Biomedical subjects

S Kagawa

Publications and source records attributed to S Kagawa.

At least 19 recordsLinked to original sources

PubMed2007

Bicalutamide 80 mg combined with a luteinizing hormone-releasing hormone agonist (LHRH-A) versus LHRH-A monotherapy in advanced prostate cancer: findings from a phase III randomized, double-blind, multicenter trial in Japanese patients.

To compare combination therapy with bicalutamide 80 mg and a luteinizing hormone-releasing hormone agonist (LHRH-A) versus LHRH-A alone in Japanese men with untreated advanced prostate cancer. A total of 205 patients with stage C/D prostate cancer were randomized to either LHRH-A+once-daily oral bicalutamide 80 mg or placebo. Primary study variables have been reported previously. Secondary variables included: time to achieve prostate-specific antigen < or = 4 ng/ml, time-to-treatment failure (TTTF), time-to-disease progression (TTP), overall survival (OS), adverse events and adverse drug reactions. Following combination therapy with bicalutamide 80 mg, there were significant (P<0.001) advantages over LHRH-A alone in terms of TTTF and TTP, but the difference in the interim OS was not statistically significant. First-line combination therapy with bicalutamide 80 mg in Japanese patients with advanced prostate cancer offers significant benefits over LHRH-A alone, with respect to TTTF and TTP. Follow-up for OS continues.

Aged↗

PubMed2006

Prostate cancer incidence varies among males from different Y-chromosome lineages.

The incidence rate of prostate cancer in African-American males is two times higher than Caucasian men and ten times higher than Japanese men. The geographical specificity of Y haplogroups implies that males from different ethnic groups undoubtedly have various Y lineages with different Y-chromosomal characteristics that may affect their susceptibility or resistance to such a male-specific cancer. To confirm this hypothesis we studied the Y-chromosomal haplogroups of 92 Japanese prostate cancer patients comparing them with randomly selected 109 unrelated healthy Japanese male controls who were confirmed to be residents of the same geographical area. Males could be classified using three binary Y-chromosome markers (sex-determining region Y (SRY), YAP, 47z) into four haplogroups DE, O2b(*), O2b1, and untagged group. Our results confirmed that prostate cancer incidence varies among males from different Y-chromosome lineages. Males from DE and the untagged haplogroups are at a significantly higher risk to develop prostate cancer than O2b(*) and O2b1 haplogroups (P=0.01), odds ratio 2.17 and 95% confidence interval (1.16-4.07). Males from haplogroup DE are over-represented in the patient group showing a percentage of 41.3%. The underlying possible causes of susceptibility variations of different Y lineages for such a male-specific cancer tumorigenesis are discussed. These findings explain the lower incidence of prostate cancer in Japanese and other South East Asian males than other populations. To our knowledge, this is the first reliable study examining the association between prostate cancer and Y-chromosomal haplogroups, comparing prostate cancer patients with carefully selected matched controls.

Carcinoma↗

PubMed2006

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis.

BACKGROUND: At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. RESULTS: Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. CONCLUSION: High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

Animals↗

PubMed2006

Nucleotide sequencing and analysis of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) of Taylorella equigenitalis, as an important pathogen for contagious equine metritis (CEM).

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184(T), Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184(T) and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184(T) and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.

Animals↗

PubMed2005

cDNA microarray analysis assists in diagnosis of malignant intrarenal pheochromocytoma originally masquerading as a renal cell carcinoma.

Intrarenal pheochromocytoma (paraganglioma) is a very rare tumour. Its diagnosis is often difficult to establish because of its rarity and its histological similarity to renal cell carcinoma (RCC). Recently, we examined the molecular signatures of different subtypes of kidney tumours by using cDNA microarray. The signature pattern for one tumour, which was originally diagnosed as granular cell RCC, was clearly distinct from that of any other subtype of kidney tumour, and led us to re-evaluate the case. Haematoxylin and eosin staining revealed histological features suggestive of pheochromocytoma, and immunohistochemical studies showed positive staining for neuroendocrine markers but not for keratin. A germline missense mutation, D119E, in the familial paraganglioma related gene succinate dehydrogenase subunit D (SDHD), was subsequently identified. The treatment modality was revised and radiotherapy was given, to which the patient responded, leading to a reduction in tumour size of 25% within the first month. To our knowledge, this is the first report of an intrarenal pheochromocytoma that was diagnosed with the assistance of cDNA microarray analysis.

Adult↗

PubMed2005

ACE inhibitor and AT1 antagonist stimulate duodenal HCO3- secretion mediated by a common pathway - involvement of PG, NO and bradykinin.

Recent study demonstrated that duodenal HCO3- secretion is affected by modulation of the renin-angiotensin system. We examined the effects of enalapril (angiotensin-converting enzyme (ACE) inhibitor) or losartan (angiotensin AT1 receptor antagonist) on duodenal HCO3- secretion in rats and investigated the mechanisms involved in the renin-angiotensin system-related HCO3- response. A proximal duodenal loop was perfused with saline, and HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Enalapril increased the HCO3- secretion in a dose-dependent manner, with a decrease in arterial blood pressure (MBP), and these effects were significantly attenuated by pretreatment with indomethacin, L-NAME and FR172357 (a selective bradykinin B2 receptor antagonist). Although losartan alone did not affect the HCO3- secretion, despite reducing MBP, the agent dose-dependently increased the HCO3- secretion in the presence of angiotensin II, and this response was totally antagonized by prior administration of FR172357, indomethacin and L-NAME. Bradykinin also dose-dependently increased the HCO3- secretion with no change in MBP, though transient, and again the effects were blocked by indomethacin, L-NAME and FR172357. Both prostaglandin (PG) E2 and the nitric oxide (NO) donor NOR-3 also increased the HCO3- secretion, the latter effect being inhibited by indomethacin. These results suggest that both an ACE inhibitor and AT1 antagonist (in the presence of angiotensin II) increase duodenal HCO3- secretion via a common pathway, involving bradykinin, NO and PGs. It is also assumed that bradykinin releases NO locally, which in turns stimulates HCO3- secretion mediated by PGs.

Angiotensin II Type 1 Receptor Blockers↗

PubMed2004

Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer.

BACKGROUND: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax). AIMS: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53. METHODS: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment. RESULTS: Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment. CONCLUSION: Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.

Adenoviridae↗

PubMed2002

Molecular genotyping of isolates of Actinobacillus actinomycetemcomitans by pulsed-field gel electrophoresis after separate digestion with Sse8387I and XhoI.

Genomic DNA from 18 Japanese clinical isolates of Actinobacillus actinomycetemcomitans was obtained from six periodontitis patients and analyzed using pulsed-field gel electrophoresis (PFGE) after separate digestion with Sse8387I and with XhoI. Three isolates from an identical patient were found to share an identical PFGE profile, and isolates from distinct patients were found to have PFGE profiles distinctly different from each other. Consequently, the 18 Japanese clinical isolates were discriminated into six distinct genotypes by means of PFGE. The genomic DNA from the other six reference strains (ATCC33384, ATCC43717, ATCC 43718, JCM2434, JCM2435 and Nig-1) was discriminated into six genotypes by the same PFGE methodology, and these six genotypes were found to be distinctly different from the six genotypes of the 18 Japanese clinical isolates described above. Serotyping demonstrated three PFGE genotypes in the serotype a strains, four the serotype b strains and three the serotype c strains. The present results clearly suggest that the PFGE procedure after separate digestion with Sse8387I and with XhoI has an excellent discriminatory power amongst strains and has a good genotypability for A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed2002

Gastric ulcerogenic responses following barrier disruption in knockout mice lacking prostaglandin EP1 receptors.

BACKGROUND/AIMS: Endogenous prostaglandins (PGs) are considered to play a pivotal role in maintaining the mucosal integrity of the stomach after injury. In the present study, we evaluated the mucosal ulcerogenic and mucosal blood flow (GMBF) responses in the stomach after damage by taurocholate (TC) in knockout mice lacking EP1 or EP3 receptors. METHODS: Under urethane anaesthesia, a mouse stomach was mounted in an ex vivo chamber, exposed to 20 mmol/L TC for 20 min and treated with 20 mmol/L HCl before and after TC. GMBF was measured with a laser Doppler flowmeter. RESULTS: Mucosal exposure to TC in wild-type mice caused a marked decrease in potential difference (PD), followed by an increase in H+ loss and GMBF. The decreased PD was gradually normalized after removal of TC from the chamber, with minimal damage in the mucosa 1 h after TC treatment. This hyperaemic response was inhibited by indomethacin, resulting in severe lesions in the mucosa without any change in PD or H+ loss. None of these responses induced by TC were altered in EP3-/- mice. However, in mice lacking EP1 receptors, TC treatment did not increase GMBF, despite causing PD reduction and acid loss, and resulted in severe damage in the mucosa. These responses were closely similar to those observed in animals pretreated with ONO-8711, a EP1 receptor antagonist. Mucosal PGE2 content was significantly increased after TC, similarly in all groups of mice. CONCLUSION: These results confirm a mediator role for PGE2 in gastric hyperaemic response following mucosal exposure to TC and suggest that endogenous PGs may contribute to maintaining mucosal integrity after barrier disruption, mainly through activation of the EP1 receptor subtype.

Animals↗

PubMed2002

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC).

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.

Campylobacter↗

PubMed2001

Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.

BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.

Adenoviridae↗

PubMed2001

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.

Adenoviridae↗

PubMed2001

Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification.

To better understand the molecular mechanisms that underlie the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC), we studied the gene expression profiles of 29 ccRCC tumors obtained from patients with diverse clinical outcomes by using 21,632 cDNA microarrays. We identified gene expression alterations that were both common to most of the ccRCC studied and unique to clinical subsets. There was a significant distinction in gene expression profile between patients with a relatively nonaggressive form of the disease [100% survival after 5 years with the majority (15/17 or 88%) having no clinical evidence of metastasis] versus patients with a relatively aggressive form of the disease (average survival time 25.4 months with a 0% 5-year survival rate). Approximately 40 genes most accurately make this distinction, some of which have previously been implicated in tumorigenesis and metastasis. To test the robustness and potential clinical usefulness of this molecular distinction, we simulated its use as a prognostic tool in the clinical setting. In 96% of the ccRCC cases tested, the prediction was compatible with the clinical outcome, exceeding the accuracy of prediction by staging. These results suggest that two molecularly distinct forms of ccRCC exist and that the integration of expression profile data with clinical parameters could serve to enhance the diagnosis and prognosis of ccRCC. Moreover, the identified genes provide insight into the molecular mechanisms of aggressive ccRCC and suggest intervention strategies.

Adenocarcinoma, Clear Cell↗

PubMed2001

Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene and transferred them into malignant cells in vitro and tumors in vivo. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of activation of pro-caspase-8 and cleavage of poly(ADP-ribose) polymerase. The intratumoral delivery elicited tumor cell apoptosis and suppressed tumor growth. In comparison with Bax gene treatment, which is toxic to normal cells, TRAIL gene treatment caused no detectable toxicity in cultured normal fibroblasts nor in mouse hepatocytes after systemic gene delivery. Furthermore, coculture of cancer cells expressing TRAIL with those expressing green fluorescent protein (GFP) resulted in apoptosis of both cells, whereas coculture of Bax-expressing cells with GFP-expressing cells resulted in the cell death of the Bax-expressing cells only, which suggested that the transfer of the TRAIL gene resulted in bystander effects. Moreover, culture of cells with medium from TRAIL-expressing cells showed the proapoptotic activity and bystander effect of the TRAIL gene to be not transferable with medium. To further demonstrate the bystander effect of the TRAIL gene, we constructed plasmid vectors encoding GFP-TRAIL or GFP-Bik chimeric proteins. Transfection of the GFP-TRAIL gene into cancer cells resulted in the death of GFP-positive cells and their neighbors, whereas transfection of the GFP-Bik gene killed GFP-positive cells only. Finally, GFP-TRAIL genes, transfected into normal human fibroblasts or bronchial epithelial cells, did not kill such cells, whereas transfected GFP-Bik genes did. Thus, the direct transfer of the TRAIL gene led to selective killing of malignant cells with bystander effect, which suggests that the TRAIL gene could be valuable for treatment for cancers. Together, these results suggest that delivering the TRAIL gene to cancerous cells may be an alternative approach to cancer treatment.

Adenoviridae↗

PubMed2001

Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer.

Using adenoviral technology, we overexpressed the proapoptotic molecules pro-caspase-3, pro-caspase-7, and Bax to induce therapeutic apoptosis of prostate cancer cell lines growing in vitro and in vivo. Because overexpressed pro-caspase-3 did not undergo autocatalytic activation in any of the five prostate cancer cell lines evaluated, this strategy was unable to engage any component of the apoptotic pathway. Overexpressed pro-caspase-7 was proteolytically cleaved in LNCaP and LnCaP-Bcl-2 cells but not in PC-3, DU-145, or TsuPr(1) cells. Cleavage was associated with engagement of many components of the apoptotic pathway, including DEVDase activity, cleavage of intracellular caspase targets such as the DNA fragmentation factor and the proapoptotic Bid, release of cytochrome c from the mitochondria to the cytoplasm, and terminal deoxynucleotidyl transferase-mediated nick end labeling. No apoptosis was observed in the cells where caspase-7 did not undergo autocatalytic activation. Searching for an approach that would more reliably induce therapeutic apoptosis of prostate cancer cell lines, we used a binary adenoviral system to overexpress the proapoptotic molecule Bax. Bax was dramatically overexpressed and caused apoptosis of every cell line infected by engaging the mitochondrial pathway, including proteolytic cleavage and catalytic activation of the caspases, cleavage of caspase substrates, release of cytochrome c from the mitochondria, and DNA fragmentation. Furthermore, three injections of the Bax overexpression system into PC-3 cell tumors in nude mice in vivo caused a 25% regression in tumor size corresponding to a 90% reduction relative to continued tumor growth in animals that received injections with the control binary system expressing Lac-Z. These experiments show that adenovirus-mediated Bax overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo by activating the mitochondrial pathway of apoptosis. On the basis of these studies, we conclude that manipulation of Bax expression is an attractive new gene therapy approach for the treatment of prostate cancer.

Adenoviridae↗

PubMed2001

Augmenting transgene expression from carcinoembryonic antigen (CEA) promoter via a GAL4 gene regulatory system.

Though extensively studied, the use of tissue- or cell-type-specific promoters to target transgene expression is hampered by their weak activity. We hypothesized that this problem could be addressed by using a GAL4 gene regulatory system, wherein a weak, tissue-specific promoter would drive expression of the GAL4/VP16 fusion protein (GV16), which in turn would transactivate a minimal synthetic promoter, GAL4/TATA (GT), upstream of a transgene. To test this hypothesis, we constructed adenoviral vectors expressing a lacZ or GV16 gene driven by a carcinoembryonic antigen (CEA) promoter (Ad/CEA-LacZ or Ad/CEA-GV16) and evaluated levels of transgene expression they produced in cultured cells and in subcutaneous tumors after intratumoral administration. In CEA-positive cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ versus Ad/CEA-LacZ increased transgene expression 20- to 100-fold. In CEA-negative cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ increased transgene expression to a much lower degree (6- to 8-fold). In addition, analysis of Bax gene-mediated cell death revealed that this system can be used to avoid Bax's toxic effects on CEA-negative cells without compromising its ability to kill CEA-positive cells in vitro and in vivo. Thus, the combination of a tissue-specific promoter with the GAL4 gene regulatory system could be useful for targeting transgene expression.

Adenoviridae↗

PubMed2001

Comparison of the value of pulsed-field gel electrophoresis, random amplified polymorphic DNA and amplified rDNA restriction analysis for subtyping Taylorella equigenitalis.

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.

Animals↗

PubMed2001

Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-field gel electrophoresis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.

Animals↗