High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction.

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 microL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguished truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

ERalpha-CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis.

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis.

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides.

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.

Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase.

To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined. E. coli harboring the vector plasmid was used as a control. Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG). TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG. TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG. Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains. These results indicate that chicken AK1 expressed in E. coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.

Thiamin-triphosphate-synthesizing activity of mutant cytosolic adenylate kinases: significance of Arg-128 for substrate specificity.

The thiamin triphosphate (TTP)-synthesizing activity and the ATP-synthesizing activity of two mutant enzymes of chicken cytosolic adenylate kinase whose Arg-128 was substituted by Trp (cAK1(Trp)) or Ala (cAK1(Ala)) were compared to those of the wild-type enzyme. The TTP-synthesizing activity of both the mutant enzymes was higher due to higher affinity to thiamin diphosphate (cAK1(Trp)) or a larger Vmax (cAK1(Ala)). The optimal pH shifted to pH 9.0 from pH 10.5. The ATP-synthesizing activity of both the mutant enzymes was, on the other hand, markedly decreased with lower affinity for ADP and lower Vmax. These results suggest that Arg-128 plays an important role in the substrate specificity of the cytosolic adenylate kinase.

Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.

Isolation and expression of a novel member of the CITED family.

Chicken CITED3 (cCITED3) is a novel gene, which is expressed in the pre-somitic mesoderm, the mesonephric tubules, the Wolffian ducts and collecting tubules of the developing urogenital system and in the cranial sensory ganglia. Sequence analysis revealed that cCITED3 encodes a protein which contains two conserved domains that have been described for members of the CITED family.

In vivo sequence changes of HIV-1 envelope V2 and V3 loops.

Human immunodeficiency virus type 1 (HIV-1) circulates in vivo as heterogeneous mixed populations (quasispecies). We analyzed the quasispecies nature of the first, second, and third hypervariable loops (V1, V2, and V3) of the gp 120 in plasma and viral isolates obtained from 19 infected individuals. Sequence analysis of 7 SI and 10 NSI isolates showed that the increased positive charge in V3 and elongation of V1 clearly correlated with viral SI capability in PBMC. Contrary to the previous report (M. Groenink et al. Science 260: 1513-1525, 1993), there appeared to be no correlation between SI or non-SI phenotype and length of V2. However, all the 5 isolates with long V2 and basic V3 showed SI phenotype, whereas 4 out of 6 isolates with short V2 and basic V3 showed NSI phenotype, still suggesting some functional cooperation of V2 and V3 on viral syncytium inducing capability. Sequence analysis of HIV-1 in plasma showed the positive correlation between V3 charge and V1 or V2 length in 4 cases. This result suggests the associated evolution of V1/V2 and V3 in these cases.

IL-4 and a glucocorticoid up-regulate CXCR4 expression on human CD4+ T lymphocytes and enhance HIV-1 replication.

CXCR4 is a key co-receptor required for the infection of T-tropic HIV-1 strain of CD4+ T lymphocytes. The regulation of this chemokine receptor was therefore studied. Th2 polarized cells expressed more CXCR4 than Th1 cells. Among a panel of cytokines and stimulants, a Th2 type cytokine interleukin-4 (IL-4) selectively up-regulated the mRNA level as well as surface protein expression of CXCR4 within 16 h. In addition, CXCR4 was also up-regulated by a glucocorticoid, dexamethasone. These treated cells became more responsive in transendothelial migration assays to the specific CXCR4 ligand, SDF-1alpha. Furthermore, up-regulation of CXCR4 was also associated with the enhancement of HIV replication in human CD4+ T lymphocytes. This study indicates the enhanced T-tropic HIV-1 infection to CD4+ T lymphocytes through up-regulation of CXCR4 by several immunomodulating agents, IL-4, and a glucocorticoid. These findings may explain the shift to T-tropic HIV-1 dominance during AIDS progression when Th2 comes to predominate.

Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis.

Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction-validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan-Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance.

Syncytium formation of human and non-human cells by recombinant vaccinia viruses carrying the HIV env gene and human CD4 gene.

We examined the ability of the various human and non-human cell lines to form syncytia upon coinfection with recombinant vaccinia viruses each carrying the HIV env and the human CD4 gene. We found that three human cell lines and one of three monkey cell lines exhibited syncytium formation, but that one human cell line, two monkey cell lines and all the rabbit and mouse cell lines examined did not. This indicated that factors other than HIV env and CD4 were participating in syncytium formation and that distribution of these factors was restricted by species and by the cell type in a species.

The induction of cataracts by HIV-1 in transgenic mice.

OBJECTIVE: To elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host-range barrier of this virus. RESULTS: mRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3-6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. CONCLUSIONS: Preferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

Identification of multiple HIV-1 cytotoxic T-cell epitopes presented by human leukocyte antigen B35 molecules.

OBJECTIVE: To identify HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by human leukocyte antigen (HLA)-B35 molecules that are associated with the accelerated progression of AIDS using a reverse immunogenetic approach. METHODS: 8-mer to 11-mer sequences carrying two anchor residues at position 2 and the carboxy-terminus were selected from HIV-1SF2 strain. Sixty-four peptides matched to these sequences were synthesized and tested by a peptide binding assay using RMA-S-B*3501 cells. Peripheral blood lymphocytes (PBL) from two HIV-1-infected donors carrying HLA-B35 were stimulated once-weekly with each HLA-B*3501 binding peptide. The CTL activity of the cultured cells for the HLA-B35-positive target cells loaded with the corresponding peptides was examined after the second and fourth stimulation. Furthermore, the CTL activity of the cultured cells possessing HLA-B*3501-restricted HIV-1 peptide-specific CTL activity were examined for the HLA-B*3501-positive target cells infected with the recombinant vaccinia virus containing corresponding HIV-1 gene. RESULTS: HIV-1 peptide-specific HLA-B*3501-restricted CTL was induced in PBL of HIV-1 infected donors by in vitro stimulation with 11 out of 27 HLA-B*3501-binding HIV-1 peptides. The specific CTL induced with 10 peptides killed the cells infected with recombinant vaccinia virus expressing the corresponding HIV-1 proteins. Out of these HIV-1 peptide epitopes, two epitopes were also presented by HLA-B51 molecules. CONCLUSION: In addition to the four HLA-B35-restricted HIV-1 CTL epitopes that have been previously reported, nine HLA-B35-restricted HIV-1 CTL epitopes were identified in the present study. These multiple epitopes will be useful in studies for immunopathogenesis of AIDS.

Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1.

OBJECTIVE: To examine the biological properties of HIV-1/SIVmac chimeric viruses from HIV-1 isolates that have different replication rates, cell tropisms and cytopathicities. DESIGN AND METHODS: Four chimeric viruses with gag, pol, vif, vpx, nef and long terminal repeats of SIVmax and vpr, tat, rev, vpu and env of various HIV-1 isolates were constructed and compared in vitro. Cynomolgus monkeys were inoculated with two chimeras that were replicative in monkey peripheral blood mononuclear cells (PBMC). RESULTS: The type-specific neutralization of the chimeras by monoclonal antibodies 0.5 beta and mu 5.5, which recognize V3 of HIV-1IIIB and HIV-1MN respectively, was observed to be similar to those of the parental viruses, HIV-1NL432, HIV-1HAN2 and HIV-1SF13. The chimeras constructed from HIV-1SF2 and HIV-1SF13, which were isolates from the same individual but from different disease stages, reflected their parental properties, that is, the isolate from the later stage was rapid-high replicating, was more cytopathic and had a wider host range. Chimeras constructed from HIV-1HAN2' HIV-1SF13 and HIV-1NL432 were infectious to macaque monkeys, although the monkeys infected with the chimera from HIV-1SF13 showed lower virus loads and shorter viremic periods than those infected with the others. CONCLUSIONS: Chimeras have in vitro properties that are similar to those of their parental HIV-1 isolates, but their growth in macaque PBMC was dependent on which HIV-1 isolate was used. Evaluation of a vaccine by challenging with viruses possessing different antigenicities has become possible in macaque monkeys using newly constructed chimeras.

Membrane modification during secretory granule formation in rat somatotrophs.

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.

MSG1 and its related protein MRG1 share a transcription activating domain.

MSG1 is a recently described melanocyte-specific nuclear protein whose biochemical function is unknown [Shioda et al. (1996) Proc. Natl. Acad. Sci. USA 93, 12298-12303]. Two human cDNA sequences found in the EST (expressed sequence tag) database were predicted to encode a small peptide (45 aa) that showed 69% identity to the C-terminal sequence of MSG1, suggesting the existence of a novel MSG1-related protein. Based on these EST sequences, we isolated a novel gene, MRG1 (MSG1-Related Gene 1), by the 5'-RACE (rapid amplification of cDNA ends) technique. The MRG1 mRNA transcript is expressed widely and encodes a nuclear protein that share two highly conserved domains, CR1 (14 aa) and CR2 (approx. 50 aa), with MSG1. The CR2 domain is significantly acidic and activates transcription in yeast cells. The full-length MSG1 and MRG1 fused to GAL4 DNA-binding domain activates transcription in mammalian cells, and this is dependent on the presence of the CR2 domain. These results suggest that MRG1 and MSG1 may function as transcription activators.

Enhanced anti-HIV-1 activity of CC-chemokine LD78beta, a non-allelic variant of MIP-1alpha/LD78alpha.

We compared the anti-HIV-1 activity of CC-chemokine LD78beta with that of MIP-1alpha, another CC-chemokine which shows 94% sequence homology with LD78beta. Despite its close similarity to MIP-1alpha, the anti-HIV-1 activity of LD78beta appeared to be nearly 10 times higher than that of MIP-1alpha. Mutagenesis of MIP-1alpha showed that the N-terminal additional tetrapeptide, which was present in LD78beta and absent in MIP-1alpha, is responsible for enhanced anti-HIV-1 activity. The N-terminal structure-function relationship of LD78beta described here will be of value in understanding the chemokine-receptor interactions and designing anti-HIV-1 compounds based on LD78beta.

Negative regulation of the anti-human immunodeficiency virus and chemotactic activity of human stromal cell-derived factor 1alpha by CD26/dipeptidyl peptidase IV.

Stromal cell-derived factor 1alpha (SDF-1alpha) is a chemokine that has been shown to prevent infection of T-tropic HIV strains and is a possible substrate of CD26/dipeptidyl peptidase IV (DPPIV). In this study, we show that SDF-1alpha was cleaved at the N-terminal region by CD26/DPPIV and as a result the inhibitory activity of SDF-1alpha against HIV infection disappeared. Moreover, the chemotactic activity of SDF-1alpha also disappeared specifically by DPPIV activity of recombinant soluble CD26. These results suggested that dissemination of T-tropic HIV strains in vivo may be facilitated by CD26/DPPIV via inactivation of functional SDF-1alpha.