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PubMed2006

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions.

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV). Like other PDVs, MdBV replicates in the ovaries of females where virions accumulate to form a suspension called calyx fluid. Wasps then inject a quantity of calyx fluid when ovipositing into hosts. The MdBV genome consists of 15 segments that range from 3.6 (segment A) to 34.3 kb (segment O). EM analysis indicated that MdBV virions contain a single nucleocapsid that encapsidates one circular DNA of variable size. We developed a semi-quantitative real-time PCR assay using SYBR Green I. This assay indicated that five (J, O, H, N and B) segments of the MdBV genome accounted for more than 60% of the viral DNAs in calyx fluid. Estimates of relative segment abundance using our real-time PCR assay were also very similar to DNA size distributions determined from micrographs. Analysis of parasitized Pseudoplusia includens larvae indicated that copy number of MdBV segments C, B and J varied between hosts but their relative abundance within a host was virtually identical to their abundance in calyx fluid. Among-tissue assays indicated that each viral segment was most abundant in hemocytes and least abundant in salivary glands. However, the relative abundance of each segment to one another was similar in all tissues. We also found no clear relationship between MdBV segment and transcript abundance in hemocytes and fat body.

Animals↗
PubMed2005

bloodthirsty, an RBCC/TRIM gene required for erythropoiesis in zebrafish.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Amino Acid Sequence↗
PubMed2005

Human herpesvirus 6 activates lytic cycle replication of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a gamma-herpesvirus consistently identified in Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. KSHV infection appears to be necessary, but not be sufficient for development of KS without other co-factors. However, factors that facilitate KSHV to cause KS have not been well defined. Because patients with KS are often immunosuppressed and susceptible to many infectious agents including human herpesvirus 6 (HHV-6), we investigated the potential of HHV-6 to influence the replication of KSHV. By co-culturing HHV-6-infected T cells with KSHV-latent BCBL-1 cell line, infecting BCBL-1 cells with HHV-6 virions, and generating heterokaryons between HHV-6-infected T cells and BCBL-1 cells, we showed that HHV-6 played a critical role in induction of KSHV replication, as determined by production of lytic phase mRNA transcripts and viral proteins. We confirmed and extended the results by using a luciferase reporter assay in which KSHV ORF50 promoter, the first promoter activated during KSHV replication, drove the luciferase expression. Besides HHV-6, we also found that cytokines such as interferon-gamma partially contributed to induction of KSHV replication in the co-culture system. These findings suggest that HHV-6 may participate in KS pathogenesis by promoting KSHV replication and increasing KSHV viral load.

Cell Cycle↗
PubMed2004

Activation of the GLI oncogene through fusion with the beta-actin gene (ACTB) in a group of distinctive pericytic neoplasms: pericytoma with t(7;12).

Activation of the GLI oncogene is an important step in the sonic hedgehog signaling pathway, and leads to, eg, tissue-specific cell proliferation during embryogenesis. GLI activity in adult tissues is restricted, but has been identified in various neoplasms, as a result of mutations in the PTCH (patched) or SMOH (smoothened) genes, encoding components of the sonic hedgehog pathway, or by amplification of GLI. Herein, we present a new mechanism of GLI activation through fusion with the beta-actin gene (ACTB) in five histologically distinctive soft tissue tumors showing a t(7;12)(p21-22;q13-15) and a pericytic phenotype. Each was composed of a perivascular proliferation of monomorphic short spindle cells that stained positively for smooth muscle actin and laminin and that showed pericytic features by electron microscopy. To date, with a median follow-up of 24 months, none has behaved in an aggressive manner. Molecular genetic analysis showed that the translocation in all cases resulted in a fusion transcript including the 5'-part of ACTB and the 3'-part of GLI. The DNA-binding zinc finger domains of GLI were retained in the fusion transcripts and it is likely that the replacement of the promoter region of GLI with that of the ubiquitously expressed ACTB gene leads to deregulation of GLI expression and its downstream target genes.

Actins↗
PubMed2002

Simian virus 40 sequences and expression of the viral large T antigen oncoprotein in human pleomorphic adenomas of parotid glands.

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.

Adenoma, Pleomorphic↗
PubMed2002

Calnexin Delta 185-520 partially reverses the misprocessing of the Delta F508 cystic fibrosis transmembrane conductance regulator.

Abnormal retention of Delta F508 CFTR (cystic fibrosis transmembrane conductance regulator) in the endoplasmic reticulum is a major cause of cystic fibrosis (CF). We show that calnexin Delta 185-520 but not calnexin can partially reverse the mislocalization of Delta F508 CFTR. This 256-amino acid protein has neither the transmembrane domain nor the P domain of calnexin. Calnexin Delta 185-520 interacted with CFTR directly, and was secreted into the extracellular compartment over time. Forty-eight hours after transfection into CHO cells, calnexin Delta 185-520 increased the conversion of immature Delta F508 CFTR into mature Delta F508 CFTR. In immortalized human CF cell lines expressing Delta F508 CFTR, a halide efflux assay showed that calnexin Delta 185-520 partially restored CFTR function. These data indicate that calnexin Delta 185-520 may give a clue to develop the therapeutic way of cystic fibrosis with Delta F508 CFTR.

Animals↗
PubMed1998

Genomic organisation, alternative splicing and primary structure of human matrilin-4.

We have recently cloned a cDNA for mouse matrilin-4. By sequence comparison we identified the 12 kb long human matrilin-4 gene as a part of a high-throughput genomic sequence (HS453C12) in the databases. Additionally we found a human matrilin-4 expressed sequence tag (H54037) in the database that had been mapped to chromosome 20q13.1-2. The gene contains 10 exons and, like the matrilin-1 gene, the human matrilin-4 gene contains an AT-AC intron between the two exons encoding the coiled-coil domain. The cDNA sequence of human matrilin-4 was determined by sequencing of RT-PCR products obtained from mRNA of the human embryonic kidney cell line HEK 293. At the amino acid level it showed an overall sequence identity to the mature mouse matrilin-4 of 91% with a maximum of 97% in the second vWFA-like module. Alternative splicing leads to three different mRNAs. They all encode the putative signal peptide, the two vWFA-like domains and the potential coiled-coil alpha-helical oligomerisation domain but differ in that either one, two or three EGF-like domains are retained in the mature mRNA. Due to a G to A mutation at the splice donor site of intron C, the third exon encodes an untranslated pseudo-exon specifying the first EGF-like domain when compared to mouse matrilin-4.

Alternative Splicing↗
PubMed2002

Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder.

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly-Lys-Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.

Amino Acid Sequence↗
PubMed2003

Papillomaviruses infect cells via a clathrin-dependent pathway.

In this study we have examined the pathway by which papillomaviruses infect cells, using bovine papillomavirus (BPV) virions and mouse C127 cells as the model system. By confocal microscopy, the entry of BPV virions, BPV virus-like particles (VLPs), and HPV16 VLPs were very similar. In dually exposed cells, HPV-16 VLPs and BPV virions colocalized intracellularly. BPV VLPs colocalized with AP-2, a clathrin adapter molecular and a marker of the clathrin-dependent endocytic pathway; and also with transferrin receptor, a marker of early endosomes; and Lamp-2, a marker of late endosomes and lysosomes. BPV infection was detected within 12 h of virion cell-surface binding, as measured by an RT-PCR assay. Infection was prevented by several pharmacologic inhibitors, including chlorpromazine, which blocks clathrin-dependent endocytosis and the lysosomotropic agent, bafilomycin A. By contrast, two inhibitors of caveolae-dependent uptake, filipin and nystatin, did not prevent BPV infection. We conclude that papillomaviruses infect cells via clathrin-dependent receptor-mediated endocytosis. Surprisingly, the kinetics of internalization were unusually slow for this mechanism, with the t(1/2) of entry of BPV-1 being approximately 4 h versus 5-15 min for a typical ligand.

Animals↗
PubMed2002

Differential expression of Kallikrein gene 5 in cancerous and normal testicular tissues.

OBJECTIVES: Kallikrein gene 5 (KLK5; formerly designated as kallikrein-like gene 2, or human stratum corneum tryptic enzyme) is one of the new members of the human kallikrein gene family on chromosome 19q13.4. Although it is expressed at low levels in various tissues, KLK5 expression is highest in the human mammary gland and testis. Previous investigations have established that the expression of KLK5 is estrogen and progestin driven in the BT-474 breast cancer cell line. In this study, we focused on KLK5 expression in normal and cancerous testicular tissue. METHODS: Fourteen matched testicular tumor and adjacent normal tissue samples were minced on dry ice and homogenized. Total RNA was extracted and mRNA was reverse transcribed. The cDNA samples were amplified by real-time quantitative polymerase chain reaction with KLK5-specific primers to compare the relative KLK5 levels in normal and cancerous testicular tissues. RESULTS: In 13 (93%) of the 14 patients, KLK5 expression in the cancerous area was significantly lower than in the adjacent, histologically confirmed, normal testicular tissue samples (P <0.001). The KLK5 level was 9.0 +/- 3.9 (mean +/- standard error, arbitrary units) in the noncancerous tissue and 4.5 +/- 2.9 in the cancerous tissue. We noted significantly lower KLK5 expression in seminomas than in nonseminomas (P = 0.009), as well as in late-stage (II/III) tumors versus early-stage (I) tumors (P = 0.026). KLK5 expression was also associated with the extent of primary tumor, with tumors with vascular/lymphatic invasion (T2/T3) expressing lower KLK5 message than did tumors limited to the testis and epididymis (T1) (P = 0.008). CONCLUSIONS: We found significantly lower expression of KLK5 in testicular tumors than in normal testicular tissue. More studies are necessary to investigate the mechanism behind this finding.

Adolescent↗
PubMed1998

FIV infection of IL-2-dependent and -independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation.

We have analyzed the ability of three molecular clones of feline immunodeficiency virus (FIV) and an ex vivo variant to infect nine distinct specific-pathogen-free feline cell lines in tissue culture. The purpose of these studies was to elucidate mechanisms by which host cells regulate the level of virus infection and expression and to assess host cell cytokine responses to virus infection. Cells used for the analyzes included four IL-2-dependent continuous T-cell lines (104-C1, 104-C7, MCH5-4 and DB FeTs) which arose from long-term passage, followed by limiting dilution cloning of peripheral blood mononuclear cells (PBMCs); two IL-2-independent T-cell lines (104-C1DL and MCH5-4DL) which originated from two of the IL-2-dependent lines, 104-C1 and MCH5-4; respectively; Crandell feline kidney cells (CrFK); G355-5 brain-derived glial cells; and the T-cell lymphoma line, 3201. Cells were infected with FIV-PPR, FIV-34TF10, FIV 34TF10orf2rep, and a variant arising from FIV-PPR during ex vivo passage on 104-C1DL cells, termed FIV-PPRglial. Infection of the IL-2-dependent T-cell line, 104-C1, by FIV-PPR resulted in the specific and distinct upregulation of cytokine expression. In particular, these cells doubled their expression of the pleiotropic cytokines, interleukin-4 and interleukin-12 after FIV infection. Interferon-gamma production also increased after infection with FIV whereas, TNFalpha expression remained constant. Also, a marked upregulation of MHC class II expression was noted post infection of MCH5-4 and 104-C1 cells with FIV-PPR. Similar results were obtained after infection with FIV-34TF10orf2rep, indicating that the upregulation of cytokine expression is not an isolate-specific phenomenon. Changes in cytokine and class II expression are similar to various reports for the in vivo cytokine alterations in FIV, SIV and HIV infections. The ex vivo infection of these cell lines offers amanipulable system to examine the mechanism(s) by which lentiviruses alter cytokine expression.

Animals↗
PubMed2001

Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach.

A detection system based on nested PCR after IC-RT-PCR (IC-RT-PCR-Nested PCR) was developed to improve indexing of Prunus necrotic ringspot virus in peach trees. Inhibitory effects and inconsistencies of the standard IC-RT-PCR were overcome by this approach. IC-RT-PCR-Nested PCR improved detection by three orders of magnitude compared with DAS-ELISA for the detection of PNRSV in leaves. Several different tissues were evaluated and equally consistent results were observed. The main advantages of the method are its consistency, high sensitivity and easy application in quarantine programs.

Bromoviridae↗
PubMed2002

Pretreatment to avoid positive RT-PCR results with inactivated viruses.

Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.

Animals↗
PubMed2002

Comparison of four reverse transcription-polymerase chain reaction procedures for the detection of dengue virus in clinical specimens.

The sensitivity of dengue virus identification by mosquito inoculation and four reverse transcription-polymerase chain reaction (RT-PCR) procedures (Am. J. Trop. Med. Hyg. 45 (1991) 418 (H); J. Clin. Microbiol. 29 (1991) 2107 (M); J. Clin. Microbiol. 30 (1992) 545 (L); and Southeast Asian J. Trop. Med. Public Health 27 (1996) 228 (Y)) were compared using coded clinical specimens derived from areas in Thailand where all four dengue serotypes circulate. The sensitivity of virus detection in serologically confirmed dengue cases was 54, 52, 60, 79, and 80% for mosquito inoculation, procedures H, M, L and Y, respectively. In comparison to clinical specimens which yielded virus isolates by mosquito inoculation, there was relatively low sensitivity in detecting each of the four dengue serotypes by PCR: procedure H-dengue 4 (25%), procedure M-dengue 3 (73%), procedure L-dengue 1 (70%), and procedure Y-dengue 1 (79%). Dengue virus was detectable by RT-PCR for more days of illness and in the presence of dengue-specific antibody when compared to virus isolated in mosquitoes. Procedures L and Y were more sensitive than mosquito inoculation or procedures H and M in detecting all four dengue serotypes in clinical specimens and may be the RT-PCR methods of choice for virus surveillance or research use.

Aedes↗
PubMed1999

Nested PCR improves detection of infectious hematopoietic necrosis virus in cells coinfected with infectious pancreatic necrosis virus.

A nested assay using the reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures coinfected with infectious pancreatic necrosis virus (IPNV). Two pairs of primers were designed: one for the amplification of glycoprotein G-specific gene RNA from IHNV (or 1512 bp fragment), and the other for the amplification of an inner 753 bp fragment using the cDNA from the G gene as substrate. Direct RT-PCR was also developed for the amplification of a VP-2 gene fragment from IPNV (613 bp fragment); this method always detected the virus IPNV in the coinfected cells tested but the amplification of IHNV was not as readily achieved. IHNV, however, was detected specifically by nested PCR in coinfected cells at a multiplicity of infection that was 1000 times lower than that of IPNV. Nested PCR was therefore more sensitive than direct RT-PCR for IHNV, and may thus be more appropriate for the detection of low infective titers of IHNV in the presence of IPNV when interference occurs.

Animals↗
PubMed1999

Inhibition of HIV-1 infection by zinc group metal compounds.

Thirty-seven metal compounds were examined for inhibitory activities against infection with human immunodeficiency virus type 1 (HIV-1). Zinc group metal compounds, namely, zinc acetate, zinc chloride, zinc nitrate, cadmium acetate and mercury chloride, showed anti-HIV-1 activities. Cadmium and mercury compounds at 1-10 microg/ml and zinc compounds at 100 microg/ml strongly inhibited HIV-1 infection, although the cadmium, mercury and zinc compounds had severe cytotoxities at 100, 100 and 1000 microg/ml, respectively. They inhibited transcription of HIV-1 RNA and HIV-1 production at concentrations at which they did not affect the growth of HIV-1-producing cells. They had little effect on syncytium formation resulting from cocultivation of uninfected with HIV-1-producing cells. Nor did they affect HIV-1 DNA synthesis following HIV-1 infection. The metal compounds may owe their anti-HIV-1 effects to inhibition of HIV-1 DNA to RNA transcription, rather than inhibition of the adsorption, penetration or reverse transcription step of HIV-1 infection.

Antiviral Agents↗
PubMed2001

Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans.

A genomic copy of a gut-expressed Haemonchus contortus candidate vaccine antigen, pepsinogen, was isolated using the polymerase chain reaction (PCR). The isolated sequence was 4 kb in length and contained eight introns ranging in size from 54 to 1475 base pairs. This sequence, together with its 3' non-coding DNA region containing a polyadenylation signal sequence, was cloned into the Bluescript SK(+) vector immediately downstream of the Caenorhabditis elegans cpr-5 gene promoter. This promoter has been shown previously to direct protein expression to the gut of C. elegans. The construct was micro-injected into DR96 unc-76(e911) mutant C. elegans together with a rescue plasmid and transgenic worms identified by reversion back to wild-type phenotype. Two transgenic lines of C. elegans were established. The presence of the injected construct and of the Haemonchus pepsinogen transcript in transgenic worms was confirmed by PCR analysis. Correct splicing of intronic sequences was observed. Immunohistochemistry showed expression of the Haemonchus pepsinogen protein in the gut of transgenic C. elegans, with reactivity evident in the larval and adult stages. Expression of the Haemonchus pepsinogen in C. elegans affirms the role of C. elegans as a model for parasitic nematodes and demonstrates its potential as a vector for expression of candidate vaccine antigens from parasitic nematodes.

Animals↗