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Y Yoshikawa

Publications and source records attributed to Y Yoshikawa.

At least 127 records · Page 7Linked to original sources
PubMed1999

New index for oxygen cost of contractility from curved end-systolic pressure-volume relations in cross-circulated rat hearts.

We have already reported the linear oxygen consumption per beat (VO(2))-systolic pressure-volume area (PVA) relation from the curved left ventricular (LV) end-systolic pressure-volume relation (ESPVR) in the cross-circulated rat heart. The VO(2) intercept (PVA-independent VO(2)) is primarily composed of VO(2) for Ca(2+) handling in excitation-contraction (E-C) coupling and basal metabolism. The aim of the present study was to obtain the oxygen cost of LV contractility that indicates VO(2) for Ca(2+) handling in E-C coupling per unit LV contractility change in the rat heart. Oxygen cost of LV contractility is obtainable as a slope of a linear relation between PVA-independent VO(2) and LV contractility. We obtained a composite VO(2)-PVA relation line at a mid-range LV volume (mLVV) under gradually enhanced LV contractility by stepwise increased Ca(2+) infusion and thus the gradually increased PVA-independent VO(2) values. As a LV contractility index, we could not use E(max) (ESP-V ratio; ESP/ESV) for the linear ESPVR because of the curved ESPVR in the rat LV. A PVA at a mLVV (PVA(mLVV)) has been proposed as a good index for assessing rat LV mechanoenergetics. Since the experimentally obtained PVA(mLVV) was not triangular due to the curved ESPVR, we propose an equivalent ESP-V ratio at a mLVV, (eESP/ESV)(mLVV), as a LV contractility index. This index was calculated as an ESP-V ratio of the specific virtual triangular PVA(mLVV) that is energetically equivalent to the real PVA(mLVV). The present approach enabled us to obtain a linear relation between PVA-independent VO(2) and (eESP/ESV)(mLVV) and the oxygen cost of LV contractility as the slope of this relation.

Animals↗
PubMed1999

Technology to obtain sustained release characteristics of drugs after delivered to the colon.

To determine the necessary technology by which sustained drug release is obtained after drug is delivered to the colon, two kinds of microcapsules were prepared and were filled in a pressure-controlled colon delivery capsule (PCDC). As a model drug 5-aminosalicylic acid (5-ASA) was used, because the target site of 5-ASA is the entire large intestine. 5-ASA was microencapsulated using a water-insoluble polymer, ethylcellulose (EC) or with pH-sensitive polymers, Eudragit L-100 or S-100 and encased in PCDC. The particle size of these microcapsules was around 800 microns and the loading efficiencies of 5-ASA were approximately 90%. In vitro dissolution tests were performed with the prepared microcapsules. The release rate of 5-ASA from the microcapsules was significantly prolonged as compared to 5-ASA powder, although there were no significant differences in the release rates between these microcapsules. By incorporating the 5-ASA microcapsules into PCDC, sustained release PCDCs for colon delivery were prepared and in vivo evaluation was performed using beagle dogs. As a fast release colon delivery system, PCDCs were prepared with 5-ASA powder suspended in suppository base. After oral administration of the test preparations to beagle dogs, plasma 5-ASA concentrations were measured and sustained release characteristics of 5-ASA from the test preparations were evaluated from the plasma 5-ASA concentration-time profiles. The first appearance time of 5-ASA into the systemic circulation after oral administration were 3 h for all the colon delivery preparations and it was thought that these test preparations were delivered to the colon. Both EC microcapsules and Eudragit S-100/RS-100 microcapsules in PCDC showed longer the mean residence time MRT, 8.2 +/- 0.6 h and 8.7 +/- 0.9 h, than Eudragit L-100/RS-100 microcapsules in PCDC where the MRT was 6.6 +/- 0.2 h. Since PCDCs containing 5-ASA powder exhibited a MRT of 7.0 +/- 1.0 h, these two types of preparations have suggested sustained release characteristics.

Algorithms↗
PubMed1999

Preparation and evaluation of once-a-day injectable microspheres of interferon alpha in rats.

Gelatin microspheres (ms) and gelatin/BSA (bovine serum albumin) or gelatin/alginate ms were prepared by encapsulating fluorescein isothiocyanate (FITC) labeled dextran or interferon alpha (IFN-alpha). Ms were obtained by an emulsion-solvent-extraction method. Gelatin and gelatin/BSA ms were obtained by treating water-in-oil (W/O) emulsions with iso-propyl alcohol. Gelatin/alginate ms having different composition (25/1, 20/1, 15/1, 10/1 and 5/1) were obtained by treating a W/O emulsion composed of gelatin and sodium alginate with 0.5 M calcium chloride solution. The average diameters of all the prepared ms were approximately 300 microns. The FITC-dextran loading efficiencies were 96.5 +/- 0.6% for gelatin ms (#1), 97.3 +/- 2.2% for gelatin/BSA ms (#2) and 68.7 +/- 2.2%, 55.0 +/- 3.9%, 47.5 +/- 3.3%, 44.4 +/- 1.2%, 27.1 +/- 2.2% for gelatin/alginate ms (#3-#7). The IFN-alpha loading efficiencies were 10.8 +/- 0.5% for gelatin/BSA ms (#8) and 22.5 +/- 1.8%, 17.6 +/- 0.9% and 14.5 +/- 0.5% for gelatin/alginate ms (#9, #10 and #11). In vitro release studies with ms containing FITC-dextran showed that the release rate of FITC-dextran from the ms decreased by the modification of gelatin ms with BSA or sodium alginate, although the effect of BSA addition to gelatin ms did not elucidate satisfactory sustained-release characteristics of FITC-dextran after subcutaneous (s.c.) injection to rats. By decreasing the formulated ratio of gelatin/alginate from 25/1 to 5/1, the mean T50%, the time when the half amount of FITC-dextran contained was released from the ms, increased from 1.7 +/- 0.1 to 13.8 +/- 3.6 h and three ms preparations (#4, #5 and #6) showed sustained-release characteristics on the serum FITC-dextran concentration-time profiles. Based on these results, three types of ms containing IFN-alpha were prepared and in vivo pharmacokinetic studies were performed in rats, where the dose of IFN-alpha was 2 x 10(4) IU/rat. By the addition of alginate to gelatin, the release rate of IFN-alpha was decreased and the serum IFN-alpha concentration-time profiles showed better sustained-release characteristics of IFN-alpha from #10 ms than the other IFN-alpha ms (#8, #9 and #11) after s.c. injection to rats.

Alginates↗
PubMed1999

Characterization of norfloxacine release from tablet coated with a new pH-sensitive polymer, P-4135F.

A new pH-sensitive polymer, P-4135F, was evaluated as a colon delivery device for norfloxacine (NFLX) which is used for the therapy of patients with Vero toxin-producing Escherichia coli gastroenteritis. P-4135F has a dissolution threshold pH of 7.2 which is higher than the conventional pH-sensitive polymers, Eudragit S100 and L100. To compare the dissolution site of P-4135F coated tablets with other enteric polymer coatings, mini-tablets containing sodium fluorescein (FL) as a model drug were prepared by coating them with the three polymers. After oral administration of FL mini-tablets to rats, the first-appearance time, Ti, of FL into the systemic circulation was measured. The Tis were 0.7+/-0.2 h for Eudragit L100, 1.8+/-0.4 h for S100 and 2.0+/-0.3 h for P-4135F. Direct inspection of the dissolution process of the FL mini-tablets after oral administration to rats was performed by abdominal incision studies. All of the coated FL mini-tablets started to dissolve in the rat ileum. The dissolution sites were identified to be proximal to the ileocecal junction for P-4135F, at the middle part of the ileum for Eudragit S100 and at the proximal part of the ileum for Eudragit L100. NFLX tablets with different membrane thicknesses of P-4135F were prepared and were orally administered to beagle dogs. The colon delivery efficiency was evaluated by measuring the Ti of NFLX into the systemic circulation. The mean Tis were 1.33+/-0.33 h for 56.8+/-0.5 microm membranes, 3.75+/-0.25 h for 64.6+/-0.7 microm membranes, 4.00+/-1.00 h for 70.5+/-0.5 microm membranes and 3.00+/-1.00 h for 74.9+/-0.4 microm membranes. By comparing the Ti, 4.33+/-0.33 h, obtained after oral administration of NFLX in a pressure-controlled colon delivery capsule, and the colon arrival time, 3.5+/-0.3 h, determined by a sulfasalazine test in beagle dogs. P-4135F coated NFLX tablets appeared to dissolve and disintegrate before reaching the colon. Studies using rats and beagle dogs have suggested that P-4135F dissolves in the lower part of the small intestine, i.e., the ileum. These studies also suggest that this new polymer will be useful for the delivery of NFLX to the lower part of the small intestine.

Acrylic Resins↗
PubMed1999

[Yellow fever].

Explore the source record for details and available documents.

Aedes↗
PubMed1999

Cyno-EBV (EBV-related herpesvirus from cynomolgus macaques) induces rabbit malignant lymphomas and their tumor cell lines frequently show specific chromosomal abnormalities.

Malignant lymphoma (ML) induction in rabbits by Epstein-Barr virus (EBV)-related herpesvirus of cynomolgus (Cyno-EBV) is reported. Twenty-seven of 30 (90%) rabbits inoculated intravenously with Cyno-EBV-producing simian (cynomolgus) lymphocyte cell line (Ts-B6) cells developed ML between 45 and 115 days after inoculation. The peroral inoculation of Ts-B6 cells induced ML in only 2 of 10 (20%) rabbits (75 to 85 days). Five of 6 (83%) rabbits injected with cell-free pellets from Ts-B6 cultures also developed ML (27 to 122 days). Antibody response to the viral capsid antigen of EBV was also detected in sera from rabbits inoculated with Ts-B6. ML of the large cell or mixed type infiltrated diffusely in many organs, frequently involving the spleen, liver, kidneys, heart, and less frequently the lungs, lymph nodes, brain, eyes, gastrointestinal tract, thymus, and bone marrow. A chromosomal analysis of five lymphoma cell lines established from tumor-bearing rabbits revealed the rabbit karyotype. Three of these cell lines showed the chromosomal abnormalities with 12q- or t (7p+:12q-). EBV-encoded small RNA-1 and EBV-associated nuclear antigen 1 were expressed in Ts-B6 cells, the tumor tissues, and all rabbit cell lines by in situ hybridization and by immunofluorescence tests, respectively. EBV DNA was also detected in Ts-B6 cells and rabbit lymphoma cell lines by polymerase chain reaction and Southern blot analysis. The Southern blots of EBV termini revealed oligoclonal bands in the Cyno-EBV-induced rabbit lymphomas. No lymphoma was induced by the inoculation of B95-8 (EBV-producing cells) or peripheral leukocytes from normal cynomolgus (controls). These data suggest that the high rate of lymphoma induction in rabbits may be caused not by human EBV (B95-8) but by Cyno-EBV from Ts-B6 cells. A sequence analysis of the IR1 (BamHIW) region of Cyno-EBV revealed that this region is quite similar to that of herpesvirus Macaca fascicularis 1, which is a causative agent for a monkey model of AIDS-related lymphomas. The present rabbit model of lymphoma with specific chromosomal abnormalities is very useful to clarify the role of EBV in human EBV-associated lymphoma and provides a means for studying prophylactic and therapeutic regimens.

Animals↗
PubMed1999

[Mediastinoscopic resection combined with a sternum lift technique in a case of thymic cyst].

We report on a case of thymic cyst which was successfully treated with mediastinoscopic resection combined with a sternum lift technique. A 62-year-old man was admitted to our hospital with a thymic cyst, which was causing chest discomfort and was increasing in size. The tumor was located above the pulmonary artery in the anterior mediastinum. He underwent mediastinoscopic resection while lifting the inferior portion of the sternum with a Laparolift. The cyst was resected sharply and bluntly with the Harmonic Scalpel without rupture. The patient was discharged on the 6th postoperative day in a good condition. Mediastinoscopic resection of a thymic cyst through a xiphoid approach is a useful surgical approach because of the absence of postoperative pain and because it allows early discharge from the hospital.

Endoscopy↗
PubMed1999

Re-replacement of the atrioventricular valve for the systemic circulation in children.

BACKGROUND AND AIM OF THE STUDY: The study aim was to determine whether re-replacement of the systemic atrioventricular (AV) valve can be achieved efficiently in children. METHODS: The systemic AV valve was re-replaced in 10 children. Initial replacement was needed for regurgitation in nine cases and congenital mitral stenosis in one case at a mean age of 3.7 +/- 3.1 years (range: 0.7-10.2 years). The initial prosthesis chosen was a mechanical valve in all cases; prosthesis size ranged from 16 mm to 27 mm. Reoperation was indicated at a mean age of 9.7 +/- 3.6 years (range: 3.5-14.8 years) because of non-structural dysfunction in five cases, prosthetic valve endocarditis in two, thrombosed valve in two, and progressive obstruction of the left ventricular outflow tract in one case. Fibrous tissues were extensively resected to enlarge the valvular orifice. A translocation maneuver was employed in five cases. RESULTS: Re-replacement using a bileaflet mechanical valve was successful, with no operative or late deaths. Up-sizing was feasible in six cases with the initial valve < or = 25 mm, the alternative prosthesis being 2-8 mm (mean 4.5 mm) larger than the initial one. Consecutive echocardiography demonstrated improved peak flow velocity across the AV valve (from 2.3 +/- 0.6 to 1.6 +/- 0.3 m/s). Catheterization showed improved mean pulmonary arterial pressure (from 32 +/- 13 to 21 +/- 3 mmHg). No change was seen in cardiac index (3.4 +/- 0.6 and 3.5 +/- 0.6 l/min/m2) or systemic ventricular ejection fraction (55 +/- 14% and 49 +/- 23%). CONCLUSIONS: Re-replacement of the systemic AV valve can be achieved efficiently, even in children, with up-sizing feasible by appropriate surgical maneuvers.

Adolescent↗
PubMed1999

Replacement of the morphologically tricuspid valve in children with discordant atrioventricular connections.

BACKGROUND AND AIM OF THE STUDY: Our clinical experience was reviewed to determine the efficacy of replacement of the atrioventricular (AV) valve for the systemic circulation in children with discordant AV connections undergoing functional biventricular repair. METHODS: Nine children underwent replacement of the morphologically tricuspid valve at the age of 10 months to 15 years. Ventriculoarterial connections were discordant in five children, and double outlet right ventricle with pulmonary stenosis or atresia in four. In all children the prosthesis chosen was a mechanical valve; valve sizes ranged from 19 mm to 31 mm. RESULTS: One patient died of ventricular failure immediately after surgery. Two patients underwent reoperation for re-replacement at eight and 68 months after the initial replacement because of non-structural dysfunction. Complete AV block occurred after intracardiac maneuvers in the non-survivor. Transient AV dissociation was noted in another patient. General conditions improved greatly after surgery in all survivors. In the morphologically right ventricle placed for the systemic circulation the end-diastolic volume fell from 327 +/- 182% (range: 109-621%) to 169 +/- 97% (range: 85-352%) of the anticipated normal value (p = 0.03), while pressure fell from 13 +/- 4 (range: 7-19) mmHg to 8 +/- 3 (range: 2-12) mmHg (p = 0.005). The ejection fraction was only marginally reduced (47 +/- 13% (range: 26-62%) preoperatively versus 34 +/- 11% (range: 20-54%) postoperatively; p = 0.13). CONCLUSIONS: In children with discordant atrioventricular connections and severe regurgitation across the morphologically tricuspid valve, the valve can be efficiently replaced for the systemic circulation.

Adolescent↗
PubMed1998

New preparation method of intestinal pressure-controlled colon delivery capsules by coating machine and evaluation in beagle dogs.

A new method for preparing large amounts of pressure-controlled colon delivery capsules (PCDCs) which employs a pharmaceutical coating machine, Hicoater-mini, has been developed. In contrast to our original method for preparing PCDCs where the inner surfaces of gelatin capsule were coated with the water-insoluble polymer ethylcellulose (EC), PCDC were directly prepared by coating the capsular shaped suppositories with EC. As a model drug, fluorescein (FL) was used in this study. FL powder was suspended with the suppository base, polyethylene glycol (PEG) 1000, at 50 degreesC, and was hardened in the capsular shape the sizes of which were #0 and #2. The capsular shaped suppositories were coated with 5% w/v ethanolic EC (7G grade) solution by a coating machine. By increasing the coating time from 55 to 75 min, the mean coating thickness of #0 PCDCs increased from 141+/-7 to 211+/-4 micrometer. In the case of #2 PDDCs, the mean coating thickness increased from 102+/-3 to 110+/-5 micrometer by increasing the coating time from 35 min to 40 min. Several kinds of #0 PCDCs having the mean EC coating membrane thickness of 141+/-7 micrometer (type 1), 166+/-4 micrometer (type 2), 188+/-4 micrometer (type 3), 211+/-4 micrometer (type 4) as well as #2 PCDCs having thickness of 102+/-3 micrometer (type 5) and 110+/-5 micrometer (type 6) were used for in vivo evaluation using beagle dogs. After oral administration of the test preparations containing 30 mg of FL, blood samples were obtained from the jugular vein and plasma FL levels were measured. The first appearance time, Ti, of FL in the plasma was used as a parameter for the estimation of the release time of FL from PCDCs in the gastrointestinal tract. The mean Ti of #0 PCDCs were 2.3+/-0.5 for type 1, 3.3+/-0.5 for type 2, 4.8+/-1.0 for type 3 and 7.8+/-1.7 h for type 4 preparations while the mean Ti of #2 PCDCs were 3.2+/-0.4 for type 5 and 3.8+/-0.4 h for type 6, respectively. There were good correlations between EC coatings.

Animals↗
PubMed1998

Different adhesive characteristics and VLA-4 expression of CD34(+) progenitors in G0/G1 versus S+G2/M phases of the cell cycle.

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.

Adult↗
PubMed1998

Induction of MHC-IIDR expression on circulating CD8+ lymphocytes in macaques infected with SIVmac239 nef-open but not with its nef-deletion mutant.

We examined the expression kinetics of activation antigens CD38 and MHC-IIDR (DR) on circulating CD8+ lymphocytes in rhesus macaques infected with pathogenic simian immunodeficiency virus strain SIVmac239 nef-open (239) or its nonpathogenic nef-deletion mutant (delta nef). In the longitudinal study, we found for the first time the induction of DR expression on CD8+ lymphocytes in 239-infected macaques. The induction of DR was in parallel with an increasing viral load and a decreasing CD4+ lymphocyte level. In the macaques with the high viral load and low CD4 level, a considerable proportion of the DR+CD8+ subpopulation was CD69+, indicating an activated state. On the other hand, no significant increase in the DR+CD8+ subpopulation level was observed in delta nef-infected macaques. These data indicate that the evaluation of activation markers such as DR and/or CD69 on circulating CD8+ cells may be valuable as a surrogate marker in the SIV-macaque model.

ADP-ribosyl Cyclase↗
PubMed1998

Selective expression of beta 7 integrin on lymphocytes undergoing apoptosis in lymphoid tissues.

It has been previously shown that the beta 7 chain of integrin forms heterodimers with the alpha 4 or alpha E chain, which plays essential roles in lymphocyte homing to mucosal lymphoid tissues. The aim of this study was to re-evaluate the possible role of the beta 7 integrin other than lymphocyte homing. We prepared spleen and lymph node lymphocytes from biopsied specimens from macaque monkeys and examined for the reactivity with a monoclonal antibody specific for the beta 7 chain. As a result, a minor population of the lymphocytes with a smaller size, which were in the early stage of apoptosis, was found to express a higher level of the beta 7 integrin than a majority of the lymphocytes with a normal size. Interestingly, the apoptotic lymphocytes expressed neither alpha 4 nor alpha E chains, suggesting that the beta 7 chain on these cells may be associated with an undefined alpha chain. These findings indicate that in the lymphoid tissues the shrunken lymphocytes undergoing apoptosis selectively express a unique beta 7 integrin.

Animals↗
PubMed1998

Evaluation of intestinal pressure-controlled colon delivery capsule containing caffeine as a model drug in human volunteers.

The delivery ability of a pressure-controlled colon delivery capsule (PCDC) containing caffeine as a test drug was evaluated after oral administration to healthy male human volunteers. The driving force causing PCDC disintegration in the intestinal tract is the physiological luminal pressure which results from peristalsis. Three kinds of PCDCs having different thickness of a water-insoluble polymer membrane was prepared by coating the inner surface of the gelatin capsules with ethylcellulose (EC). The mean thickness were 40 +/- 1 (SE) for type 1, 44 +/- 1 for type 2 and 50 +/- 1 micron for type 3 PCDC, respectively. Caffeine was dissolved with a suppository base (PEGs 400 and 1000) and the capsules were filled. Doses were 15, 45 or 75 mg. After blank saliva samples were obtained, test preparations were orally administered to the volunteers and saliva samples were collected for 1 min intervals hourly from 1 to 10 h in the fasted state study, and from 1 to 20 h and at 25 h in the fed state study. Caffeine concentrations in the saliva samples were analyzed by HPLC. The maximum salivary caffeine excretion rate increased as the oral caffeine dose increased. The maximum salivary caffeine excretion rate increased predominantly compared to the pre-dose level in 75 mg dose study. Therefore, all following studies were performed with this dose. The first appearance time of caffeine into the saliva, TI, was used as a parameter to estimate the disintegration time of test preparations in the gastrointestinal tract. The mean TI of types 1, 2, and 3 PCDCs were 3.0 +/- 0.4, 4.0 +/- 0.4 and 4.5 +/- 0.3 h, respectively. After oral administration of 75 mg caffeine in pain gelatin capsule as a reference preparation, caffeine appeared in the saliva within 0.5 h. The mean hardness of the PCDCs were 1.05 +/- 0.10 (type 1), 1.55 +/- 0.06 (type 2) and 2.08 +/- 0.15 newton (type 3), respectively. There were good correlations between three parameters: EC coating membrane thickness, hardness and TI (determination coefficient r2 = 0.935 between TI and thickness, r2 = 0.998 between thickness and hardness, r2 = 0.958 between hardness and TI). The effect of food intake on the delivery ability was examined with type 3 PCDCs. Food intake prolonged the mean TI, from 4.5 +/- 0.3 to 7.8 +/- 1.3 h. This increase is thought to be ascribed to prolonged gastric emptying time. Comparison with reported colon arrival times indicates that the type 3 PCDC functions in colon delivery of caffeine and is thought to be applicable to other drugs.

Adult↗
PubMed1998

Simian T cell leukemia virus type I-induced malignant adult T cell leukemia-like disease in a naturally infected African green monkey: implication of CD8+ T cell leukemia.

Spontaneous T cell leukemia was found in an African green monkey (Cercopithecus aethiops, AGM) naturally infected with simian T cell leukemia virus type I (STLV-I). The hematological features and the evidence for monoclonal integration of provirus DNA in the leukemic cells revealed that the leukemia was an ATL-like disease. The expression of surface markers on the leukemic cells indicated that they were defined as an activated CD8+ T cell subset. Together with the finding that seven in vitro spontaneously STLV-I-transformed cell lines were CD4-CD8+, it is likely that CD8+ T cells are transformed by STLV-I in AGMs, in contrast with human ATL. Finally, we assessed characteristics of the CD8 chains on these transformed cells. The result indicated that the leukemic cells expressed only the alpha chains but not the beta chains. However, in the case of in vitro-transformed cell lines the expression pattern of the CD8 chains varied in individual monkeys. Thus, STLV-I may preferentially transform CD8+ (both alphaalpha+ and alphabeta+) T cells in AGMs.

Adult↗
PubMed1998

Enhanced invasion of Tyzzer's organism into cultured mouse hepatocytes by cytochalasin D.

The effects of cytoskeleton inhibitors on the invasion of Tyzzer's organism, an obligate intracellular bacterium, into cultured mouse hepatocytes were examined by double immunofluorescence observation and plaque assay. The two techniques gave comparable results. Invasion of bacteria was significantly enhanced by cytochalasin D, a microfilament disrupting drug, and markedly suppressed by vinblastine, a microtubule inhibitor. Another microtubule inhibitor, colchicine, did not show any substantial effect. However, the cytoskeletal system of cultured mouse hepatocytes was sensitive to these three drugs, as judged by inhibition of FITC-dextran uptake. These results suggest that Tyzzer's organisms invade host cells by a unique mechanism that is suppressed by the normal functions of host cell microfilaments.

Animals↗
PubMed1998

Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning.

Differential display (DD) has been used extensively to detect differentially expressed genes. However, the low reproducibility of displayed bands makes verification by Northern blot difficult and the technique is labor-intensive. This report describes a fluorescent DD with a ROX (carboxy-X-rhodamine)-labeled anchor primer and a revised RT-PCR, utilizing AMV reverse transcriptase, a more thermostable reverse transcriptase than Mu-MLV, and optimized concentrations of dNTPs and of MgCl2. Our technique yielded clear fingerprints with high reproducibility. Further, we have developed a method of rapid screening to select the cDNA fragments of interest in excised bands from a polyacrylamide gel without cloning. This method consists of electrophoresis with an agarose gel containing a base-specific DNA ligand to separate the equally sized fragments differing in base composition, and side-by-side comparison of the reamplified products from the experimental and control lane. Most of the cDNA fragments selected by this protocol provided readable sequences by direct sequencing and were confirmed to exhibit differential expression by Northern blot analysis or semiquantitative RT-PCR.

Base Sequence↗