Isolation of novel microsatellite markers from Paralichthys lethostigma (Paralichthyidae) and their cross-species application in Pleuronectiformes.

We investigated the genetic diversity of the southern flounder Paralichthys lethostigma. Microsatellite-enriched libraries were constructed and novel microsatellite markers were developed and applied for genetic detection of wild populations. Cross-species amplification was also conducted in five pleuronectiforme species. Of 45 randomly selected and sequenced clones, 43 contained a CA or GA repeat motif. Fourteen pairs of primers were designed to investigate the polymorphism and genetic structure of a wild population collected from North Carolina State coastal waters. Two loci were monomorphic and 12 loci were polymorphic. The number of alleles per polymorphic locus ranged from 2 to 16, with an average of 7.3, and the expected heterozygosity per locus ranged from 0.10 to 0.92, with an average of 0.58. Cross-species amplification showed that most of the markers could successfully amplify Paralichthys olivaceus DNAs, few markers amplified in Verasper variegatus and Verasper moseri, and none of them could amplify Scophthatmus maximus and Cynoglossus semilaevis DNAs. The isolated polymorphic markers would be useful for the genetic breeding and assessment of genetic variation within the genus Paralichthys.

Isolation and characterization of polymorphic microsatellite markers from Coilia ectenes.

Coilia ectenes (Jordan and Seale 1905) is an important anadromous species that is an important resource at risk of extinction because of over-fishing, pollution, and coastal construction. To evaluate the genetic diversity of C. ectenes for use in breeding programs, elite microsatellite-enriched libraries were constructed and novel microsatellite markers were developed, and applied to genetically detect wild populations. Out of 92 randomly selected and sequenced clones, 89 contained a CA or GA repeat motif. Twenty-two pairs of primers were designed to investigate the polymorphism and genetic structure of a wild population collected from the Yellow River estuary, China. It was found that 2 loci were monomorphic and 20 loci were polymorphic. The number of alleles per polymorphic loci ranged from 3 to 13, with an average of 7.9. The expected heterozygosity per locus ranged from 0.05 to 0.89, with an average of 0.68. The isolated polymorphic markers are expected to be of use in future genetic breeding programs for C. ectenes, and in the assessment of genetic variation within this species.

Atm is dispensable for p53 apoptosis and tumor suppression triggered by cell cycle dysfunction.

Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM's role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model.

No requirement for V(D)J recombination in p53-deficient thymic lymphoma.

The p53 tumor suppressor is activated in response to a variety of cellular stress signals, although specific in vivo signals that trigger tumor suppression are unknown. In mouse thymocytes, where p53 inactivation leads to tumorigenesis, several observations suggest that V(D)J recombination of T-cell receptor (TCR) loci could provide a DNA damage signal triggering p53-dependent apoptosis and tumor suppression. Inactivation of p53 would allow V(D)J driven mutation of additional cancer genes, facilitating tumorigenesis. Here, we show that mice with a p53 deficiency in thymocytes and unable to carry out V(D)J recombination are not impaired in the development of thymoma. Recombination-activating gene (RAG) deficiencies were introduced into both p53-/- mice and TgTDeltaN transgenic mice, a strain in which 100% of the mice develop thymoma due to thymocyte-specific inactivation of p53 by a simian virus 40 T-antigen variant. V(D)J recombination was dispensable for tumorigenesis since thymomas developed with or without the RAG-1 or RAG-2 gene, although some delay was observed. When V(D)J recombination was suppressed by expression of rearranged TCR transgenes, 100% of the TgTDeltaN mice developed thymoma, surprisingly with reduced latency. Further introduction of a RAG deficiency into these mice had no impact on the timing or frequency of tumorigenesis. Finally, karyotype and chromosome painting analyses showed no evidence for TCR gene translocations in p53-deficient thymomas, although abundant aneuploidy involving frequent duplication of certain chromosomes was present. Thus, contrary to the current hypothesis, these studies indicate that signals other than V(D)J recombination promote p53 tumor suppression in thymocytes and that the mechanism of tumorigenesis is distinct from TCR translocation oncogene activation.

Ion specificity in fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles.

Fusion of phosphatidylcholine-phosphatidic acid mixed lipid vesicles has been studied under the influence of the divalent Ca2+, Mg2+, Cd2+, and Ba2+, which range in size from much smaller to much larger, than Ca2+. Fusion has also been studied under the influence of Eu3+, which has a similar radius but different charge. The effects of these ions are reflected in the different degrees of fusion determined by changes in vesicle size, and in the varying fusion rates monitored by 1H-NMR spectroscopy. Cd2+, which has an ionic radius similar to Ca2+, exhibits the same effect on fusion as Ca2+, while other ions show lower efficiencies. This suggests that the vesicle fusion intermediate has a geometry ideally suited to the binding of Ca2+ or Cd2+.

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipids vesicles: a proton nuclear magnetic resonance study.

The kinetics of Ca2+-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance has been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125--300 A. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates of Ca2+ as well as an vesicle concentration. For vesicle concentrations in the range of 3 . 10(-6)--10(-5) M and Ca2+ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1--10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.

Fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles.

Ca2+-induced transformation of phosphatidylcholine-phosphatidic acid vesicles to larger bilayer structures has been examined using nuclear magnetic resonance, electron microscopy, gel permeation and radioisotope tracer techniques. For concentrated vesicle preparations where phosphatidic acid content remains less than 50% of total lipid, transformation to larger well defined unilamellar structures can be induced. The size of the product formed is dependent on phosphatidic acid content and on Ca2+ content when Ca2+ levels are between 0.3 and 1.0 mol ratios with respect to phosphatidic acid. During transformation bilayer composition remains unchanged and internal contents are retained in the final structure. These properties are indicative of concerted two vesicle and multiple vesicle fusions. The controllable and concerted fusions make the phosphatidic acid system suitable for further mechanistic studies.

Structural properties of a Ca2+-phosphatidic acid complex: small angle X-ray scattering and calorimetric results.

The structures of Ca2+ and Cd2+ complexes with dipalmitoyl- and dimyristoylphosphatidic acids have been investigated by differential scanning calorimetry and small angle X-ray diffraction. The lipids are found to form complexes with Ca2+ which exhibit no thermal phase transitions between temperatures of 25 and 90 degrees C. Transition enthalpies of residual uncomplexed lipid extrapolate to zero near a 1 : 1 lipid to cation ratio indicating a 1 : 1 complex stoichiometry. Ca2+ and Cd2+ complexes appear isomorphous in the X-ray data and show lamellar phases with short repeat distances (51.5 A (5.15 nm) and 58.0 A (5.80 nm) for dimyristoyl and dipalmitoyl homologs, respectively). The data are discussed in terms of structures in which ions bridge phosphates on adjacent bilayers.

Cytokines induced by Sendai virus in human peripheral blood leukocytes.

Human peripheral blood leukocytes (hPBL) are a rich source of natural leukocyte interferon (IFN-alpha) when treated with Sendai virus. Sendai virus treatment of hPBL will also result in significant production of several chemokines and cytokines such as macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, RANTES, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8, in a time-dependent way. A significant amount of MCP-1 is constitutively produced in overnight culture of leukocytes. The most abundant cytokine is IFN-alpha, which is induced to its maximum level approximately 11-15 h after addition of Sendai virus. The amount of IFN-alpha induced at 15 h after Sendai virus treatment is more than 16-fold higher than those of MIP-1alpha, MIP-1beta, and RANTES. IFN-alpha is also induced more than 60-fold higher than TNF-alpha and IL-8. The amount of IL-6 induced is approximately 400-fold less than IFN-alpha. Limited amounts of other cytokines such as IL-1alpha, IL-1beta, macrophage colony-stimulating factor, TNF-beta, and IFN-gamma are also induced in Sendai virus-treated hPBL. No measurable amount of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, leukemia inhibitory factor, IL-2, IL-3, IL-4, IL-5, IL-7, IL-10, IL-11, or IL-12 was induced in the supernatant of Sendai virus-treated hPBL.

A population survey on the prevalence of diabetes in Kin-Hu, Kinmen.

OBJECTIVE: To investigate the prevalence of non-insulin-dependent-diabetes mellitus in Kin-Hu, Kinmen. RESEARCH DESIGN AND METHODS: This is a community-based population survey. The target population are residents > or = 30 years of age in Kin-Hu, Kinmen, according to the official household registry in 1990. Face to face interviews were conducted by the Yang-Ming Crusade in 1991 using a structured questionnaire. Fasting blood samples were drawn by public health nurses, and a 75-g oral glucose tolerance test was performed for definite diagnosis of diabetes. RESULTS: There were 4,097 eligible subjects (2,026 men and 2,071 women), and 3,236 had complete fasting plasma glucose data (1,536 men and 1,700 women). The response rate was 79% (76% for men and 82% for women). The age-specific response rates were 81% for the 30- to 39-year and 50- to 59-year age-groups, 84% for the 40- to 49-year age-group, and 69% for the > or = 60-year age-group. The crude prevalence of diabetes in Kin-Hu was 6.5% (2.0% previous and 4.5% new). With the standard world population of Segi, the age-adjusted prevalence rate was 4.9% (4.5% for men and 5.4% for women). The prevalence rate of diabetes increased significantly with age. The prevalence of previously diagnosed diabetes accounted for less than one third of the total rate. CONCLUSIONS: The population survey in Kin-Hu, Kinmen, had a high response rate of 79%. The crude prevalence rate of diabetes was 6.5%, and the age-adjusted prevalence rate was 4.9%. The low ratio of previously diagnosed to newly diagnosed diabetic cases may be due to lack of public awareness and medical services in this community.

Identification of interferon-alpha 7, -alpha 14, and -alpha 21 variants in the genome of a large human population.

The genes for type I interferon (IFN), which include 14 IFN-alpha genes, 1 IFN-beta gene, 1 IFN-omega gene, and a number of IFN-omega pseudogenes, are clustered on human chromosome 9. Among IFN-alpha genes, a number of variants have been reported. Three variants of IFN-alpha 7 (IFN-alpha 7a, IFN-alpha 7b, and IFN-alpha 7c) and IFN-alpha 14 (IFN-alpha 14a, IFN-alpha 14b, and IFN-alpha 14c) and two variants of IFN-alpha 21 (IFN-alpha 21a and IFN-alpha 21b) are identified. The variants differ from each other by base changes in the coding region and can be distinguished by selective restriction enzyme analysis and DNA sequencing. We have used polymerase chain reaction (PCR) with IFN species-specific oligonucleotide primers for amplification of IFN-alpha 7, IFN-alpha 14, and IFN-alpha 21 gene sequences. Genomic DNA obtained from over 28,000 normal healthy individuals were collected in six pools for PCR amplification. To identify the presence of variant sequences, the resulting PCR products of specific IFN-alpha genes were analyzed by restriction endonuclease digestion and DNA sequencing, with a limit of detection of minor components to 1% and 10%, respectively. The results show that only one variant form for each of IFN-alpha 7, IFN-alpha 14, and IFN-alpha 21, namely, IFN-alpha 7a, IFN-alpha 14c, and IFN-alpha 21b, is detectable in the genomic DNA of the population examined. Similar results were obtained from the analysis of a human myeloblastoid cell line, KG-1.

Both variant forms of interferon-alpha4 gene (IFNA4a and IFNA4b) are present in the human population.

Alpha interferons (IFN-alpha) are a class of cytokines with various activities that are used as therapeutic agents for treatment of cancer and viral and immune disorder diseases. At least 13 IFN-alpha genes and 1 IFN-alpha pseudogene have been identified, which are clustered on human chromosome 9. Among the known IFN-alpha species, a number of allelic variants have been reported. Two variants of IFN-alpha4 (IFN-alpha4a and IFN-alpha4b) are known, which differ from each other by changes in their coding regions at nucleotide positions 220 and 410 and can be distinguished by selective restriction enzyme analysis. We have developed oligonucleotide primers for specific amplification of IFN-alpha4 gene fragments using the polymerase chain reaction (PCR). Genomic DNA obtained from over 28,000 normal healthy individuals and six human cell lines were used in this study. The resulting PCR products were analyzed by restriction endonuclease digestion and DNA sequencing to identify the presence of variant sequences. The results show that the DNA sequences for both variants of IFN-alpha4 are found in the population in nearly equal proportion. Individuals with either homozygous (e.g., alpha4a/alpha4a or alpha4b/alpha4b) or heterozygous (i.e., alpha4a/alpha4b) IFN-alpha4 genes were detected. Among the cell lines, KG-1, EB-3, and HTB-10 cells contain the genes for IFN-alpha4a only, whereas U-937, Namalwa, and Daudi cells contain the genes for both IFN-alpha4a and IFN-alpha4b.

Interferon-alpha 8b is the only variant of interferon-alpha 8 identified in a large human population.

Three variants of human interferon (IFN)-alpha 8a gene, that is, IFN-alpha 8b, and IFN-alpha 8c, have been reported previously. They differ from each other by changes in their coding region at nucleotide positions 359-360, 372, and 550. Human genomic DNA obtained from over 28,000 healthy blood donors and from 4 human cell lines was used in the polymerase chain reaction (PCR) designed for specific amplification of the IFN-alpha 8 gene fragments. The resulting PCR product was analyzed by (1) restriction endonuclease digestion, (2) DNA sequencing, and (3) allele-specific secondary PCR amplification. Only one sequence for IFN-alpha 8 was identified, and that was for IFN-alpha 8b. The sequences for IFN-alpha 8a and IFN-alpha 8c were not detected after PCR amplification either in the pooled leukocytes obtained from > 28,000 individuals or in cell lines tested. These data suggest that the naturally occurring variant or allele for IFN-alpha 8 in the population is IFN-alpha 8b. IFN-alpha 8a and IFN-alpha 8c variants were consistently below the level of detection of the assays and, if present at all in the population, are very rare.

IFN-alpha1a gene is the major variant in the North American population.

Thirteen interferon (IFN)-alpha functional genes have been reported. A number of these genes have allelic members (variants). In the case of IFN-alpha1, two variants, IFN-alpha1a and IFN-alpha1b, are known. The variants differ from each other by one base change in the coding region, leading to a single change in amino acid sequence and the presence of a restriction site. We have developed oligonucleotide primers for amplification of IFN-alpha1 gene(s) using polymerase chain reaction (PCR). Genomic DNA, obtained from over 23,000 normal healthy individuals and from four human cell lines, were used as templates in PCR to amplify the IFN-alpha1 gene sequences. The resulting PCR products were analyzed by restriction endonuclease digestion and DNA sequencing to identify the presence of variant sequences. The results show that IFN-alpha1a is predominant in the genomic DNA of the population examined. Among the cell lines studied, IFN-alpha1a is the only variant found in U-937 and Namalwa cells, whereas KG-1 cells have only IFN-alpha1b, and EB-3 cells have both IFN-alpha1a and IFN-alpha1b in the genome.

A new allele of interferon-alpha17 gene encoding IFN-alpha17b is the major variant in human population.

Thirteen interferon (IFN)-alpha functional genes have been reported. Among these, a number of genes have allelic members (variants). In the case of IFN-alpha17, five variants, IFN-alpha17a, IFN-alpha17b, IFN-alpha17c, IFN-alpha17d, and IFN-alphaT, are known. The variants differ from each other by base changes in the coding region, leading to differences in amino acid sequences. We have developed oligonucleotide primers for amplification of IFN-alpha17 gene(s) using polymerase chain reaction (PCR). Genomic DNA, obtained from over 28,000 normal healthy individuals and from four cell lines, were used as templates in PCR to amplify the IFN-alpha17 gene sequences. The resulting PCR products were analyzed by restriction endonuclease digestion and DNA sequencing to identify the presence of variant sequences. The results show that a new variant of IFN-alpha17 is abundantly present (approximately 70%) along with another variant, possibly IFN-alpha17c (approximately 30%), in the genomic DNA of the population examined. This new variant, the protein product of which is identical to IFN-alpha17b, differs from the gene for IFN-alpha17b by a point mutation. We have named it IFN-alpha17b', which is the only variant found in U-937, KG-1, and EB-3 cell lines. Namalwa cells have IFN-alpha17b' and, possibly, IFN-alpha17c in equal proportions.

Different relative activities of human cell-derived interferon-alpha subtypes: IFN-alpha 8 has very high antiviral potency.

Interferon-alpha (IFN-alpha) subtypes were separated by HPLC from the IFN mixtures produced by virus-stimulated human lymphoblastoid cells and leukocytes. Together with preparations of lymphoblastoid IFN and recombinant IFN-beta, these were tested in three human tumor cell lines derived from liver, lung, and neuroblasts. Their relative antiviral activities differed markedly: subtype IFN-alpha 8 was the most potent and IFN-alpha 1 the least. The results were broadly similar in all three cells, with some minor differences. when the same preparations were tested for inhibition of thymidine incorporation, the relative activities were quite different: subtypes IFN-alpha 10, IFN-alpha 17, IFN-alpha 21, and IFN-alpha 5 were now the most active, and IFN-alpha 2 was the least active. IFN-alpha 1 and IFN-alpha 8 had comparable intermediate activity. Thus, the differences in activity were not caused by degradation of some subtypes during their separation. IFN-alpha 8 not only had the greatest antiviral activity but also, like IFN-beta, induced an antiviral state in U1 mutant cell lines, which lack the tyrosine kinase, Tyk2, required for signal transduction by other IFN-alpha subtypes.

Interferon-alpha 2 variants in the human genome.

Variants of human leukocyte interferon alpha 2 (IFN-alpha 2a, alpha 2b, and alpha 2c) differ from each other by changes in their coding regions at nucleotide positions 137 and 170. As a result of these nucleotide variations, the DNA sequences of the three variants can be distinguished by selective restriction enzyme analysis. Human genomic DNA obtained from over 28,000 normal healthy individuals was used as templates in the polymerase chain reaction (PCR) to amplify the human IFN-alpha 2 gene sequence. The resulting PCR products were analyzed with restriction nucleases to identify the specific IFN-alpha 2 variant sequences present in the genomic DNA of the population examined. The results show that IFN-alpha 2b was detected as the predominant species and IFN-alpha 2c as a very minor species (< 0.1%). The IFN-alpha 2a gene was not detected in this population.

Regulation and characterization of the interferon-alpha present in patients with advanced human immunodeficiency virus type 1 disease.

To examine a possible association between plasma viremia and interferon-alpha (IFN-alpha) in patients with the acquired immunodeficiency syndrome (AIDS), we performed IFN plasma immunoadsorption by apheresis (IFN-alpha apheresis) in four volunteers with AIDS who had sustained levels of endogenous plasma IFN-alpha. IFN-alpha apheresis with two plasma volume exchanges was performed daily for 5 days. Clinical signs and symptoms and hematologic, virologic, and immunologic parameters were monitored. Two subjects developed anemia from phlebotomy, and one had a catheter++-associated bacteremia. The IFN-alpha apheresis was effective only in transiently removing IFN-alpha: depletion of IFN-alpha led only to its rapid reconstitution. Cell-associated HIV-1 was unchanged, but three of four subjects had a modest decrease in culturable plasma virus burden following the procedures. The recovery of in vivo HIV-1-related IFN-alpha by apheresis allowed its biologic and biochemical characterization. The HIV-1 IFN-alpha showed characteristics on ELISA, western blot, and biologic assays similar to two subspecies of the natural protein. The natural, recombinant, and HIV-1-induced IFN-alpha s demonstrated nearly identical antiviral activities. The HIV-1 IFN-alpha eluted from the column was not acid labile. The inability of large amounts of plasma IFN-alpha found in some patients with AIDS to affect viral burden likely reflects properties of the virus or of host factors independent of IFN-alpha.

Detection of picogram amounts of nucleic acid by dot blot hybridization.

An increasing number of human proteins isolated from cell sources are being produced for pharmaceutical use. Consequently, federal agencies have required the quantitative determination of residual nucleic acids that copurify with the potential protein products. We have conducted these assays in connection with our application for licensure of Alferon Injection. We report a sensitive dot blot hybridization assay that was used to quantitate picogram (or less) amounts of nucleic acids which copurified with human proteins isolated from recombinant (S. cerevisiae) or natural (leukocytes) sources.