of fyke-net data from wetlands along Lakes Huron and Michigan to describe yellow perch distribution in drowned river mouth (DRM) and coastal fringing systems. Principal components analysis and multi-response permutation procedures indicated that DRM wetlands (yellow perch CPUE = 0.2) were eutrophic systems that often exhibit high temperatures and periods of hypoxia, whereas coastal fringing wetlands (yellow perch CPUE = 32.1) were less productive. Among the coastal fringing systems, Saginaw Bay (Lake Huron), displayed characteristics of being more productive and had more yellow perch. Most yellow perch captured in Saginaw Bay were age-0, suggesting that it was an important nursery habitat. Among DRM ecosystems, we found that the downstream lake macrohabitats contained more yellow perch than upstream wetlands; however, there was no significant difference in abiotic characteristics to explain the higher catches in lakes. We hypothesize that yellow perch were more prevalent in wetlands with intermediate productivity during summer because these systems provide abundant food resources without the harsh conditions associated with highly eutrophic wetlands.     

Proteolytic Activity of Microorganisms Isolated from Freshwater Fish

A raw fish-juice was prepared and sterilized through the use of (60)Co gamma-irradiation. It was evaluated for suitability in an agar medium for testing the proteolytic activity of bacteria isolated from fish. Microorganism proteolytic activity was also detected by conventional methods with skim milk-agar. We tested 1,145 isolates from fresh and spoiling irradiated (0.0, 0.3, and 0.6 Mrad) yellow perch fillets for proteolytic activity, by the use of both media. Most isolates that showed proteolytic activity exhibited this activity in both media. A few isolates showed proteolytic activity only in one medium or the other. Proteolysis was found mainly among bacteria isolated from nonirradiated perch fillets. Nonproteolytic organisms were slightly more abundant than were proteolytic ones throughout refrigerated storage (6 days); the latter constituted 48% of the total organisms. Irradiation eliminated essentially all proteolytic bacteria when the fillets were stored at 1 C. However, some proteolytic bacteria survived for a few days after irradiation when the fillets were stored at 5 C.     

苎麻细胞质雄性不育“三系”ISSR 特异片段克隆和序列分析

利用ISSR 分子标记技术对苎麻细胞质雄性不育“三系”mtDNA 进行多态性分析; 在选用的38 个ISSR引物中, 有6 个引物的扩增产物在不育系、保持系和恢复系之间存在差异。对这些特异性片段进行克隆和序列测定, 结果表明: 片段21-MS 全长956 bp , 包含一个525 bp 的完整编码区, 共编码174 个氨基酸。片段31-M􊄯R 全长778 bp , 包含一个404 bp 的不完整编码区, 共编码134 个氨基酸; 其核苷酸和氨基酸序列与已报道的多种植物中的番茄红素β-环化酶基因分别存在71%～76%和73%～77%的同源性。     

苎麻细胞质雄性不育"三系"ISSR特异片段克隆和序列分析

利用ISSR分子标记技术对苎麻细胞质雄性不育"三系"mtDNA进行多态性分析;在选用的38个IS-SR引物中,有6个引物的扩增产物在不育系、保持系和恢复系之间存在差异。对这些特异性片段进行克隆和序列测定,结果表明:片段21-MS全长956bp,包含一个525bp的完整编码区,共编码174个氨基酸。片段31-M/R全长778bp,包含一个404bp的不完整编码区,共编码134个氨基酸;其核苷酸和氨基酸序列与已报道的多种植物中的番茄红素β-环化酶基因分别存在71～76和73～77的同源性。     

Unidentified cells reside in fish skeletal muscle

Cell cultures were established from the skeletal muscle tissue of 6–13 months old rainbow trout and 12–14 months old yellow perch. Approximately 27,000 ± 5,000 cells/g (trout; N = 5) and 5,000 ± 1,200 cells/g of tissue (perch; N = 4) were obtained. Isolation and propagation were qualitatively greater for both species when the cells (younger fish producer more cells than older fish) were exposed to DMEM + 15% FBS, rather than L-15 + 15% FBS, at 20 °C (trout) and at 24 °C (yellow perch). Two morphologically distinct cell types were observed in cultures of both species, some of which eventually formed very small myotubes, which displayed immunocytological reactivity for myogenin, myosin heavy chain, and α-actinin; the second population of cells remained unstained. Successful cryopreservation was achieved using a 5% DMSO and 95% serum mixture, but post-thawing viabilities were low 5–27% (trout) and 14–30% (perch). Further research is needed in order to determine cell type specificity of isolated cells.     

Ubiquitin-calmodulin conjugating activity from cardiac muscle

Enzyme activity capable of covalently linking ubiquitin to bovine calmodulin in an ATP-dependent manner has been detected in rabbit cardiac muscle demonstrating that this enzyme occurs not only in reticulocytes but also in other tissues and possibly all tissues and cells which contain calmodulin as intracellular Ca2+-acceptor protein. This is of special interest since a ubiquitin-dependent proteolytic activity could previously not be detected in cardiac muscle. The name ubiquityl-calmodulin synthetase [uCaM-synthetase, ubiquityl:calmodulin ligase (EC 6.3.?.?)] is therefore suggested for this enzyme. In crude cardiac muscle extracts uCaM-Synthetase displays a specific activity of 93 nUnits/mg in comparison to reticulocyte lysate with 270 nUnits/mg as measured by the fluphenazine-Sepharose affinity adsorbent test (FP-test). Analysis of the ubiquitination product (125I-uCaM) by polyacrylamide electrophoresis in the presence of SDS followed by autoradiography reveals a major double band with molecular masses of 27 and 29 kDa (mono-ubiquitination products) respectively. In addition two novel minor bands (17 and 20 kDa) of smaller molecular mass than the monoubiquitination products were detected. These are probably proteolytic breakdown products of uCaM. A model is suggested for a specific function of this synthetase in the Ca2+-dependent breakdown of calmodulin in vertebrate (eukaryotic) cells.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.