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Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

Abstract Background Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats. Results Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001). Conclusion The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control. [ABSTRACT FROM AUTHOR]

Untreatable epileptic seizure challenges clinical and experimental researchers. P glycoprotein (Pgp) acts as efflux pump for hydrophobic substrates in normal tissues, is encoded by mdr1a/b genes and is a putative candidate that explains the lack of response to drug treatment. We study Wistar Audiogenic Rats (WAR), epileptic rats genetically selected for audiogenic seizures. The antiepileptic drug Phenytoin did not block seizures in up to 50% of the WAR, suggesting that WARs could be a suitable model for drug resistance studies. The present work aims to optimize an absolute quantitative PCR (AQPCR) for mdr1a/b mRNA expression in sound-stimulated WARs (n=6) compared to sound-stimulated Wistar (WS; n=6) and to Wistar controls (WC; n=5). RNA was extracted from skeletal muscle (skm) and liver, and primers were designed for a 84 bp mdr1 mRNA target. S26 ribosomal amplicon target was used to normalize AQPCR. Reverse transcription of 1 µg total RNA was followed by AQPCR of 2 µl of cDNA from each reaction. Protocols used were as specified by ABI 7000 SDS Sybr Green kit. The dissociation curve yielded an amplicon Tm of 79.1 °C. The standard curve ranging from 1.8 to 1.8x10-7 ng showed linear regression (r²=0.9934), with a slope = -2.91 (E=100%). Mdr1 cDNA expression (ng/µg total RNA) was 0.004, 0.013, 0.463 for skm and 0.555, 1.581, 0.622 for liver of WC, WS and WAR, respectively. The AQPCR shows differences between tissues in the three groups and should be applied to future mdr1 gene expression studies in Phenytoin resistant WAR. [ABSTRACT FROM AUTHOR]

Canine distemper is a systemic viral disease characterized by immunosuppression followed by secondary infections. In most cases the clinical diagnosis of distemper is difficult to confirm. Considering the great infectious potential of the disease, the possibility of carrying out an antemortem diagnosis of distemper is important. Absolute quantitative real time PCR (AQPCR) was used for detection and diagnosis of canine distemper virus (CDV) in blood of 5 asymptomatic dogs and vaccine samples. CDV-specific oligonucleotide primers of 20 and 21 nt length were designed for the amplification of an 84 bp target CDV nucleocapsid protein coding region fragment. Another target fragment of 93 bp of canine â-actin was utilized as endogenous control. After conventional reverse transcription at 45°c, cDNA samples were used for AQPCR in ABI Prism 7000 SDS with the PE Biosystems SYBR®Green and protocol. Standard calibration curves were determined for both CDV and â-actin target amplicons. Quantitative results were generated by ABI-Prism Software. Dissociation curves for target fragments confirmed the AQPCR specificity. Slopes for calibration curves from both CDV and â-actin in the range of -3.2 demonstrated a reaction efficiency of 100% and a high linearity with r² = 0.989. Positive controls confirmed that the product detected was CDV, and negative (no template) controls showed no CDV PCR products. The AQPCR detected 3 positives in 5 dogs asymptomatic for CDV. The present protocol detected as little as 10-8 ng of CDV cDNA in asymptomatic dogs, and may be suitable for future viral quantification and epidemiologic studies. (Sponsored by CNPq-Brasil). [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) causes canine distemper diseases in dogs and in other carnivores. CDV is characterized by fever, nasal discharge, encephalomyelitis, conjunctivitis, gastroenteritis and pneumonia. The cellular pathogenesis includes mainly complications in the CNS and immunosuppressive systems, which are not completely understood. Several viruses have been shown recently to cause cell death by means of apoptosis induction. Dogs naturally infected with CDV show apoptosis in lymphoid tissues and cerebellum. However, apoptotic pathway mechanisms for CDV are not well determined. Our goal was to develop a sensitive method for diagnosis of CDV. We used quantitative PCR to investigate expression of the pro-apoptotic caspases 3 and 8, and antiapoptotic Bcl-2, in leukocytes of infected dogs. CDV diagnosis was performed by gel-based RT-PCR, using previously characterized primers. Six CDV positives and 6 negative samples were selected for apoptotic gene expression comparison by a Real Time PCR SYBR®GREEN protocol. Caspase -3 cDNA expression (ng/IJg total RNA) was 4.7 times higher in the CDV positive (0.14) than in the CDV negatives (0.03); Caspase -8 was 6.2 times higher in the CDV positive (0.52) than in the CDV negatives (0.083); and Bcl-2 levels did not change in CDV positives (0.035) and negatives (0.037). Results demonstrated that CDV infection induced transcription of caspases -3 and -8, while Bcl-2 expression did not change. This suggests that CDV might cause immune suppression induced by apoptosis in leukocytes via caspase-3 activation, but the main cascade for this induction may occur due to activation of the caspase-8 pathway. [ABSTRACT FROM AUTHOR]

Mandible condyle remodeling is a great challenge on craniofacial growth studies. The great majority of the reports deals with growing period. However, there is a great necessity to clarify the importance of functional stimulation on adult mandible condyle remodeling. By using an adult mouse model, we investigated the influence of mandible forwarding on condyle remodeling and gene expression by bone forming cells. Tomographic and scintigraphic evaluations showed sagittal growth and cell activity enhancement. RT-PCR showed that Type I collagen, osteocalcin and osteonectin expression level can be altered. We showed that functional stimulation is necessary to maintain the regular gene expression by condyle bone forming cells in adult mice. It opens new frame for further investigations aiming new clinical approaches to temporomandibular joint problems treatment, as well as mandible retrusion treatment.

Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.