Mutagenesis of avian carcinoma virus MH2: only one of two potential transforming genes (delta gag-myc) transforms fibroblasts.

Avian carcinoma virus MH2 contains two potential transforming genes, delta gag-mht and delta gag-myc. Thus, MH2 may be a model for two-gene carcinogenesis in which transformation depends on two synergistic genes. Most other directly oncogenic viruses contain single, autonomous transforming (onc) genes and are models for single-gene carcinogenesis. To determine which role each potential onc gene of MH2 plays in oncogenesis, we have prepared deletion and frameshift mutants of each of the two MH2 genes by in vitro mutagenesis of cloned proviral DNA and have tested transforming function and virus production in cultured primary quail cells. We have found that mht deletion mutants and wild-type virus transform primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mht deletion mutants and by wild-type MH2 are similar yet vary considerably. Nevertheless, typical mutant transformed cells can often be distinguished from cells transformed by wild-type MH2. We conclude that the delta gag-myc gene transforms primary cells by itself, without the second potential onc gene. This myc-related gene is the smallest that has direct transforming function. delta gag-mht is without detectable transforming function but may affect transformation by delta gag-myc. Thus, MH2 behaves like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be a model for two-gene carcinogenesis. Further work is necessary to determine whether the delta gag-mht gene possibly enhances oncogenic function of delta gag-myc or has independent oncogenic function in animals.     

Proteins specified by avian erythroblastosis virus: coding region localization and identification of a previously undetected erb-B polypeptide.

Avian erythroblastosis virus (AEV) induces erythroblastosis and sarcomas in chickens. Two domains within the viral genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. By use of hybridization-arrested translations and hybridization-selections of mRNA, we have mapped on the viral genome the polypeptides specified by the erb domains. The results of hybridization-arrest with DNA representing the spliced 5' leader region of the AEV mRNA suggested that the authentic product of the erb-B domain was a 61,000 molecular weight protein, not a 41,000 molecular weight polypeptide previously identified.     

Angiotensin II is a necessary component for the development of hypertension in the two kidney, one clip rat

The current study was undertaken to define the role of the renin-angiotensin system in the development of hypertension in the two kidney, one clip Goldblatt rat. Captopril was administered orally (100 mg/kg/day) to two groups of rats (n = 8 each) 24 hours before and each day after unilateral renal artery clipping (0.2 mm internal diameter): the drug was given for either 16 weeks (group I) or 24 weeks (group II). Sham-operated (n = 5) and Goldblatt (n = 8) rats not receiving captopril were prepared for comparisons of plasma renin activity and systolic blood pressure. Indomethacin (20 mg/kg/day subcutaneously) was administered for 48 hours concomitantly with captopril to the rats in group I. In group II, systolic blood pressure was monitored for 7 weeks after cessation of captopril. Continual captopril administration to Goldblatt rats completely prevented the rise in systolic blood pressure, a rise that was observed in Goldblatt rats not receiving captopril. Whereas systolic blood pressure of captopril-treated rats approximated 100 mm Hg throughout the study, that of Goldblatt rats not receiving the drug increased to nearly 180 mm Hg within 6 weeks after clipping. Systolic blood pressure of sham-operated rats remained normal. Indomethacin did not change systolic blood pressure in the drug-treated rats in group I. On cessation of captopril therapy in group II, systolic blood pressure increased gradually in a manner that paralleled the development of the disease in the Goldblatt rats that did not receive captopril. Plasma renin activity was determined in Goldblatt and sham-operated rats at either 16 weeks (group I) or 24 weeks (group II) after clipping; the rats from either group with mild hypertension (systolic blood pressure less than 180 mm Hg) had normal plasma renin activity whereas those with severe hypertension (systolic blood pressure greater than 180 mm Hg) had greatly elevated plasma renin activity. In summary, captopril can completely prevent the increase in systolic blood pressure for up to 24 weeks in Goldblatt rats, and this hypotensive effect is not mediated by the prostaglandins. It is concluded that the renin-angiotensin system is a necessary component of the hypertensive process in this experimental model.     

The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).     

Disease-modifying anti-rheumatic drugs are only one of a number of potential causes of myelosuppression: a careful drug history is necessary to elucidate the cause of an adverse event

SIR, We read with interest the article by Lim et al. [1] concerningmethotrexate (MTX)-induced pancytopenia, and present here threecases of drug-induced myelosuppression that illustrate thatdisease-modifying antirheumatic drugs (DMARDs) are not alwaysto blame. Case 1 was a 76-yr-old lady who was treated with MTX for herrheumatoid arthritis (RA). A routine blood test on 1 July 2004showed that her haemoglobin (Hb) had fallen over 1 month from11.1 g/dl to 8.4 g/dl, the white blood count (WBC) from     

Three populations of cells with dendritic morphology exist in peripheral blood, only one of which is infectable with human immunodeficiency virus type 1.

Conflicting data have been reported with regard to the infectability, dysfunction, and depletion of dendritic cells (DCs) in human immunodeficiency virus (HIV) disease. These discrepancies could potentially be explained by the existence of multiple subsets of cells with dendritic morphology in peripheral blood. The isolation of DCs in humans is accomplished through negative selection until a morphologically pure population is obtained. Recently, DC precursors purified from peripheral blood by negative selection have been observed to develop into functionally and morphologically mature DCs. In this report we identify three populations of cells in peripheral blood that have or can develop a dendritic morphology. The first population, when allowed to mature in culture, develops a dendritic morphology and gains the expression of HB15, a marker of DCs in blood, thymus, skin, and lymphoid organs. The second population expresses HB15 and has the phenotypic and morphologic characteristics of mature DCs. The third population is morphologically very similar to mature DCs but does not share the same T-cell-stimulatory activity and is the only population that is infectable with HIV. Understanding the heterogeneity of cells of dendritic lineage and/or morphology in the peripheral blood will aid in understanding their role as antigen-presenting cells in general and as potential participants in the immunopathogenesis of HIV disease.     

口蹄疫病毒P1+3CD基因重组腺病毒的构建

目的构建包含有O型口蹄疫病毒(FMDV)强毒结构蛋白(P1)基因及弱毒非结构蛋白(3CD)基因的重组腺病毒,为进一步研制FMD活载体疫苗奠定基础。方法通过RT-PCR方法扩增得到含有FMDV P1、3CD编码区的目的基因。P1基因经BglⅡ和XhoⅠ,3CD基因经XhoⅠ和XbaⅠ双酶切后与经BglⅡ和XbaⅠ双酶切处理的腺病毒穿梭载体pAd Track-CMV连接。将获得的重组穿梭质粒与腺病毒骨架载体在大肠杆菌内同源重组获得重组腺病毒质粒。线性化后的重组腺病毒质粒转染293细胞,通过观测细胞病变及报告基因绿色荧光蛋白的表达鉴定重组的腺病毒。结果在pAd Track-CMV载体中成功克隆了P1+3CD基因,并与腺病毒骨架载体在大肠杆菌中实现了同源重组。线性化后的重组腺病毒质粒转染293细胞后可观测到典型的细胞病变与绿色荧光蛋白的表达。结论成功获得了含有FMD P1+3CD基因的重组腺病毒,为FMD活载体疫苗的研制提供了依据。     

A Rous sarcoma virus provirus is flanked by short direct repeats of a cellular DNA sequence present in only one copy prior to integration.

The Rous sarcoma virus (RSV)-transformed rat cell line RSV-NRK-2 contains a single complete RSV provirus. We have obtained recombinant lambda clones that contain both ends of the RSV provirus and the flanking rat sequences. The provirus is integrated in unique DNA and is present in only one of the two homologous chromosomes. The rat sequences into which the RSV provirus integrated were also cloned from the RSV-NRK-2 cell line. The sequences of the regions involved in the recombination event have been determined and compared. Our data suggest that, compared with the sequence of viral DNA in the large circular form of unintegrated viral DNA, the provirus lacks two base pairs at each end and that the provirus is flanked by a six-base-pair direct repeat of cellular DNA. This six-base-pair repeat was apparently created during the integration event because this sequence was present only once at the integration site before the provirus was inserted. A survey of eight other independent RSV transformed rat cell lines demonstrates that, in agreement with earlier results, the RSV proviruses have entered different segments of rat cell DNA. We have also determined the sequence of a second virus DNA-host cell DNA junction from a second RSV-transformed rat cell line (RSV-NRK-4) and find that there are no obvious similarities between the two integration sites or between the integration sites and the termini of viral DNA.     

Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.     

Vesicular stomatitis virus glycoprotein is necessary for H-2-restricted lysis of infected cells by cytotoxic T lymphocytes.

Vesicular stomatitis virus (VSV) elicited cytotoxic thymus-derived lymphocytes (CTLs) in mice of the BALB/c and three congenic strains (BALB.b, BALB.k, BALB.HTG). CTL lysis of VSV-infected fibroblasts from the four strains was restricted by the target cells' major histocompatibility complex (H-2). Target cells were also infected with two temperature-sensitive mutants of VSV, tsM and tsG in which, respectively, the viral matrix protein and glycoprotein are not expressed at 39 degrees (restrictive temperature) on the infected cell's surface membrane. At the restrictive temperature, cells infected with wild-type VSV or tsM were lysed by CTLs, but cells infected with tsG were not. The requirement for the glycoprotein on the target cell was also evident from the ability of antisera to the glycoprotein to block completely CTL lysis of VSV-infected cells.     

Localization of connexin26 and connexin32 in putative CO(2)-chemosensitive brainstem regions in rat.

Recent studies have suggested that cell-to-cell coupling, which occurs via gap junctions, may play a role in CO(2) chemoreception. Here, we used immunoblot and immunohistochemical analyses to investigate the presence, distribution, and cellular localization of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) in putative CO(2)-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed that both Cx subtypes were expressed in putative CO(2)-chemosensitive brainstem regions; however, regional differences in expression were observed. Immunohistochemical experiments confirmed Cx expression in each of the putative CO(2)-chemosensitive brainstem regions, and further demonstrated that Cx26 and Cx32 were found in neurons and Cx26 was also found in astrocytes in these regions. Thus, our findings suggest the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction proteins Cx26 and Cx32, at least in part, form the neuroanatomical substrate for this gap junctional communication, which is hypothesized to play a role in central CO(2) chemoreception.     

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.