Ultra-sensitive detection of papaya ringspot virus using single-tube nested PCR.

Aphid-transmitted papaya ringspot virus (PRSV) is the greatest disease threat to the commercial production of papaya worldwide. Specific ultrasensitive assays are important for the early detection of PRSV in the field. We have developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of PRSV. The annealing temperatures and concentrations of both primer pairs were optimized to reduce potential competition between primer sets in STNP. The assay is more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. STNP is capable of detecting PRSV in plants 7 days post-inoculation, whereas RT-PCR and ELISA are capable of detecting PRSV 14 to 21 days post-inoculation. This new assay can also detect PRSV from virus infected but asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV infection rates in the field and in imported plant samples. Keywords: early detection; quarantine protocols.

First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2).

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerase chain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primers were designed to anneal at higher temperatures than the nested primers to prevent primer competition in consecutive amplification reactions. To reduce potential competition further, the outer primers were used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of this assay are much greater than PCR using only a single primer-pair. A TaqMan(®) probe was also designed for use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR are reduced handling time and prevention of cross contamination compared to regular nested PCR in which the reactions are carried out in two separate tubes.

Pineapple bacilliform CO virus: Diversity, Detection, Distribution, and Transmission.

Members of the genus Badnavirus (family Caulimovirdae) have been identified in dicots and monocots worldwide. The genome of a pineapple badnavirus, designated Pineapple bacilliform CO virus-HI1 (PBCOV-HI1), and nine genomic variants (A through H) were isolated and sequenced from pineapple, Ananas comosus, in Hawaii. The 7,451-nucleotide genome of PBCOV-HI1 possesses three open reading frames (ORFs) encoding putative proteins of 20 (ORF1), 15 (ORF2), and 211 (ORF3) kDa. ORF3 encodes a polyprotein that includes a putative movement protein and viral aspartyl proteinase, reverse transcriptase, and RNase H regions. Three distinct groups of putative endogenous pineapple pararetroviral sequences and Metaviridae-like retrotransposons encoding long terminal repeat, reverse-transcriptase, RNase H, and integrase regions were also identified from the pineapple genome. Detection assays were developed to distinguish PBCOV-HI1 and genomic variants, putative endogenous pararetrovirus sequences, and Ananas Metaviridae sequences also identified in pineapple. PBCOV-HI1 incidences in two commercially grown pineapple hybrids, PRI 73-114 and PRI 73-50, was 34 to 68%. PBCOV-HI1 was transmitted by gray pineapple mealybugs, Dysmicoccus neobrevipes, to pineapple.

First Report of Pepper mottle virus Infecting Tomato in Hawaii.

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5'- CACTCCCTATTATCCAGG(T)16-3') following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.

First Report of Iris yellow spot virus in Onion in Hawaii.

Onion (Allium spp.) production in Hawaii is mostly comprised of green onion and the locally prized sweet bulb onions (Allium cepa L.) that include short- and medium-day cultivars. Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) is an important constraint to bulb and seed onion production in many onion-growing regions of the continental United States and the world (3). In June 2010, straw-colored, diamond-shaped lesions with occasional green islands were observed on leaves of sweet onion 'Linda Vista' in an insecticide trial on Maui for onion thrips (Thrips tabaci) control. Collapse and lodging occurred when lesions on leaves were severe. Seven bulbs with green leaves exhibiting lesions were collected from this onion field in the Pulehu Region of the lower Kula District on Maui. Leaf samples that included a lesion or were within 1 cm of a lesion were found to be positive in indirect ELISA with IYSV-specific polyclonal antisera (2). A405nm readings after 1 h ranged from 0.263 to 2.067 for positive samples and 0.055 to 0.073 for healthy onion controls. Four samples that were prepared from leaf tissue several centimeters away from a lesion tested negative in ELISA. Such uneven virus distribution in the plants has been previously reported (4). In July 2010, symptomatic sweet onion from a commercial farm in upper Kula, Maui at the 1,060 to 1,220 m (3,500 to 4,000 foot) elevation tested positive for IYSV by ELISA. Green onion samples collected from a commercial farm in Omaopio, Maui, located approximately 0.8 km (0.5 mile) north of Pulehu, have tested negative, suggesting distribution may be limited at this time. RNA was isolated from leaf tissue from the seven 'Linda Vista' sweet onions collected from the Maui insecticide trial. Reverse transcription (RT)-PCR with forward and complementary primers 5'-CTCTTAAACACATTTAACAAGCAC-3' and 5'-TAAAACAAACATTCAAACAA-3' flanking the nucleocapsid (N) gene encoded by the small RNA of IYSV was conducted as previously described (1). Amplicons approximately 1.1 kb long were obtained from all seven symptomatic onion samples but not from healthy samples or water controls. Sequencing of selected amplicons confirmed IYSV infection. Three sequence variants (GenBank Accession Nos. HM776014-HM776016) were identified from two RT-PCR reactions. Phylogenetic analyses of the three sequence variants with the neighbor-joining procedure available through NCBI-BLASTn Tree View showed that the highest nucleotide identities of 97 to 98% were shared with IYSV isolates from New Zealand (EU477515), Nevada (FJ713699), and northern California (FJ713700). Phylogenetic analyses with the N-gene showed the sequences from Hawaii are most closely related to isolates from the western United States, Texas, and New Zealand. To date, to our knowledge, IYSV has not been detected on the islands of Kauai, Oahu, Molokai, or Hawaii. The distribution and economic consequences of this disease to Hawaii's onion production are under investigation. References: (1) H. R. Pappu et al. Arch Virol. 151:1015, 2006. (2) H. R. Pappu et al. Plant Dis. 92:588, 2008. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) T. N. Smith et al. Plant Dis. 90:729, 2006.

First Report of Banana bract mosaic virus in Flowering Ginger in Hawaii.

Flowering ginger, Alpinia purpurata (Vieill.) K. Schum., is a popular cut flower and tropical landscape plant in Hawaii. In Hawaii, ginger flowers, including red and pink cultivars, are grown as field crops with an estimated annual sales of more than $1.6 million (USD) in 2006 (2). In June 2009, a commercial ginger flower grower from Waimanalo, Oahu, Hawaii reported plants with symptoms that included severe mosaic and stripes on the leaves. Flowers showed significant cupping and browning and growers report a reduction in size and shelf life. Symptomatic ginger was also identified at the Lyon Arboretum in Honolulu. Double-stranded RNAs (dsRNAs) were isolated from pooled leaf samples collected from 42 symptomatic plants at two locations on the island of Oahu to further characterize the pathogen associated with the symptomatic ginger. dsRNAs of approximately 0.7, 1.1, 1.8, 2.2, and 12 kb were present in the extractions from symptomatic plants but not in extractions from asymptomatic plants. Partial cloning and sequence analysis of the dsRNA revealed 95 to 98% nucleotide identity to sequences of P1, HC-Pro, C1, 6K2, VpG, NIb, and CP genes and the 3' untranslated region (total approximately 6 kb) of Banana bract mosaic virus (BBrMV). Total RNAs were also isolated from the symptomatic and asymptomatic plants from the Waimanalo farm and Lyon Arboretum. These RNA isolations were used in reverse transcription (RT)-PCR with primers Bract N1: 5'-GGRACATCACCAAATTTRAATGG-3' and Bract NR: 5'-GTGTGCYTCTCTAGCCCTGTT-3' (1), to amplify a 279-bp conserved region of the coat protein of BBrMV. Amplicons of the appropriate size were obtained from 38 of the symptomatic plants, whereas none were obtained from asymptomatic controls. RT-PCR amplicons of arbitrarily selected samples were cloned into pGEM-T Easy, sequenced, and found to be 99% identical to corresponding sequences of BBrMV. Furthermore, using double-antibody sandwich-ELISA assay and antibodies (3), we developed a system that can specifically detect BBrMV in infected flowering ginger plants and not in healthy appearing ginger. To our knowledge, this is the first report of BBrMV in flowering ginger in Hawaii. Further research is needed to determine if BBrMV infecting ginger poses a threat to banana, edible ginger, and other closely related ornamentals in Hawaii. References: (1) M. L. Iskra-Caruana et al. J. Virol. Methods 153:223, 2008. (2) Statistics of Hawaii Agriculture (2006). HDOA/USDA (NASS). 96, 2008. (3) J. E. Thomas et al. Phytopathology 87:698, 1997.

Spatial and Temporal Incidences of Pineapple mealybug wilt-associated viruses in Pineapple Planting Blocks.

A complex of Pineapple mealybug wilt-associated viruses (PMWaVs) that can infect pineapple (Ananas comosus) is correlated with reduced yields and mealybug wilt of pineapple. The incidences of PMWaV-1 and PMWaV-2 at planting, fruit harvest, the beginning of the ratoon crop, and ratoon fruit harvest were determined for end, side, and central regions of planting blocks in eight commercial fields. Differences in virus incidence for the three regions at ratoon harvest were highly significant (P = 0.0018). Central regions of planting blocks had lower virus incidences at the time of ratoon fruit harvest. Collection of propagation material from the central regions of planting blocks will help to minimize PMWaV incidence in fields planted with this material.

First Report of Tomato yellow leaf curl virus in Hawaii.

Tomato yellow leaf curl disease, caused by the begomovirus Tomato yellow leaf curl virus (TYLCV; family Geminiviridae), is an economically important disease of tomato (Solanum lycopersicum L.) that can be very destructive in tropical and subtropical regions (1). In October 2009, tomato plants showing stunted new growth, interveinal chlorosis, and upward curling of leaf margins were reported by a residential gardener in Wailuku, on the island of Maui. Similar symptoms were observed in approximately 200 tomato plants at a University of Hawaii research farm in Poamoho, on the island of Oahu in November 2009. The similarity between these symptoms and those of tomato yellow leaf curl disease and the presence of whiteflies (Bemisia spp.), the vector of TYLCV, suggested the causal agent was a geminivirus such as TYLCV. Total nucleic acids were extracted from a tomato plant sample from Wailuku and Poamoho and used in a PCR assay with degenerate primers PAR1c715 and PAL1v1978 for geminivirus detection (4). The ~1.5-kbp amplicon expected to be produced from a geminivirus template was generated from the symptomatic tomato plant samples but not from a greenhouse-grown control tomato plant. The amplicons were cloned by the pGEM-T Easy vector (Promega, Madison, WI). Three clones from each sample were sequenced, revealing 97 to 99% nucleotide identity to TYLCV sequences in GenBank and a 98.9% nucleotide identity between the Wailuku (Accession No. GU322424) and Poamoho (Accession No. GU322423) isolates. A multiplex PCR assay for the detection and discrimination between the IL and Mld clades of TYLCV was also performed on these isolates (2). A ~0.8-kbp amplicon was generated from both isolates confirming the presence of TYLCV and their inclusion into the TYLCV-IL clade (2). Seven symptomatic and three asymptomatic tomato plant samples from Poamoho were tested for TYLCV using a squash-blot hybridization assay (3) utilizing a digoxigenin-labeled probe derived from the ~1.5-kbp PCR amplicon. All symptomatic tomato plants and one asymptomatic tomato plant were found to be infected with TYLCV. How the virus entered Hawaii and how long it has been present is unknown. The most plausible route is through infected plant material such as an asymptomatic alternative host rather than viruliferous whiteflies. It appears TYLCV is not a recent introduction into Hawaii since the Wailuku gardener observed similar disease symptoms for a few years before submitting samples for testing. In January 2010, TYLCV was also detected in two commercial tomato farms on Oahu, posing a serious threat to the state's $10 million annual tomato crop. References: (1) H. Czosnek and H. Laterrot. Arch. Virol. 142:1392, 1997. (2) P. Lefeuvre et al. J. Virol. Methods 144:165, 2007. (3) N. Navot et al. Phytopathology 79:562, 1989. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Complete nucleotide sequence and genome organization of pineapple mealybug wilt-associated virus-1.

Pineapple mealybug wilt-associated virus-1 (PMWaV-1; family Closteroviridae, genus Ampelovirus) belongs to a complex of mealybug-transmissible viruses found in pineapple worldwide. In this study, the complete genome of PMWaV-1 was sequenced and found to be 13.1 kb in length, making it the smallest in the family. The genome encoded seven open reading frames (ORFs) and was unusual for an ampelovirus due to the lack of an intergenic region between the RdRp and p6 ORFs, an ORF encoding a relatively small coat protein (CP), and the absence of an ORF encoding a coat protein duplicate (CPd). Phylogenetic analyses placed PMWaV-1, plum bark necrosis stem pitting-associated virus and some grapevine leafroll-associated viruses in a distinct clade within the genus Ampelovirus.

First Report of Phytoplasmas Infecting Watercress in Hawaii.

Symptoms of leaf yellowing, reduced leaf size, and witches'-brooms have recently been observed affecting watercress (Nasturtium microphyllum Boen. × Rcbh.) in Hawaii. These symptoms are followed by the collapse of affected plants. This condition has led to 80 to 90% losses for one of the largest watercress farms on Oahu and is now affecting other watercress farms in the area. Nutritional deficiencies or toxicities, water salinity, and insect or mite feeding damage were investigated but could not be implicated in the etiology of this syndrome. Eighteen watercress plants with early yellowing or advanced symptoms and nine symptomless plants were analyzed for phytoplasma infection using polymerase chain reaction (PCR) assays with primer pairs P1/Tint or P1/P7 (4). Amplicons of the expected sizes were produced from all symptomatic plants, whereas no products were amplified from symptomless plants. Sequence analysis of the cloned PCR products confirmed their phytoplasma origin and indicated that the watercress was infected with a phytoplasma most similar to SAY (2), a severe strain of western aster yellows phytoplasma previously classified as a 16SrI-B group member (3). Leafhoppers collected from an affected watercress planting have been identified as the aster leafhopper (Macrosteles quadrilineatus Fbs.) This species is the most efficient vector of the aster yellows phytoplasma and had not been previously recorded in Hawaii. The only other phytoplasma disease known in Hawaii prior to this report is Dodonaea yellows (1), which affects one of the most common native plants (Dodonaea viscosa (L.) Jacq.) in dry upland forests on all the islands. Dodonaea yellows, however, has been attributed to an X-disease (16SrIII) group phytoplasma. The occurrence of an aster yellows group phytoplasma in watercress, a previously unrecorded host, and the presence of a very efficient vector, M. quadrilineatus, poses a serious threat to the production of other vegetable and floral crops in Hawaii. References: (1) W. Borth et al. Plant Dis. 79:1094, 1995. (2) C. Kuske and B. Kirkpatrick. Int. J. Syst. Bacteriol. 42:226, 1992. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) C. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Effects of synthetic cecropin analogs on in vitro growth of Acholeplasma laidlawii.

Four synthetic peptides (Peptidyl MIMs; Demeter Biotechnologies, Inc.) were evaluated for their in vitro activity against Acholeplasma laidlawii. Fifty percent effective concentration values ranged from 1 to 15 microM. Three of these compounds are more lethal than cecropin B against A. laidlawii.

Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II.

Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.

Degradation of amyloid beta-protein by a serine protease-alpha2-macroglobulin complex.

Progressive cerebral deposition of the amyloid beta-peptide (Abeta) is an early and constant feature of Alzheimer's disease. Abeta is derived by proteolysis from the beta-amyloid precursor protein. beta-Amyloid precursor protein processing and the generation of Abeta have been extensively characterized, but little is known about the mechanisms of degradation of this potentially neurotoxic peptide. We identified and purified a proteolytic activity in culture medium that can degrade secreted Abeta but not larger proteins in the medium. Detection of the activity in conditioned medium required the presence of fetal bovine serum and the passage of the cells with a pancreatic trypsin preparation. Its inhibitor profile showed that the activity was a serine protease other than trypsin or chymotrypsin. The protease occurs as a stable approximately 700-kDa complex with the inhibitor, alpha2-macroglobulin (alpha2M), that retains activity against small substrates such as Abeta. Its NH2-terminal sequence suggests that the protease is previously unidentified. Our results indicate that the Abeta-degrading protease we have detected is a non-trypsin component of a pancreatic trypsin preparation or else derives from a zymogen in serum that is activated by a protease in the latter preparation. Because Abeta-bearing plaques in Alzheimer's disease brain contain both alpha2M and receptors of alpha2M-protease complexes, the same or a similar alpha2M-protease complex could arise in vivo and play a role in Abeta clearance.

Homocysteine and hemostasis: pathogenic mechanisms predisposing to thrombosis.

Growing evidence suggests that moderately elevated levels of homocysteine are associated not only with arterial thrombosis and atherosclerosis but also with venous thrombosis as well. We have reviewed recent studies that indicate that homocysteine inhibits several different anticoagulant mechanisms that are mediated by the vascular endothelium. The protein C enzyme system appears to be one of the most important anticoagulant pathways in the blood. Homocysteine inhibits the expression and activity of endothelial cell surface thrombomodulin, the thrombin cofactor responsible for protein C activation. Homocysteine inhibits the antithrombin III binding activity of endothelial heparan sulfate proteoglycan, thereby suppressing the anticoagulant effect of antithrombin III. Homocysteine also inhibits the ecto-ADPase activity of human umbilical vein endothelial cells (HUVECS). Because ADP is a potent platelet aggregatory agent, this action of homocysteine is prothrombotic. Homocysteine also interferes with the fibrinolytic properties of the endothelial surface because it inhibits the binding of tissue plasminogen activator. Homocysteine stimulates HUVEC tissue factor activity. We have found that lipoprotein(a) [Lp(a)] also stimulates HUVEC tissue factor activity. The combination of Lp(a) plus homocysteine induced more tissue factor activity than either agent alone. These disruptions in several different vessel wall-related anticoagulant functions provide plausable mechanisms for the occurrence of thrombosis in hyperhomocysteinemia.

THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-beta 1 via increased expression of urokinase and the urokinase receptor.

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor-beta 1 (TGF-beta 1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-beta 1 induction of uPA expression by THP-1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF-beta 1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF-beta 1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-beta 1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF-beta 1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity. Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-beta 1. The increase in membrane-uPA activity expressed by TGF-beta 1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-beta 1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF-beta 1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-beta 1-treated cells. However, no change in immunoreactive membrane-bound plasmin(ogen) was observed. In addition, binding of 125I-Lys-plasminogen to THP-1 cells was not affected by TGF-beta 1 treatment. We conclude that TGF-beta 1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression.

Lipoprotein(a), plasmin modulation, and atherogenesis.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein however the mechanisms by which Lp(a) promote the atherosclerotic process are not clear. The apolipoprotein(a) portion of Lp(a) shares partial homology with plasminogen, a finding that has stimulated numerous studies. Lp(a) binds to fibrin and the affinity between fibrin surfaces and Lp(a) appears to be related to the state of oxidation of the lipoprotein particle. Lp(a) also effects fibrin-dependent plasminogen activation. Recent findings suggest that dependent plasminogen activation. Recent findings suggest that depending upon the in vitro conditions, Lp(a) either promotes or inhibits plasmin formation. Lp(a) also inhibits cell-surface dependent plasmin generation that is associated with an inhibition of transforming growth factor-beta (TGF-beta) production in cell coculture systems. Lp(a) stimulates smooth muscle cell migration and proliferation as a secondary response to this decrease in TGF-beta concentration. Studies in transgenic mice containing the human apolipoprotein(a) gene, document that both plasmin and TGF-beta formation in the media of the aorta is markedly decreased in the presence of apo(a). Thus the atherogenicity of Lp(a) may be mediated, in part, through its modulation of plasmin and TGF-beta production in the blood vessel wall.

Regulation of macrophage receptor-bound plasmin by autoproteolysis.

The activation of plasminogen by macrophage is regulated by their expression of receptors for urokinase and plasmin(ogen). In these studies we have examined plasmin(ogen) binding to adherent human THP-1 macrophage. Plasmin bound to the THP-1 cells in a time- and dose-dependent manner (Kd 15.8 +/- 6.2 nM; Bmax 1.4 +/- 0.3 x 10(6)/cell). The lysine analog epsilon-aminocaproic acid competitively inhibited plasmin binding. The fraction of membrane-bound plasmin, however, became increasingly resistant to displacement with epsilon-aminocaproic acid. Over a 24-h period, membrane-bound plasmin activity fell 80% despite the presence of catalytically active plasmin in the incubation media. The loss of receptor-bound plasmin activity was not due to proteolytic alterations of its receptor since 125I-Lys-plasminogen bound to THP-1 cells pretreated with plasmin with similar affinity as to untreated cells. Following a 24-h incubation of 125I-Lys-plasminogen or 125I-plasmin with THP-1 cells, several degradative fragments were apparent in their conditioned media. The smaller degradative fragments (28 and 36 kDa) lacked cell binding activity and were demonstrated to be active by casein-zymography. A 48-kDa fragment bound to cells in a lysine-dependent manner but was not active. In contrast, phenylmethylsulfonyl fluoride-inactivated 125I-plasmin retained its binding activity over 24 h, and degradative fragments were not present in the conditioned media. The binding of 125I-Lys-plasmin(ogen) to THP-1 cells was also examined in the presence of excess alpha 2 plasmin inhibitor. Despite the absence of fluid-phase plasmin activity, membrane-bound 125I-Lys-plasmin(ogen) decreased over 24 h. At 24 h a radiolabeled 48-kDa fragment was observed in the conditioned media and together with 125I-Lys-plasmin(ogen) was bound to cells. Unlike 125I-Lys-plasmin, the 48-kDa fragment did not form a complex with alpha 2 plasmin inhibitor. Thus, autoproteolysis of receptor-bound plasmin results in fragments with truncated physiologic properties that possess either cell binding or catalytic activities. We propose that autoproteolysis is a mechanism for regulating membrane-bound plasmin activity.