Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe.

Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.

Whole-body positron emission tomography with 18F-fluorodeoxyglucose is an effective method to detect extra-pelvic recurrence in uterine sarcomas.

PURPOSE OF INVESTIGATION: To assess the clinical use of F-18-fluorodeoxyglucose positron emission tomography (FDG-PET) in the post-therapy surveillance of uterine sarcoma. METHODS: Eight whole-body FDG-PET studies were performed in seven women with previously treated uterine sarcoma. Conventional image studies (computed tomography) and physical examinations were performed for follow-up. All FDG-PET studies were indicated to localize suspected recurrences noted by conventional methods. RESULTS: The per case sensitivity of the FDG-PET studies and CT scans was 85.7% (6/7) and 100% (7/7), respectively (p = 0.174). FDG-PET was able to detect seven extrapelvic metastastic sites below the diaphragm (7/7, sensitivity: 100%), including the liver, spleen, paraaortic lymph node, spine and paracolic gutter, as well as pulmonary lesions in five patients, while the CT scan detected only three lesions (3/7, sensitivity: 42.9%; p = 0.070). FDG-PET detected only four recurrent pelvic lesions (4/6) and CT scan detected six (6/6) recurrent pelvic lesions (66.7% vs 100%, p = 0.455). CONCLUSIONS: The FDG-PET showed a better detection rate than the abdominal CT scan for extrapelvic metastatic lesions and a similar detection rate as well as abdominal CT scan. FDG-PET can serve as a useful detection tool for patients with uterine sarcomas because nearly 80% of recurrence involve an extrapelvic site.

Macrophages derived from bone marrow modulate differentiation of myeloid dendritic cells.

Dendritic cells (DC) are specialized antigen-presenting cells. Bone marrow monocytes have been widely used to generate murine myeloid DC. We found that mouse macrophages derived from bone marrow CD11b+ monocytes influenced the differentiation of these precursors into DC. Modulation of differentiation was demonstrated by the down-regulation of CD11c, CD40, and CD86 expression and by IL-12 production. DC differentiated in the presence of conditioned medium from bone marrow-derived macrophage culture (MCM) had impaired ability to stimulate proliferation of, and IFN- gamma production by, allogeneic CD4+ T cells. This inhibition of DC differentiation was mainly mediated by secretory products from macrophages but not by cell-cell contact. MCM contained higher concentrations of macrophage-colony-stimulating factor (M-CSF), IL-10, and TGF- beta1, whereas IL-6 remained unchanged compared with conditioned medium from fresh monocytes. M-CSF may be the major mediator in MCM inhibiting DC differentiation. This study demonstrates an important influence of bone marrow-derived macrophages on DC precursors during DC differentiation.

Expression of capsid proteins and non-structural proteins of waterfowl parvoviruses in Escherichia coli and their use in serological assays.

While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.

[Availability of pediatric and neonatal intensive care units in the city of São Paulo]. / Disponibilidade de unidades de terapia intensiva pediátrica e neonatal no município de São Paulo.

OBJECTIVE: To describe the health care service provided in pediatric intensive care units in the city of São Paulo, by identifying and describing the units and analyzing their geographic distribution. METHODS: A descriptive cross-sectional study was carried out during a two-year period (August 2000 to July 2002). Data were collected through questionnaires answered by medical directors of each pediatric and neonatal intensive care unit. RESULTS: São Paulo is served by 107 pediatric and neonatal intensive care units, of which 85 (79.4%) completed and returned the questionnaire. We found a very unequal distribution of units as there were more units in places with the least pediatric population. Regarding to pediatric intensive care units specialization, 7% were pediatric, 41.2% were neonatal and 51.7% were mixed (pediatric and neonatal). Regarding hospital funds, 15.3% were associated with philanthropic institutions, 37.6% were private and 47% were public. A total of 1,067 beds were identified, of which 969 were active. The ratio bed/patient aged 0-14 was 1/2,728, varying from 1/604 at health districts-I to 1/6,812 at health districts-III. The units reported an average of 11.7 beds (2 to 60). The neonatal intensive care unit had a median of 16.9 beds per unit and pediatric intensive care units a median of 8.5 beds/unit. CONCLUSION: In São Paulo, we found an uneven distribution of pediatric and neonatal intensive care units among the health districts. There was also an uneven distribution between public and private units, and neonatal and pediatric ones. The current report is the first step in the effort to improve the quality of medical assistance in pediatric and neonatal intensive care units in São Paulo.

Disponibilidade de unidades de terapia intensiva pediátrica e neonatal no município de São Paulo / Availability of pediatric and neonatal intensive care units in the city of São Paulo

OBJETIVO: Caracterizar a assistência de saúde prestada em tratamento intensivo pediátrico e neonatal no município de São Paulo através da identificação, descrição e distribuição geográfica das unidades. MÉTODOS: Estudo descritivo, tipo transversal, onde foram estudadas as unidades de terapia intensiva pediátrica e neonatal do município de São Paulo, no período de agosto de 2000 a julho de 2002. A coleta dos dados foi realizada por meio de questionário preenchido pelo coordenador médico de cada unidade. RESULTADOS: Foram listadas 107 unidades de terapia intensiva pediátricas e neonatais no município de São Paulo. Oitenta e cinco (79,4 por cento) unidades forneceram os dados, constituindo a população de estudo. Observou-se maior número de unidades de terapia intensiva em Núcleos Regionais de Saúde com menor população pediátrica. Quanto à faixa etária, 7 por cento eram exclusivamente pediátricas, 41,2 por cento neonatais, e 51,7 por cento mistas. Em relação ao mantenedor: 47 por cento eram públicas, 37,6 por cento privadas, e 15,3 por cento filantrópicas. Identificamos 1.067 leitos, estando 969 em atividade. A razão leito/paciente de 0 a 14 anos foi de 1:2.728, variando de 1:604 (Núcleo Regional de Saúde - I) a 1:6.812 (Núcleo Regional de Saúde - III). O número de leitos por unidade variou de 2 a 60, com média de 11,7 (unidades de terapia intensiva neonatais: 16,9; mistas: 8,5). CONCLUSÃO: No município de São Paulo, observou-se uma distribuição desproporcional das unidades de terapia intensiva pediátrica e neonatal entre os cinco Núcleos Regionais de Saúde. Houve também uma distribuição desproporcional entre unidades de terapia intensiva públicas e privadas e entre neonatais e pediátricas. Esse estudo foi o primeiro esforço na busca por melhor qualidade na assistência intensiva pediátrica e neonatal no município de São Paulo.

Nucleotide sequences of goose circovirus isolated in Taiwan.

We report the complete nucleotide (nt) sequences of eleven goose circovirus (GoCV) isolated in Taiwan. Nine out of the eleven isolates had a genome size of 1821 nt, whereas the remaining two isolates have a size of 1820 nt. Sequence comparisons of the eleven Taiwanese GoCV isolates and a German isolate revealed that these viruses could be divided into three distinct genetic groups. Group I contains the German isolate, group II contains three Taiwanese isolates, and group III contains eight Taiwanese isolates. Nucleotide differences between viruses of different genetic groups ranged from 7.0-7.7%, whereas the differences within the same group were only 0.2-1.0%. The most diversified sequences were found at a region between nt 27-72 of the viral genome, which corresponded to the right one-third of the 5' intergenic region. Open reading frame analysis shows that the genome of all Taiwanese GoCV isolates could encode four proteins: V1 (Rep, 293 amino acids), V2 (37 amino acids), C1 (capsid, 250 amino acids), and C2 (99 amino acids). The sizes of V1, C1 and C2 proteins of all Taiwanese isolates and the German GoCV isolates were identical. However, the size of V2 protein (37 amino acids), although identical in all Taiwanese isolates, was much smaller than that of the German isolate (120 amino acids). Moreover, the initiation codon of the V2 ORF of three Taiwanese isolates was ATA rather than ATG. Our result indicates that GoCV of multiple genetic groups might have been circulating in Europe and Asia, and these viruses differ in their nucleotide sequences, sizes of the genome, and sizes of the V2 ORFs.

Health-risk assessment for workers exposed to polycyclic aromatic hydrocarbons (PAHs) in a carbon black manufacturing industry.

This study was established to assess workers' health-risks posed by PAHs exposures via both routes of inhalation and dermal contact. Personal inhalation exposure sampling was conducted on eight wet pelletizing workers and 22 packaging workers, by using a sampling train comprising an IOM personal inhalable aerosol sampler followed by an XAD-2 sorbent tube. Two workers were randomly selected from both exposure groups, and dermal exposures assessed by using soft polypropylene pads attached to the skin for nine different body surface areas for each selected worker. All personal inhalation and dermal samples were analyzed for 21 polycyclic aromatic hydrocarbon (PAH) species, and then converted to benzo[a]pyrene equivalent (BaPeq) concentrations by using the list of toxic equivalent factors (TEFs) suggested by Nisbet and LaGoy [Regul Toxicol Pharmocol 16 (1992) 290]. The resultant inhalation and dermal BaPeq exposure levels were used to estimate lifetime risks for lung cancer and skin cancer by using the BaP unit risks of 7.0 x 10(-2) (microg/m3)(-1) and 37.47(mg/kg bodyweight/day)(-1), respectively. Results show the personal inhalation BaPeq exposure levels for pelletizing and packaging workers were 622 and 774 ng/m3, respectively. The corresponding lifetime lung cancer risks estimated for both exposure groups were 4.35 x 10(-2) and 5.42 x 10(-2) respectively. For dermal exposures, results show personal dermal BaPeq exposure levels for both exposure groups were 0.664 and 0.847 microg/kg per day, respectively. The corresponding estimated lifetime skin cancer risks were 1.13 x 10(-3) and 1.56 x 10(-3), respectively. Although the estimated skin cancer risks were lower than the corresponding lung cancer risks for both exposure groups, however, both were higher than the designated significant risk level (= 10(-3)) which was defined by the US Supreme Court in 1980. Considering the bioavailability of particle-bound PAHs still remains unknown, the health risks obtained from this study could be overestimated and thus require further investigation.

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones.

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.

Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks.

There are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin-neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.

Identification and subtyping of avian influenza viruses by reverse transcription-PCR.

Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1-H15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running simultaneously 15 RT-PCR reactions, each using a set of primers specific to one HA-subtype. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined. The result of HA-subtyping was then confirmed by sequence analysis of the PCR product. A total of 80 strains or isolates of avian influenza viruses were subtyped by this RT-PCR procedure, and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates.

Eficácia e segurança do uso inalatório da adrenalina-L na laringite pós-intubação utilizada em associação com a dexametasona / Efficacy and safety of nebulized L-epinephrine associated with dexametasone in post-intubation laryngitis

Objetivo: avaliar a eficácia e a segurança do uso de adrenalina-L inalada na laringite pós-intubação como droga suplementar à dexametasona. Método: estudo prospectivo, duplo-cego, controlado por placebo, randomizado, com duas coortes de pacientes com laringite pós-intubação de grau 3 a 6, aferido pelo escore de downes e Raphaelly, durante dois anos. Os grupos, A e B, receberam dexametasona por via endovenosa e realizaram duas inalaçòes com soro fisiológico, mas nestas apenas o grupo B recebeu adrenalina-L. A eficácia da adrenalina-L foi avaliada através da comparação do escore de Downes e Raphaelly. os efeitos colaterais da adrenalina-L foram avaliados pela ocorrência de arritmia cardíaca, elevação da pressão arterial e da frequência cardíaca médias no grupo B, em relação ao grupo A. Resultados: foram selecionados 22 pacientes para o grupo A (escore médio=4,8) e 19 para o grupo B (escore médio=5,2) após o início do tratamento, três pacientes do grupo A alcançaram o escore 8 e foram reintubados; este grupo também apresentou escore clínico médio maior do que o grupo B, durante as primeiras duas horas do protocolo, resultados não estatisticamente significantes. Não foram constatados efeitos colaterais atribuíveis à adrenalina. Os índices gasométricos foram adequados nos dois grupos, porém melhores no grupo controle

Academic leadership style predictors for nursing faculty job satisfaction in Taiwan.

The purpose of this study was to examine the influence of nursing deans' and nursing directors' transformational and transactional leadership styles on nursing faculty job satisfaction in baccalaureate and associate degree nursing programs in Taiwan. The study provides a mechanism by which nursing deans or nursing directors can obtain feedback from nursing faculty about leadership styles. Such feedback can then serve as the basis for further development of academic nursing leadership potential in Taiwan. The theory of transformational versus transactional leadership style guided this study. A cross-sectional mailed survey design was conducted. A convenience sample of 233 nursing faculty participated in this study. Idealized influence, intellectual stimulation, and contingent reward leadership styles significantly and positively predicted job satisfaction. However, active management-by-exception significantly and negatively predicted job satisfaction. Nursing leaders should implement effective leadership styles. Study implications for nursing education administration, limitations, and recommendations for future studies were discussed.

Monoclonal antibodies against different epitopes of nonstructural protein sigmaNS of avian reovirus S1133.

Ten monoclonal antibodies (MAbs) were prepared against the nonstructural protein sigmaNS of avian reovirus S1133. Eight of them were selected for two-way competitive binding assay after coupling with horseradish peroxidase. The results allowed the definition of three epitopes, designated A, B, and C. Blocking assay of poly(A)-Sepharose binding activity of sigmaNS with MAbs indicated that MAb recognizing epitope B was able to block poly(A) oligomer binding, suggesting that epitope B is involved in ssRNA binding of sigmaNS. An immuno-dot binding assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to protein sigmaNS in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitopes B and C was not affected. The reactivity of MAbs recognizing epitope A was fully abolished by denaturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent; however, the recognition by MAbs of epitopes B and C is not conformation-dependent. In addition, the results from the cross-reactivity of MAbs to heterologous avian reovirus strains suggest that the three epitopes are highly conserved among these virus strains.

Biochemical analysis of fructose-1,6-bisphosphatase import into vacuole import and degradation vesicles reveals a role for UBC1 in vesicle biogenesis.

When Saccharomyces cerevisiae are shifted from medium containing poor carbon sources to medium containing fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Here, we show that FBPase import is independent of vacuole functions and proteasome degradation. However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p. A strain containing a deletion of the UBC1 gene exhibited defective FBPase import. Furthermore, FBPase import was inhibited when cells overexpressed the K48R/K63R ubiquitin mutant that fails to form multiubiquitin chains. The defects in FBPase import seen for the Deltaubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the Deltaubc1 mutant, the level of the Vid vesicle-specific marker Vid24p was reduced in the vesicle fraction, suggesting that UBC1 is required for either Vid vesicle production or Vid24p binding to Vid vesicles. However, the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles, indicating that ubiquitin chain formation is dispensable for Vid24p binding to these structures. Our results support the findings that ubiquitin conjugation and ubiquitin chain formation play important roles in a number of cellular processes including organelle biogenesis.

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.

Experimental infections of rabbits with rabbit haemorrhagic disease virus monitored by polymerase chain reaction.

Adult and 4-5-week-old rabbits were inoculated subcutaneously with rabbit haemorrhagic disease virus (RHDV). Samples were prepared from various tissues at intervals postinoculation (PI) for the detection of viral RNA and antigens. Using a haemagglutination test (HAT), viral antigens were detected in the liver, bile and spleen of the adult rabbits at and after 36 h PI. The reverse transcription-polymerase chain reaction (RT - PCR) showed that RHDVRNA was present in the liver, bile and spleen as early as 18 hours PI, whereas lung, kidney, thymus, mesenteric lymph node and buffy coat were found to be positive after more than 26 hours PI. In addition, viral RNA in urine and faeces showed a variable positivity at and after 36 hours PI. In the young rabbits, RT - PCR showed that RHDVRNA was present as early as 1 day PI in the liver, bile, spleen and buffy coat; whereas lung, kidney, thymus, mesenteric lymph node and faeces were found to be positive at and after 2 days PI. Bile and spleen were the only samples in which viral RNA was detected throughout the length of the experiment. Virus was not reactivated in six recovered virus-inoculated rabbits treated with dexamethasone or a classical swine fever virus vaccine. Using a haemagglutination inhibition test and an ELISA, antibody titres increased rapidly from one week PI onwards, peaked at approximately three weeks of age, and were maintained throughout the length of the experiment.

Negative regulation of the antimetastatic gene Nm23-H1 by thyroid hormone receptors.

Metastasis of various malignant cells is inversely related to the abundance of the Nm23-H1 protein. The possible role of thyroid hormones in tumor metastasis has now been investigated by examining the effect of T3 on the expression of the Nm23-H1 gene. Human hepatoma HepG2 cells, in which endogenous thyroid hormone receptor subtype alpha1 (TRalpha1) is expressed at a low level, were stably transfected, either with expression plasmids encoding wild-type TRalpha1 or a dominant negative mutant of TRalpha1, or with the empty vector (yielding HepG2-Wt, HepG2-Mt, and HepG2-Neo cells, respectively). Immunoblot analysis revealed that exposure of HepG2-Wt and HepG2-Neo cells, but not HepG2-Mt cells, to T3-induced time-dependent decreases in the abundance of Nm23-H1 messenger RNA and protein, with the extent of these effects correlating with the level of expression of TRalpha1. An in vitro assay also revealed that T3 induced a marked increase in the invasive activity of HepG2-Wt cells; it induced a smaller increase in that of HepG2-Neo cells but had no effect on that of HepG2-Mt cells. Finally, the promoter region of Nm23-H1 spanning nucleotides -471 to -437 (relative to the transcriptional initiation site) inhibited the expression of a downstream reporter gene, in a T3-dependent manner, in COS-1 cells also transfected with an expression plasmid encoding TRalpha1 or TRbeta1. The DNA binding domain of TRbeta1 was required for this inhibitory effect. These results indicate that T3, acting through TRs, inhibits transcription of Nm23-H1, and that this effect is mediated by a negative regulatory element in the promoter region of the gene. Thus, it is possible that T3 promotes tumor metastasis by inducing down-regulation of Nm23-H1 expression.

Overexpression of thyroid hormone receptor beta1 is associated with thyrotropin receptor gene expression and proliferation in a human thyroid carcinoma cell line.

To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a well-differentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of beta subtype thyroid hormone receptor (TRbeta) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TRbeta1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TRbeta1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TRbeta1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TRbeta1. These findings suggest that TRbeta1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.

A homopolymer stretch composed of variable numbers of cytidine residues in the terminal repeats of infectious laryngotracheitis virus.

A homopolymer stretch composed of variable numbers of cytidine residues was found within the inverted terminal repeats of infectious laryngotracheitis virus (ILTV). A polymerase chain reaction procedure was developed to amplify a 750-bp fragment containing this homopolymer stretch. This fragment was then sequenced directly to determine the number of repeated cytidine residues in this homopolymer stretch, which could be used for strain differentiation. By this procedure, vaccine strains of tissue culture origin could be differentiated into two types: type I contains eight repeated cytidine residues, whereas type II contains 10 such residues. Vaccine strains of chicken embryo origin could also be divided into two types: type I contains mainly 11 repeated cytidine residues, whereas type II contains 15-21 such repeats. In comparison, two of the five field isolates examined contain 12-13 repeats; the other three field isolates contain 15-19 repeats, which were similar to the type II chicken-embryo-origin vaccines. The number of repeated cytidine residues described here could serve as a marker for the strain differentiation and epidemiologic study of ILTV.