Liquid and Solid Hybridization Methods to Detect RNAs.

Northern blotting (NB) has been a long-standing method for RNA detection. However, its labor-intensive nature, reliance on high-quality RNA, and use of radioactivity have diminished its appeal over time. Nevertheless, the emergence of microRNAs (miRNAs) has reignited the demand for sensitive and quantitative NB techniques. We have recently developed cost-effective and rapid protocols for RNA detection using solid and liquid hybridization (LH) techniques which exhibit high sensitivity without the need for radioactive or specialized reagents like locked nucleic acid (LNA) probes. Our assays incorporate biotinylated probes and improved techniques for probe hybridization, transfer, cross-linking, and signal enhancement. We demonstrate that while NB is sensitive in detecting mRNAs and small RNAs, our LH protocol efficiently detects these as well as miRNAs at lower amounts of RNA, achieving higher sensitivity comparable to radiolabeled probes. Compared to NB, LH offers benefits of speed, sensitivity, and specificity in detecting mRNAs, small RNAs, and miRNAs.

Molecular Analysis of Copy-Back Defective Interfering RNAs of Morbilliviruses.

Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated by denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 sec, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the reproducible, statistically significant reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of the poly(A) binding protein, PABPC1, with polyadenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, and cost-effective method to enable researchers to analyze expression, processing, binding, and decay of RNAs.

Measuring Poly-Adenosine Tail Length of RNAs by High-Resolution Northern Blotting Coupled with RNase H Cleavage.

The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points.

Replication of Recombinant Flock House Virus RNA Encapsidated by Turnip Yellow Mosaic Virus Coat Proteins in Nicotiana benthamiana

It was previously observed that recombinant flock house virus (FHV) RNA1 was efficiently packaged into turnip yellow mosaic virus (TYMV), provided that the TYMV coat protein (CP) sequence was present at the 3′-end. FHV RNA encapsidated by TYMV CPs also had a four-nucleotide extension at the 5′-end. Since even a short extension at the 5′- and 3′-ends of FHV RNA1 inhibits replication, we examined whether the recombinant FHV RNA is indeed capable of replication. To this end, we introduced constructs expressing recombinant FHV RNAs into the plant Nicotiana benthamiana. Northern blot analysis of inoculated leaves suggested abundant production of recombinant FHV RNA1 and its subgenomic RNA. This demonstrated that recombinant FHV RNA with terminal extensions at both ends was competent for replication. We also showed that the recombinant FHV RNA can express the reporter gene encoding enhanced green fluorescent protein.

Viral Effects of a dsRNA Mycovirus (PoV-ASI2792) on the Vegetative Growth of the Edible Mushroom Pleurotus ostreatus

A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.

The current state of play of research on the social, political and legal dimensions of HIV / O estado atual da pesquisa sobre as dimensões sociais, políticas e legais do HIV / Estado actual de la investigación sobre las dimensiones sociales, políticas y legales del VIH

This paper offers a critical overview of social science research presented at the 2014 International AIDS Conference in Melbourne, Australia. In an era of major biomedical advance, the political nature of HIV remains of fundamental importance. No new development can be rolled out successfully without taking into account its social and political context, and consequences. Four main themes ran throughout the conference track on social and political research, law, policy and human rights: first, the importance of work with socially vulnerable groups, now increasingly referred to as "key populations"; second, continued recognition that actions and programs need to be tailored locally and contextually; third, the need for an urgent response to a rapidly growing epidemic of HIV among young people; and fourth, the negative effects of the growing criminalization of minority sexualities and people living with HIV. Lack of stress on human rights and community participation is resulting in poorer policy globally. A new research agenda is needed to respond to these challenges.

Este artigo oferece uma perspectiva crítica da pesquisa em ciências sociais apresentada na Confe-rência Internacional de AIDS de Melbourne, Aus-trália, em 2014. Em tempos de grandes avanços no campo biomédico, a natureza política do HIV permanece de importância fundamental. Nenhuma inovação será bem-sucedida na prática se desconsiderar o contexto sociopolítico e suas consequências. Quatro temas emergiram da Conferência nos campos do direito, dos direitos humanos e da pesquisa social e política: (1) a importância do trabalho com grupos socialmente vulneráveis, crescentemente chamado de "populações chaves"; (2) o reconhecimento de que ações e programas devem ser sob medida para cada local e contexto; (3) a urgência da resposta a uma epidemia crescendo muito rapidamente entre adolescentes; (4) o efeito negativo da crescente criminalização de minorias sexuais e pessoas vivendo com HIV. Globalmente, a falta de ênfase nos direitos humanos e da participação comunitária tem como resultado políticas públicas de pior qualidade. Precisamos de uma nova agenda de pesquisa para responder a esses desafios.

El artículo ofrece una perspectiva crítica de la investigación en ciencias sociales, presentada en la Conferencia Internacional de SIDA en Melbourne (Australia), 2014. En tiempos de enormes avances biomédicos, la naturaleza política del VIH sigue siendo muy importante. Ninguna innovación será exitosa sin considerar el contexto sociopolítico y sus consecuencias. Cuatro temas surgieron de la conferencia en el campo legal y derechos humanos, además de investigación social y política: (1) la importancia del trabajo con grupos socialmente vulnerables, crecientemente denominados "poblaciones claves"; (2) el reconocimiento de que las acciones y programas deben ser adaptados a un contexto local; (3) la urgencia de una respuesta a una epidemia con crecimiento rápido entre adolescentes; (4) el efecto negativo de la creciente criminalización de las minorías sexuales y personas viviendo con VIH. Globalmente, un limitado énfasis en los derechos humanos y la participación comunitaria tiene como consecuencia peores políticas públicas. Necesitamos una nueva agenda de investigación para responder a estos desafíos.

Neurological examination: pioneering authors and their books / Exame neurológico: autores pioneiros e seus livros

The objective of this article is to highlight some of the most important pioneering books specifically focused on the neurological examination and their authors. During the XIX Century, Alexander Hammond, William Gowers and Charles Mills pioneered the neurological literature, followed in the XX Century by Aloysio de Castro, Monrad-Krohn, Derek Denny-Brown, Robert Wartenberg, Gordon Holmes, and Russel DeJong. With determination and a marked sense of observation and research, they competently developed and spread the technique and art of the neurological exam.

O objetivo deste artigo é destacar alguns dos primeiros e mais importantes livros-texto interessados em difundir o ensino do exame neurológico e seus autores. Durante o século XIX, Alexander Hammond, William Gowers e Charles Mills foram pioneiros na literatura neurológica, seguidos por Aloysio de Castro, Monrad-Krohn, Derek Denny-Brown, Robert Wartenberg, Gordon Holmes e Russel DeJong no século XX. Com determinação, grande senso de observação e pesquisa, eles competentemente disseminaram a técnica e a arte de se realizar o exame neurológico.

Abnormal heart rate variability and atrial fibrillation after aortic surgery / Variabilidade anormal da frequência cardíaca e fibrilação atrial após cirurgia aórtica

Introduction: Complete denervation of transplanted heart exerts protective effect against postoperative atrial fibrillation; various degrees of autonomic denervation appear also after transection of ascending aorta during surgery for aortic aneurysm. Objective: This study aimed to evaluate if the level of cardiac denervation obtained by resection of ascending aorta could exert any effect on postoperative atrial fibrillation incidence. Methods: We retrospectively analysed the clinical records of 67 patients submitted to graft replacement of ascending aorta (group A) and 132 with aortic valve replacement (group B); all episodes of postoperative atrial fibrillation occurred during the 1-month follow-up have been reported. Heart Rate Variability parameters were obtained from a 24-h Holter recording; clinical, echocardiographic and treatment data were also evaluated. Results: Overall, 45% of patients (group A 43%, group B 46%) presented at least one episode of postoperative atrial fibrillation. Older age (but not gender, abnormal glucose tolerance, ejection fraction, left atrial diameter) was correlated with incidence of postoperative atrial fibrillation. Only among a subgroup of patients with aortic transection and signs of greater autonomic derangement (heart rate variability parameters below the median and mean heart rate over the 75th percentile), possibly indicating more profound autonomic denervation, a lower incidence of postoperative atrial fibrillation was observed (22% vs. 54%). Conclusion: Transection of ascending aorta for repair of an aortic aneurysm did not confer any significant protective effect from postoperative atrial fibrillation in comparison to patients with intact ascending aorta. It could be speculated that a limited and heterogeneous cardiac denervation was produced by the intervention, creating an eletrophysiological substrate for the high incidence of postoperative atrial fibrillation observed. .

Introdução: Denervação completa do coração transplantado exerce efeito protetor contra a fibrilação atrial no pós-operatório; vários graus de denervação autonômica aparecem também após a transecção da aorta ascendente durante a cirurgia de aneurisma da aorta. Objetivo: Este estudo teve como objetivo avaliar se o nível de denervação cardíaca obtida por ressecção da aorta ascendente poderia exercer algum efeito sobre a incidência de fibrilação atrial no pós-operatório. Métodos: Foram analisados retrospectivamente os prontuários de 67 pacientes submetidos a enxerto de substituição de aorta torácica (grupo A) e 132 com a substituição da valva aórtica (grupo B). Foram relatados todos os episódios de fibrilação atrial pós-operatória ocorridos durante 1 mês de seguimento. Parâmetros de variabilidade da frequência cardíaca foram obtidos a partir de 24 h de gravação do Holter; dados clínicos, ecocardiográficos e de tratamento também foram avaliados. Resultados: No geral, 45% dos pacientes (grupo A 43%, grupo B 46%) apresentaram pelo menos um episódio de fibrilação atrial no pós-operatório. Idade mais avançada (mas não gênero, tolerância à glicose anormal, fração de ejeção, diâmetro do átrio esquerdo) foi correlacionada com a incidência de fibrilação atrial pós-operatória. Apenas em um subgrupo de pacientes com transecção aórtica e sinais de maior desarranjo autonômico (parâmetros de variabilidade da frequência cardíaca abaixo da mediana e a média de frequência cardíaca acima do percentil 75), indicando possivelmente denervação autonômica mais profunda, foi observada menor incidência de fibrilação atrial pós-operatória (22% vs. 54%). Conclusão: Transecção da aorta ascendente para correção de um aneurisma da aorta não confere qualquer efeito protetor significativo de fibrilação atrial no pós-operatório em comparação com pacientes com aorta ascendente intacta. Pode-se especular que uma denervação cardíaca limitada e heterogênea foi produzida pela ...

Constitutive and Inducible Expression of Invasion-related Factors in PC-3 Prostate Cancer Cells

BACKGROUND: Tumor growth and invasion are interconnected with the tumor microenvironment. Overexpression of genes that regulate cancer cell invasion by growth factors, cytokines, and lipid factors can affect cancer aggressiveness. A comparative gene expression analysis between highly invasive and low invasive cells revealed that various genes are differentially expressed in association with invasive potential. In this study, we selected variant PC-3 prostate cancer cell sublines and discovered critical molecules that contributed to their invasive potential. METHODS: The high invasive and low invasive variant PC-3 cell sublines were obtained by serial selection following Matrigel-coated Transwell invasion and were characterized by Transwell invasion, luciferase reporter assay, and Rhotekin pull-down assay. Lysophosphatidic acid (LPA) was added to the cultures to observe the response to this extracellular stimulus. The essential molecules related with cancer invasiveness were detected with Northern blotting, quantitative reverse transcription-polymerase chain reaction, and cDNA microarray. RESULTS: Highly invasive PC-3 cells showed higher nuclear factor kappa B (NF-kappaB), activator protein 1 (AP-1) and RhoA activities than of low invasive PC-3 cells. LPA promoted cancer invasion through NF-kappaB, AP-1, and RhoA activities. Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation. CONCLUSIONS: The results suggest that the target molecules are involved in invasiveness of prostate cancer. These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

TGF-beta Suppresses COX-2 Expression by Tristetraprolin-Mediated RNA Destabilization in A549 Human Lung Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor beta (TGF-beta) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-beta can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-beta-responsive and overexpress COX-2. MATERIALS AND METHODS: Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-beta. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. RESULTS: We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-beta. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-beta, suggesting that TGF-beta-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-beta rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-beta. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-beta. CONCLUSION: The results of this study show that TGF-beta down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.

Pathology of dogs in Campo Grande, MS, Brazil naturally co-infected with Leishmania infantum and Ehrlichia canis / Patologia de cães naturalmente coinfectados por Leishmania infantum e Ehrlichia canis em Campo Grande, MS, Brasil

Different parasites that commonly occur concomitantly can influence one another, sometimes with unpredictable effects. We evaluated pathological aspects of dogs naturally co-infected with Leishmania infantum and Ehrlichia canis. The health status of the dogs was investigated based on histopathological, hematological and biochemical analyses of 21 animals infected solely with L. infantum and 22 dogs co- infected with L. infantum and E. canis. The skin of both groups showed chronic, predominantly lymphohistioplasmacytic inflammatory reaction. The plasmacytosis in the lymphoid tissues was likely related with the hypergammaglobulinemia detected in all the dogs. The disorganization of extracellular matrix found in the reticular dermis of the inguinal region and ear, characterized by the substitution of thick collagen fibers for thin fibers, was attributed to the degree of inflammatory reaction, irrespective of the presence of parasites. In addition, the histopathological analysis revealed that twice as many dogs in the co-infected group presented Leishmania amastigotes in the ear skin than those infected solely with Leishmania, increasing the possibility of becoming infected through sand fly vectors. Our findings highlight the fact that the health of dogs infected concomitantly with L. infantum and E. canis is severely compromised due to their high levels of total plasma protein, globulins, alkaline phosphatase and creatine kinase, and severe anemia.

A infecção simultânea por parasitas de diferentes espécies pode resultar em alterações imprevisíveis. O presente estudo avaliou a patologia de cães naturalmente coinfectados por Leishmania infantum e Ehrlichia canis. A saúde dos cães foi investigada pelas análises histopatológicas, hematológicas e bioquímicas de 21 cães infectados somente por L. infantum e 22 cães coinfectados por L. infantum e E. canis. Observou-se uma reação inflamatória crônica, predominantemente linfohistioplasmocítica, na pele dos dois grupos. A plasmocitose, encontrada nos tecidos linfóides, provavelmente estava relacionada com a hipergamaglobulinemia observada em todos os cães amostrados. A desorganização da matriz extracelular da derme da região inguinal e da orelha, demonstrada pela substituição das fibras de colágeno espessas por fibras finas, foi relacionada com o grau de reação inflamatória, independente da presença de parasitas. Ainda, observamos duas vezes mais animais do grupo coinfectado apresentando formas amastigotas na pele de orelha pela histopatologia comparado ao número de cães infectados apenas por Leishmania, tornando-os desta forma mais infectivos aos vetores. Nossos resultados ressaltam que a saúde de cães coinfectados estava severamente comprometida devido aos altos níveis de proteína plasmática total, globulinas, fosfatase alcalina, creatina quinase e anemia acentuada.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

Genome Size Constraint in Replication and Packaging of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.

Expression of mitogen activated protein kinase phosphatase-1 in pancreatic cancer / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the expression of mitogen activated protein kinase phosphatase-1(MKP-1)in pancreatic cancer.</p><p><b>METHODS</b>Totally 60 cases of normal pancreas, chronic pancreatitis(CP), and pancreatic cancer tissues were collected by operation in our hospital. Pancreatic tissues were analyzed by Northern blot analysis and Western blot analysis. Meanwhile, MKP-1 expression was detected in 6 pancreatic cancer cell lines by Western blot analysis.</p><p><b>RESULTS</b>Northern blot analysis of total RNA revealed relatively low MKP-1 mRNA expression in 7 of 20(35%)normal pancreatic samples. In the remaining 13 samples, the MKP-1 mRNA was absent to faint detectable. In 7 of the 20 CP samples, MKP-1 was demonstrated moderate to high expression. In contrast, 12 of 20(60%)pancreatic cancer samples MKP-1 mRNA was expressed at high levels, whereas in the remaining 8 cancer tissues this mRNA moiety was present at low to moderate levels. Densitometric analysis with normalization to 7S revealed that the median level of MKP-1 mRNA in CP and cancerous tissues was increased by 6.2 folds(P=0.035)and 8.1 folds(P=0.016)in comparison with the median level in the normal pancreatic samples, respectively. Overexpression of MKP-1 was also found in 6 pancreatic cancer cell lines, in which the expression of MKP-1 was slightly lower in one pancreatic cancer cell line but high in the remaining 5 cell lines.</p><p><b>CONCLUSIONS</b>MKP-1 is over-expressed in pancreatic cancer, CP tissues, and pancreatic cell lines. It is speculated that MKP-1 may play an important role in tumorigenesis of pancreatic cancer.</p>

Expression of the S glycoprotein of transmissible gastroenteritis virus (TGEV) in transgenic potato and its immunogenicity in mice / 대한수의학회지

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-S0.7) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-S0.7 gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-S0.7 protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-S0.7 antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

Toxicarioside A, isolated from tropical Antiaris toxicaria, blocks endoglin/TGF-β signaling in a bone marrow stromal cell line

OBJECTIVE@#To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells.@*METHODS@#HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-β were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-β (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells.@*RESULTS@#Compared with the not treated (0 μg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-β expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells.@*CONCLUSIONS@#Our results indicate that toxicarioside A can influence bone marrow stromal HS-5's function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Cyclosporin A Inhibits Albumin Synthesis in Huh7 Cells

BACKGROUND/AIMS: Hypoalbuminemia occurs frequently in renal transplant recipients immediately after renal transplantation. We studied the regulation of hepatic albumin synthesis by cyclosporin A (CsA) in Huh7 cells. METHODS: Huh7 cells were incubated with various concentrations of CsA for 4, 8, 16, and 24 hours. Albumin was measured in Huh7 cell-conditioned medium by sandwich enzyme-linked immunosorbent assay and Western blot. Albumin mRNA expression was analyzed by Northern blotting in CsA-treated cells. RESULTS: CsA (10(-7)-10(-4) M) inhibited albumin synthesis in Huh7 cells in a dose- dependent manner. A Western blot analysis for albumin in the conditioned medium released from CsA-treated (10(-7)-10(-5) M) cells also showed significant inhibition of albumin synthesis in a dose-dependent manner. Vehicle (olive oil) did not affect albumin synthesis. In contrast, a Northern blot analysis revealed no inhibition of albumin mRNA expression by CsA at any time point from 1-24 hours, indicating that the inhibition of albumin synthesis occurred at the translational level. CONCLUSIONS: Our results suggest that inhibition of hepatic albumin synthesis by high dose CsA contributes to the hypoalbuminemia in renal transplant recipients.

Spaceflight alters the gene expression profile of cervical cancer cells / 癌症

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.