Deming Zhao

QUANTIFICATION OF PRION GENE EXPRESSION IN BRAIN AND PERIPHERAL ORGANS OF GOLDEN HAMSTER BY REAL-TIME RT-PCR

ABSTRACT Determination of tissue-specific expression of cellular prion protein (PrPc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of PrP mRNA in golden hamsters, a popular experimental model for studying mechanisms of transmissible spongiform encephalopathies (TSE), by real-time RT-PCR. Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters. PrP mRNA copy numbers were determined using absolute standard curve method with real-time quantitative PCR instrument. It was found that high mRNA levels were present in all four regions of the brain examined, including obex, neocortex, cerebellum, and thalamus. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moderate levels of the expression; and kidney showed the lowest expression. Our result is consistent with the potential involvement of different organs in prion diseases and offers essential data for further study of TSE mechanism in this animal model.

The quantification of prion gene expression in sheep using real-time RT-PCR

Determination of the transcription level of cellular prion protein (PrPC) is essential for understanding its poorly explained role in organisms. Scrapie in sheep is the prototype of all prion diseases. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail. Herein we report on measurement of sheep PrP mRNA using absolute quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR). Total RNA was isolated from seven different regions of the central nervous system (CNS) and six peripheral organs of 18 sheep and PrP mRNA was quantified by real-time RT-PCR using an externally calibrated standard curve constructed with the recombinant PrP plasmid. The results showed that high levels of PrP mRNA were expressed in all seven regions of the brain examined, with obex and neocortex expressing the highest PrP, followed by cerebellum, spinal cord, hippocampi, conarium and thalamus, In peripheral organs examined, lymph node showed a level of PrP expression similar to that in overall brain, whereas spleen, heart, liver and lung showed moderate level of expression and kidney showed the lowest expression. Our study provided the first quantitative, tissue-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.

Altered expression of the prion gene in rat astrocyte and neuron cultures treated with prion peptide 106-126.

SummaryNeuronal degeneration and astrogliosis are hallmarks of prion disease. Synthetic prion protein (PrP) peptide 106–126 (PrP106–126) can induce death of neurons and proliferation of astrocytes in vitro and this neurotoxic effect depends on the expression of cellular PrP (PrPC) and is hence believed to be PrPC -mediated. To further elucidate the involvement of PrPC in PrP106–126-induced neurotoxicity, we determined the expression of PrP mRNA in primary culture of rat cortical neuron cells, cerebellar granule cells, and astrocytes following treatment with 50μM of PrP106–126 scrambled PrP106–126 by quantitative real-time RT-PCR. As shown by MTT test, PrP106–126 induced significant death of neuron cells and marked proliferation of astrocytes after 10 days of treatment. Under the same treatment regimens, the level of PrP gene expression was significantly down-regulated in cortical neuron cell cultures and cerebellar granule cell cultures and was up-regulated in astrocyte cultures. The altered PrP gene expression occurred as early as 3 days after the treatment. After 10 days of treatment, while the cultured cortical neurons underwent further apoptosis, their expression of PrP gene started to recover gradually. These findings indicate that PrP 106–126 regulates transcription of the PrP gene and this activity is associated with its neurotoxicity in primary rat neuronal cultures.

Comparative analysis of the prion protein open reading frame nucleotide sequences in peacock and parakeet.

The open reading frame of peacock and parakeet prion protein (PrP) genes was cloned and sequenced. The peacock and parakeet PrP genes consisted of 833 and 866 nucleotides encoding 266 and 277 amino acids, respectively (GenBank Accession numbers AY365065 and AY365066). Sequence analysis showed that the peacock and parakeet PrP genes had 93.67% homology to each other, 94.04% and 99.64% homology to the chicken PrP gene and 46.0% and 42.1% similarity to the mammalian PrP genes, respectively. The structural features of all known mammalian and avian PrPs, including N-terminal signal peptides, tandem repeats, conserved hydrophobic region, disulfide bridges and glycoinositol phospholipid anchor, were also found in peacock and parakeet PrPs. The parakeet and peacock PrPs, however, differed in the hexarepeat region, with the peacock having six and the parakeet having seven hexarepeats. The phylogenetic analysis showed that the PrP genes in 52 species were clustered into 2 distinct lineages, the avian and the mammalian. The peacock and parakeet PrP genes belonged to the same lineage but the peacock PrP was sub-classed with the pigeon PrP and the parakeet PrP was sub-classed with the duck and chicken PrPs. The present work added two more species data to the collection of the PrP genes and supported the previous findings that the PrP genes are highly conserved across species.

Single nucleotide polymorphisms of the prion protein gene (PRNP) in Chinese pig breeds.

Abstract:  Prion diseases (transmissible spongiform encephalopathies, TSE), as a group of fatal neurodegenerative diseases, have affected humans and a variety of other mammals. Although no natural TSE have been documented in pigs, appropriate precautions need to be taken to prevent the iatrogenic spread of prion disease through pig‐to‐human xenotransplantation. Polymorphisms within the open reading frame (ORF) of the single‐copy gene of prion protein (PRNP) are associated with susceptibility to scrapie in sheep and variant Creutzfeldt‐Jacob disease in humans. We screened polymorphisms in the PRNP gene of 64 China Experimental Minipigs and Beijing Large White pigs. Our findings suggest that the porcine PRNP gene is highly homogenous. The amino acid sequences of the mature prion protein of all samples tested were identical. Four single nucleotide polymorphisms (G11A, G615C, G684A, T726G) in the ORF of the porcine PRNP gene were found, and the G→C nucleotide substitution resulted in a serine to asparaginate amino acid substitution at codon 4. We conclude that pigs raised under specific pathogen‐free conditions, with the exclusion of rendered mammalian material for at least two generations, will have little risk of being infected with a TSE, and even less possibility of transmitting prion disease to humans through xenotransplantation.

Amino acid sequence of the Pekingese dog prion protein gene.

Abstract:  Prion diseases are neurodegenerative disorders in humans and various animals associated with a proteinaceous infectious pathogen, designated prion. Canine species seem to be resistant to the infection and no natural prion disease has been documented in dogs. The polymorphisms within the open reading frame of the prion protein gene are associated with the susceptibility of the species, and species barriers, to prion diseases. In the present study, the open reading frame of the prion protein (Prnp) gene from 16 Pekingese dogs was cloned and screened for polymorphisms. One nucleotide polymorphism (G489C) was found; the G to C nucleotide substitution results in a glutamic to aspartic acid change at codon 163. The amino acid sequence of the Pekingese Prnp gene showed the highest homology with that of greyhounds (AF042843), when compared with other Prnp genes in GenBank. Glu/Asp163 and asparagine 107 in canine prion protein genes were replaced by asparagine and serine, respectively, in all the prion protein genes examined. These substitutes might in turn affect the potential intermolecular interactions critical for cross‐species transmission of prion disease and might influence the canine susceptibility to prion infection.

Expression patterns of Doppel gene in golden hamster: quantification using real-time RT-PCR.

Doppel (prion-like protein, Dpl) may act as a useful molecular marker in tumor diagnosis and in tumor grade definition, as over-expression of Dpl protein has been found in tumors with different histologic origin. Accordingly, the quantitative analysis of the expression of Dpl in different tissues is essential for understanding its role in tumor progression and cancer diagnostic. Herein we report Dpl mRNA quantification in golden hamster by calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different organs of golden hamster in different stages of development: from neonatal to adult golden hamster. Highest level of Dpl mRNA was detected in the testis, and lower levels of Dpl mRNA were detected in the following tissues: spleen, heart, bone marrow, skeletal muscles and neocortex (only in neonatal hamster). The expression of Dpl was not detected in kidney, liver and lung. This is the first study to report the expression of Dpl in bone marrow of murine and the difference of expression levels of Dpl in testis between adult and neonatal hamsters.

Comparative analysis of the prion protein gene sequences in African lion.

The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR

The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.