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.

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.

A dynamical systems approach to manual tracking performance.

Manual tracking performance was investigated from the perspective of dynamical systems theory. The authors manipulated the type of visual display, the control system dynamics, and the frequency of the sinusoidal input signal to examine couplings with various phases between the visual signal and control movements. Analyses of the system output amplitude ratio and relative phase showed that participants (N = 24) performed poorly with 90 degrees relative phase coupling. All the couplings became less stable as the movement frequency increased. The authors developed an adaptive oscillator model with linear damping to describe the coupled system consisting of the human performer, the visual display, and the control system dynamics. A geometric account of the stability of performance at different relative phases is also presented.

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.

Extravascular administration of interferon alfa-N3 increases serum exposure and 2-5(A) synthetase activity.

The objective of this study was to evaluate the pharmacokinetics, pharmacodynamic response, and safety of single intravenous (i.v.), intramuscular (i.m.), and subcutaneous (SQ) doses of interferon alfa-n3. Six healthy adults received 10 million units of i.v., i.m., and SQ interferon alfa-n3 in a randomized three-period crossover fashion. Serum interferon alfa-n3 concentrations and 2'-5'-oligoadenylate synthetase (2-5[A] synthetase) activity in peripheral blood mononuclear cells were determined after each dose. Extravascular administration significantly increased mean serum interferon alfa-n3 AUC values (1152 +/- 214, 944 +/- 209, and 576 +/- 188 U.h/mL, p < 0.001, with SQ, i.m., and i.v. administration, respectively) and 2-5(A) synthetase activity at 36 and 48 hours after dosing. Mild to moderate flu-like symptoms were reported by all 6 subjects, with no route-related difference in type or incidence. Interferon alfa-n3 is generally well tolerated by the i.v., i.m., and SQ routes, with i.m. and SQ administration maximizing serum exposure and 2-5(A) synthetase activity.

A new contained human immunodeficiency virus type 1 host cell system for evaluation of antiviral activities of interferons and other agents in vitro.

HIV-host infection systems in vitro are important in the pre-clinical assessment of anti-retroviral drug activity. The present report describes the development of a new HIV-host model comprised of an epithelial cell line of HeLa lineage (HeLa-1), transfected with expression vectors bearing tat and rev (TART) genes of HIV-1 as well as the CD4 receptor gene, and HIV-1(delta Tat/Rev), a biologically contained strain of HIV-1 deleted in tat and rev. Measurement of infectivity, by syncytium formation and reverse transcriptase assay, revealed that HeLa-1 is infected with HIV-1(deltaTat/Rev). This virus failed to productively infect the TART-deficient CD4-positive HeLa cells, confirming its contained, non-infectious nature. The HeLa-1/HIV-1deltaTat/Rev system was used to measure the anti-retroviral activity of a human leukocyte-derived interferon (IFN-alphan3) preparation, several nucleoside analogs, and protease inhibitors. The HeLa-1/ HIV-1(deltaTat/Rev model provides a biologically contained system for the study of the HIV pathogenesis and the relative and combined therapeutic effects of anti-retroviral agents in vitro.

Critical role for Atm in suppressing V(D)J recombination-driven thymic lymphoma.

Chromosome translocations involving T cell receptor (TCR) loci have been found in tumors from Ataxia telangiectasia (AT) patients and in mouse Atm-/- thymoma, suggesting the involvement of V(D)J recombination in these malignancies. By introducing a RAG-1 deficiency into Atm-/- mice in the presence of a TCR transgene, we show that V(D)J recombination is critical for thymoma development in these mice. Therefore, aberrant V(D)J recombination, normally suppressed by Atm, facilitates tumorigenic events leading to cancer. Because V(D)J recombination is dispensable for lymphomagenesis upon p53 deficiency, this study also indicates that Atm and p53 function by distinct mechanisms in suppressing thymoma.

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.

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.

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.

Inhibitory effect of interleukin-10 on human leukocyte interferon-alpha production by Sendai virus.

Treatment of human peripheral blood leukocytes (hPBL) with Sendai virus induces significant production of human interferon-alpha (IFN-alpha). Addition of human recombinant interleukin-10 (IL-10) to hPBL in vitro prior to treatment with Sendai virus resulted in considerable inhibition of IFN-alpha production. Downregulation of IFN-alpha production was IL-10 concentration-dependent and observed at IL-10 concentrations of as low as 0.05 ng/ml, with a median effective dose (ED50) of about 5 ng/ml. Inhibition of IFN-alpha production by IL-10 occurred at an early stage of Sendai virus induction. The inhibitory effect of IL-10 on leukocyte interferon production was specific and blocked by pretreatment with neutralizing polyclonal anti-IL-10 antibody. This downregulatory effect is at the transcriptional level, since IL-10 inhibits IFN-alpha mRNA accumulation upon Sendai virus treatment. These data suggest that leukocyte IFN-alpha production is a highly regulated process that is modulated by cytokines such as IL-10 during early immunological response to infection.

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.

[Studies on chemical constituents of Kadsura Longepedunculata].

The chemical constituents of Kadsura Longepedunculata collected from Zhejiang province were studied. Six compounds were isolated and their structures were elucidated based on physical and spectral analysis. Among them, one is a new compound, which is named kadsulactone acid (I) and the others are kadsulactone (II), (+)-anwulignan (III), mesodihydroguaiaretic acid (IV), d-epidalbacin(V) and beta-sitosterol (VI).

Tempo, Rhythm, and Aging in Golf.

Older (n = 12) and younger (n = 12) golfers attempted to hit a golf ball into a target net a short distance away. An accelerometer attached to the back of the clubhead measured the applied force. In contrast to the more typical finding of slower perceptual-motor performance by older adults, older golfers reached their peak downswing force earlier in the shot and also exhibited a trend toward a faster overall speed or tempo of the shot. Additionally, older golfers exhibited greater changes in applied force and greater variability. A pattern of divergence among the force-time histories from multiple shots suggested that the overall person-plus-golf-club dynamics were unstable during a part of the shot. Older adults may be slower in controlling this instability. Half of the participants heard a tone whose pitch was proportional to their force. These participants had a slower follow-through; however, they did not make significantly more or fewer shots than participants who had not been presented with the tone. Analyses of the temporal covariation among the backswing, downswing, and follow-through favored a chain-like temporal structure over a hierarchical, proportional structure. The pattern of covariation suggests that the tempo and rhythm of the shot are not independent and that changing one's tempo may disrupt rhythm.

Treatment of chronic hepatitis C with interferon alfa-n3: a multicenter, randomized, open-label trial.

We studied the antiviral effectiveness and safety of interferon alfa-n3, a natural alpha interferon which contains multiple interferon species, in the treatment of previously untreated patients with chronic hepatitis C. Seventy-seven patients were randomized to receive either 1.0, 2.5, 5.0, or 10.0 million units (MU) of interferon alfa-n3 three times a week for 24 weeks and were then followed for an additional 24 weeks. At the end of therapy, 67% of patients in the 10 MU group normalized serum alanine transaminase (ALT) levels and 59% had no hepatitis C virus (HCV) RNA detected by polymerase chain reaction. At the end of the follow-up period, 44% of patients in the 10 MU group maintained normal ALT, and 24% had nondetectable HCV RNA. Lower doses were much less effective. Interferon alfa-n3 was well tolerated and no patient developed neutralizing anti-interferon antibodies during or after the treatment period. Interferon alfa-n3 appears to be effective against hepatitis C virus and deserves further study in larger randomized controlled trials.

Interferon-alpha neutralizing antibodies in HIV and chronic HCV patients treated with natural-source human leukocyte-derived interferon-alpha n3.

Human leukocyte-derived IFN-alpha n3 (Alferon N Injection) was administered subcutaneously to treat 20 patients with asymptomatic human immunodeficiency virus type 1 (HIV-1) and 141 patients with chronic hepatitis C virus (HCV) infections. The treatment of HIV-1 and HCV patients, previously untreated with any IFN preparations, did not result in development of neutralizing antibodies to IFN-alpha n3. Among 69 HCV refractory patients who were unresponsive to previous treatment with rIFN-alpha 2b, 2 had neutralizing antibodies to rIFN-alpha 2b prior to IFN-alpha n3 therapy, with no or limited cross-reactivity to IFN-alpha n3. After retreatment with IFN-alpha n3, both patients had detectable neutralizing titers to IFN-alpha n3. Additionally, 2 other patients developed low and transient neutralizing titers to IFN-alpha n3. Interferon subtype specificity of these antibodies was tested against RP-HPLC purified fractions of IFN-alpha n3, as well as rIFN-alpha 2b and rIFN-alpha 8b. Sera from patients previously treated with rIFN-alpha 2b with high antibody titers to rIFN-alpha 2b strongly reacted with the natural IFN-alpha 2b, and to a limited extent with other iFN-alpha subtypes. Neutralizing activity against IFN-alpha 2b was significantly competed out by the presence of a small amount of other interferon subtypes present in IFN-alpha n3. One patient with prior presence of antibodies to IFN-alpha 2b developed a high antibody titer to IFN-alpha 8b with limited reactivity to IFN-alpha n3. Two of the HCV refractory patients with prior neutralizing antibodies to rIFN-alpha 2b responded to IFN-alpha n3 therapy. These data suggest that the presence of neutralizing antibodies to individual IFN-alpha species will not significantly diminish the biological activity and the clinical efficacy of multi-species IFN-alpha n3.

Quantifying the performance limitations of older and younger adults in a target acquisition task.

In a stationary target acquisition task, both 65-year-old and 20-year-old adults exhibited a negatively accelerated curvilinear relationship between the spatial variability of submovement endpoints and average submovement velocity. For high velocities, the variability was greater for the older adults. This elevated motor noise is considered a primary cause of their slower performance. Both age groups also exhibited a linear relationship between submovement duration and the logarithm of submovement relative accuracy. A stochastic model indicates that the two age groups were similar in the strategies they used to compose single movements from a variety of submovements. However, when performing sequences of movements containing varied target distances, older adults exhibited a repetition effect whereas younger adults exhibited a contrast effect. Older adults may plan movements individually, whereas younger adults plan sequences.

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.

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.