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Min-Liang Wong

Publications and source records attributed to Min-Liang Wong.

14 recordsLinked to original sources
PubMed2005

Prognostic factors associated with survival two years after surgery in dogs with malignant mammary tumors: 79 cases (1998-2002).

OBJECTIVE: To identify prognostic factors for female dogs that have undergone surgical removal of malignant mammary tumors. DESIGN: Retrospective case series. ANIMALS: 79 female dogs with malignant mammary tumors. PROCEDURE: Information obtained from the medical records included breed, age, sex, tumor size (maximum diameter), number and location of affected mammary glands, time between tumor identification and surgical removal, radiographic evidence of distant metastasis, surgical procedure, ovariohysterectomy (OHE) status, histologic classification of the tumor, and survival time. RESULTS: Results of univariate analyses indicated that clinical stage, tumor size, OHE status, metastasis to adjacent lymph nodes or distant sites, and histologic classification of the tumor were significantly associated with survival 2 years after surgery. Tumors > or = 5 cm in diameter and tumors that had been identified > 6 months before surgery were more likely to metastasize to adjacent lymph nodes. Ovariohysterectomy was more beneficial in dogs with complex carcinomas than in dogs with simple carcinomas. In multivariate analyses, clinical stage, tumor size, and OHE status were significantly associated with survival 2 years after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that tumor stage, tumor size, and OHE status were significant prognostic factors associated with survival 2 years after surgery in dogs with malignant mammary tumors. Further, either dogs with tumors > or = 5 cm in diameter or dogs with tumors present for > 6 months prior to surgery had a higher risk of having lymph node metastases.

Animals↗
PubMed2005

Inhibition of lytic infection of pseudorabies virus by arginine depletion.

Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation. Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine (or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work, we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase (enzyme catalyzing the conversion of arginine into ornithine and urea) treatment whereas this inhibition can be reversed by exogenous arginine, suggesting that arginine is essential for PRV proliferation. Western blotting was conducted to study the effect of arginine depletion on the levels of structural proteins of PRV in virus-infected cells. Four PRV structural proteins (gB, gE, UL47, and UL48) were chosen for examination, and results revealed that the levels of viral proteins were obviously reduced in long time arginase treatment. However, the overall protein synthesis machinery was apparently not influenced by arginase treatment either in mock or PRV-infected cells. Analyzing with native gel, we found that arginase treatment affected the mobility of PRV structural proteins, suggesting the conformational change of viral proteins by arginine depletion. Heat shock proteins, acting as molecular chaperons, participate in protein folding and translocation. Our results demonstrated that long time arginase treatment could reduce the expression of cellular heat shock proteins 70 (hsc70 and hsp70), and transcriptional suppression of heat shock protein 70 gene promoter was one of the mechanisms involved in this reduced expression.

Animals↗
PubMed2005

Phylogenetic analysis of canine parvovirus VP2 gene in Taiwan.

Canine parvovirus (CPV) is a non-enveloped virus with a single-stranded DNA genome and causes infectious enteritis in dog. In this study, 36 isolates of CPV infection were obtained in Taichung, Taiwan from 2003 to 2004. Using primers that can distinguish subtypes of CPV, we amplified part of viral VP2 gene by polymerase chain reaction (PCR) and the PCR product was sequenced; results demonstrated that two isolates could be classified as type 2a of CPV and the others were type 2b. The complete coding region of VP2 gene of type 2b was also sequenced, and phylogenetic analysis of these DNA sequences revealed that our Taichung isolate was close to the V-120, FPV-314, 97-008, Taiwan 9, LCPV-T1, and T4 isolates; however, because of the degeneracy of codons, the amino acid sequences of Taichung isolate was similar to that of the 97-008 isolate from Japan. It is known that two important amino acid residues (Asn-426 in type 2a and Asp-426 in type 2b; Ile-555 in type 2a and Val-555 in type 2b) are the determinants for the discrimination of type 2a or type 2b. After scrutinizing the complete VP2 gene of our Taichung isolate, we found the VP2 protein of the Taichung isolate did possess this molecular feature of type 2b virus. Previous studies reported that type 2a virus was the major type in Taiwan; our finding showed that CPV type 2b was the predominant type in the middle part of Taiwan. Moreover, a unique Ala-489 in VP2 of our Taichung isolate was found, contrasting to a Val-489 in the VP2 of other strains.

Amino Acid Sequence↗
PubMed2005

On the TATA box and transcriptional start site of gE gene of pseudorabies virus: a comparison of three methods.

Using the upstream region of glycoprotein E gene of pseudorabies virus (PRV) as a model, a method based on reverse transcriptase-polymerase chain reaction (RT-PCR) for the determination of transcriptional start site was developed. The conventional primer extension method was used to determine the start site. Comparing the results obtained by these two methods, a good agreement on the location of start site was achieved. In addition, a computer program was used to predict the transcriptional start site, and the predicted site was found to be close to the two sites obtained by experiments. Based on the transcriptional start site data and on the general knowledge of the eukaryotic gene, the TATA box of gE gene was assigned at approximately 33 bp upstream to the start site.

Animals↗
PubMed2004

Functional analysis of virion host shutoff protein of pseudorabies virus.

During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient transfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo.

Amino Acid Sequence↗
PubMed2004

Regulation of the vhs gene promoter of pseudorabies virus by IE180 and EP0, and the requirement of a Sp1 Site for the promoter function.

The virion host shutoff (vhs) protein is a virion component of Alphaherpesviruses, including pseudorabies virus. In this work, the upstream sequences of vhs gene of pseudorabies virus (TNL strain) was cloned and sequenced. We linked the upstream sequences of vhs gene to the CAT reporter gene and examined the promoter function of this region. The immediate-early protein IE180 of Pseudorabies Virus (PRV) is expressed immediately after infection and plays a vital role in the regulation of other viral genes. Our results demonstrated that the vhs promoter was regulated by the IE180 in a dosage-dependent manner; the vhs promoter was stimulated by low concentration of IE180 but suppressed by high concentration of IE180. Mutational analysis indicated that the only IE180 binding site at the vhs promoter was not essential for its function; however, a Sp1 binding site (15 bp downstream to TATA box) was critical to its function. In addition, the result of cotransfection demonstrated that early protein 0 (EP0) of PRV, another protein with transcriptional function, inhibited the activity of the vhs promoter.

Animals↗
PubMed2004

Examination of meat components in commercial dog and cat feed by using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique.

It has been shown that certain slow neurological diseases such as bovine spongiform encephalopathy (also known as "mad cow" disease) could be transmitted through contaminated food intake by animals; therefore, the examination of meat components in commercial feeds is important for the control of the disease in public health. The combination of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique was applied to examine the meat components in dog and cat commercial feeds. The partial nucleotide sequence (359 bp) of animal mitochondrial cytochrome b (cytb, CYT) gene was amplified by PCR and then digested with restriction enzyme Alu I or Mbo I. In this work, eight brands of commercial dog and cat feeds available in Taiwan were examined. All brands of dog feeds that were tested contained meat from four different animals (cattle, pig, goat and chicken). In cat feeds, the chicken meat was found in five out of eight brands.

Animal Feed↗
PubMed2003

Lytic infection of pseudorabies virus in the presence of spermine, spermidine, or DFMO.

The effects of polyamines (spermine or spermidine) and DFMO (an ornithine decarboxylase inhibitor) on the infection of LM (tk-) cells by pseudorabies virus (PRV) were investigated. Results from radioactive methionine labeling showed that the synthesis of viral proteins was not affected; however, the expression of a distinctive cellular protein ( approximately 27 kDa) was induced after the treatment of spermine or spermidine. Using plaque assay, we found that the plaque formation of PRV was not affected by these three reagents either. Furthermore, the effects of these drugs on the transcription of PRV immediate-early gene (IE) promoter were examined by CAT assay, and results showed weak stimulation of transcription by these drugs. Taken together, our results demonstrated that lytic infection of PRV was not influenced by addition of exogenous polyamines or depletion of endogenous polyamines; this conclusion was similar to earlier studies by using herpes simplex virus.

Animals↗
PubMed2002

Cloning and regulation of the promoter of pseudorabies virus (TNL strain) glycoprotein E gene.

The nucleotide sequence upstream to the glycoprotein E (gE) gene of pseudorabies virus (PrV, TNL strain) was cloned from the genomic virus DNA by polymerase chain reaction (PCR) and its DNA sequences were determined. The DNA segment, which was supposed to contain the gE promoter, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter gene and the resulting plasmid was named pgEp-B-CAT. To examine the promoter function of this upstream sequence of gE gene, we transfected pgEp-B-CAT DNA into L-M cells and the promoter activity was analyzed by CAT assay. Results showed that our DNA fragment could exhibit promoter activity. Furthermore, we transfected L-M cells with pgEp-B-CAT for 48 h, then superinfected cells with pseudorabies virus, and performed CAT assay. It was found that PrV superinfection could slightly enhance the activity of gE promoter, suggesting that factors produced during viral infection could stimulate the promoter. To explore the possible mechanism of regulation at transcriptional level, the pgEp-B-CAT plasmid were cotransfected with eukaryotic vectors expressing viral regulatory proteins IE or EP0, and results indicated that the gE promoter was activated by IE protein whereas it was inhibited by EP0 protein. Moreover, the effect of exogenous IE or EP0 on the protein level of gE in PrV-infected cells was examined; conclusion similar to that of CAT assay were obtained.

Animals↗
PubMed2002

A TEF-1-element is required for activation of the promoter of pseudorabies virus glycoprotein X gene by IE180.

The pseudorabies virus (PRV) immediate-early regulatory protein IE180 is able to transactivate the viral early and late genes. Using chloramphenicol acetyltransferase (CAT) assay, we investigated the transactivation function of IE180 to the promoter of PRV glycoprotein X (gX) gene, and our results showed that IE180 could significantly increase the expression of CAT gene which was under the control of gX promoter. To further identify the activation domains of IE180 protein that interact with the gX promoter sequences, various truncated mutants of IE180 gene and gX promoter gene were constructed and analyzed by CAT and gel retardation assay. Results revealed that the N-terminal amino acid residues from 133 to 736 of IE180 could interact with the binding site of transcriptional enhancer factor-1 (TEF-1) that resides in the gX promoter. Formation of protein-DNA complexes between the IE180 protein and the TEF-1 element of the gX promoter was observed using electrophoretic mobility shift assay (EMSA) as well as Southwestern blot analysis. These results indicated that a direct interaction occurred between IE180 and the TEF-1 element; and this interaction was abolished if the TEF-1 element was mutated. The association of IE180 with the TEF-1 element was further confirmed by the supershift of EMSA complexes using IE180 specific antibody. Taken together, our results suggested that formation of a complex between the IE180 protein and TEF-1 element in the gX promoter region was involved in the transcriptional regulation of the gX gene.

Animals↗
PubMed2002

Suppression of promoter activity of the LAT gene by IE180 of pseudorabies virus.

The latency-associated transcript (LAT) gene is the only viral genomic region that is abundantly transcribed during pseudorabies virus (PrV) latent infection. The mechanism of reactivation of PrV from latency remains unknown. To analyze the regulation mechanism of the LAT promoter, we constructed a series of recombinant vectors in which various sequences upstream of LAT were linked to the chloramphenicol acetyltransferase (CAT) gene. Transcriptional efficiency was examined by cotransfection with plasmids carrying the PrV IE, EP0, or gD gene, respectively. Results showed that the activity of PrV LAT promoter was dramatically repressed by the IE180 protein and a TATA box and a putative IE180 binding site within the promoter were involved in this repression. To dissect the functional domains of IE180, we compared the relative repressive abilities of IE180 variants to the LAT promoter by transient transfection assays. Mutational analysis demonstrated that almost the whole IE180 (amino acid residues 1-1440) are essential for its repression to LAT promoter. To explore the possible mechanism of repression, an electrophoretic mobility shift assay (EMSA) using nuclear extracts from neuronal cells was performed and formation of protein-DNA complexes between IE180 and the oligonucleotide probe (-46 to -19, relative to the start site of LAT transcription) was demonstrated. The association of IE180 with the region encompassing the putative IE180 binding site and the TATA box upstream of PrV LAT gene was further confirmed by supershift of EMSA complexes using IE180 specific antibody. Thus, our results suggested that IE180 repressed the LAT promoter via an interaction between IE180, LAT promoter and cellular protein(s).

Animals↗