Patch clamp studies on V-type ATPase of vacuolar membrane of haploid Saccharomyces cerevisiae. Preparation and utilization of a giant cell containing a giant vacuole.
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A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole. Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems. (+info)
Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect.
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BACKGROUND: The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine. We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma. METHODS: We introduced a plasmid vector containing the E. coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line. Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells. RESULTS: Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001). Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals. Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001). In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect. CONCLUSIONS AND IMPLICATIONS: Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma. (+info)
Thiopurine methyltransferase activity and its relationship to the occurrence of rejection episodes in paediatric renal transplant recipients treated with azathioprine.
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AIMS: Azathioprine is a prodrug commonly used in combination therapy to prevent allograft rejection after renal transplantation. After conversion to 6-mercaptopurine, the drug is metabolized into 6-thioguanine nucleotides (6-TGN) and catabolized by thiopurine methyltransferase (TPMT), an enzyme under monogenic control. The aim of this study was to evaluate the inter- and intraindividual variability of red blood cell thiopurine methyltransferase and 6-TGN concentrations and their relationship to the clinical effects of azathioprine in paediatric patients. METHODS: In the present study, the inter- and intraindividual variations in red blood cell TPMT activity and 6-TGN concentrations and their relationship to the actions of azathioprine were evaluated during the first year after renal transplantation in 22 paediatric patients. RESULTS: 6-TGN concentration reached steady-state values after 6 months and correlated negatively with TPMT activity (P=0.004). Initial TPMT activity (median: 20.8 nmol h-1 ml-1, range 7.8-34.6) and 6-TGN concentration at steady-state (median: 80 pmol 8 x 10(8-1) cells, range not detected to 366) were not related to the occurrence of rejection episodes during the period of the study. In contrast, TPMT activity and the percentage difference in TPMT activity from the day of transplantation determined at month 1 were higher in the patients with rejection episodes by comparison with those that did not reject during the first 3 months or the first year following transplantation (P<0.005). CONCLUSIONS: We report a relationship between TPMT activity and occurrence of rejection in paediatric kidney transplant patients undergoing azathioprine therapy. These data suggest a link between high red blood cell TPMT activity and poor clinical outcome probably caused by rapid azathioprine catabolism. (+info)
Effects of angiotensin II on remodelling of the airway and the vasculature in the rat.
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Airway remodelling occurs in chronic asthma. Angiotensin II promotes growth in cardiovascular remodelling. Since the renin-angiotensin system is activated in acute severe asthma, we hypothesized that angiotensin II has a role in airway remodelling. A total of 14 young male Wistar rats were randomly divided into two groups. All received 2-week infusions of bromodeoxyuridine, and the experimental group also received angiotensin II. Blood pressure rose in the angiotensin II-infused group [mean levels: pre-infusion, 134.9 (S.D. 14.7) mmHg; post-infusion, 197.1 (22.5) mmHg], and expression of renin mRNA in the renal juxtaglomerular cells was suppressed in these animals. The proportion of bromodeoxyuridine-positive cell nuclei was no different in the airways of control and angiotensin II-infused animals for smooth muscle [mean bromodeoxyuridine index: control, 8. 6% (S.E.M. 1.1%); angiotensin II, 9.3% (1.1%)], epithelium [control, 16.7% (2.3%); angiotensin II, 16.0% (2.2%)] and adventitia [control, 26.4% (2.2%); angiotensin II, 26.6% (2.4%)]. In the arteries, bromodeoxyuridine indices were higher in the angiotensin II-infused rats [18.4% (2.3%)] than in the control animals [9.4% (2.8%)], but no difference was found in the veins [12% (2.9%) and 11.4% (2.6%) respectively]. Morphometry of the airway wall and mesenteric vasculature was no different in the two groups. Therefore a 2-week infusion of angiotensin II increases blood pressure and DNA synthesis in the mesenteric arteries, but does not cause airway remodelling, in the rat. (+info)
Contraction-stimulated muscle glucose transport and GLUT-4 surface content are dependent on glycogen content.
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The influence of muscle glycogen content on basal and contraction-induced glucose transport and cell surface GLUT-4 content was studied in rat skeletal muscle. Wistar rats were preconditioned by a combination of swimming exercise and diet, resulting in 40% lower (LG) or threefold higher (HG) muscle glycogen content compared with nonexercised controls (NG). At rest and during contractions, 2-deoxy-D-glucose uptake in perfused fast-twitch muscle, but not slow-twitch muscle, was significantly lower in HG compared with LG. Cell surface GLUT-4 content in the fast-twitch plantaris was 994 +/- 180, 1,173 +/- 311, and 2,155 +/- 243 dpm/g in the basal condition and increased (P < 0.05) to 2,285 +/- 239, 3,230 +/- 464, and 4,847 +/- 654 dpm/g during contractions with HG, NG, and LG, respectively, the increase being significantly smaller in HG compared with LG. The contraction-induced increments in glucose transport and in cell surface GLUT-4 content were negatively correlated with the initial glycogen content (P <0.01). In conclusion, glucose transport and cell surface GLUT-4 content in resting and contracting fast-twitch muscle are dependent on the muscle glycogen content. (+info)
Sponge delivery variables and tissue levels of 5-fluorouracil.
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AIM: To study how the delivery of 5-fluorouracil (5-FU) to ocular tissues is affected by altering delivery variables. METHOD: Sponge(s) soaked in radiolabelled 5-FU were placed between the conjunctiva and sclera of pig eyes. Application time, sponge size, sponge make (Altomed, Weck, Merocel), and 5-FU concentration were varied. Conjunctival and scleral tissue levels were determined in samples taken from the application site. RESULTS: Dose-response curves for scleral and conjunctival 5-FU levels against application time showed increasing tissue levels that reached a plateau after 2-3 minutes. Application beyond 3 minutes did not increase tissue levels. There was no difference in tissue levels between 7x4 and 3. 5x2 mm sponges. Altomed sponges produced 5-FU tissue levels that were twice as high as those obtained with Weck-cell (p<0.01) or Merocel (p<0.02) sponges. Changing the 5-FU concentration from 25 mg/ml to 6.25 mg/ml reduced the conjunctival concentration by a factor of 3.5 (p<0.003). CONCLUSION: Application time up to 3 minutes, sponge make, and 5-FU concentration can have a large effect on the tissue delivery of 5-FU. Application time beyond 3 minutes, using 3.5x2 mm or 7x4 mm sponges, and replacing sponges every minute did not have a significant effect on tissue levels. This study models the effect that different variables can have on the ocular tissue levels of an antimetabolite applied intraoperatively. (+info)
The nuclear orphan receptor COUP-TFI is required for differentiation of subplate neurons and guidance of thalamocortical axons.
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Chicken ovalbumin upstream promotor-transcription factor I (COUP-TFI), an orphan member of the nuclear receptor superfamily, is highly expressed in the developing nervous systems. In the cerebral cortex of Coup-tfl mutants, cortical layer IV was absent due to excessive cell death, a consequence of the failure of thalamocortical projections. Moreover, subplate neurons underwent improper differentiation and premature cell death during corticogenesis. Our results indicate that the subplate neuron defects lead to the failure of guidance and innervation of thalamocortical projections. Thus, our findings demonstrate a critical role of the subplate in early corticothalamic connectivity and confirm the importance of afferent innervation for the survival of layer IV neurons. These results also substantiate COUP-TFI as an important regulator of neuronal development and differentiation. (+info)
Asymmetric growth and development of the Xenopus laevis retina during metamorphosis is controlled by type III deiodinase.
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During the metamorphosis of the Xenopus laevis retina, thyroid hormone (TH) preferentially induces ventral ciliary marginal zone (CMZ) cells to both increase their proliferation and give rise to ipsilaterally projecting ganglion cells. Here we show that dorsal CMZ cells express type III deiodinase (D3), an enzyme that inactivates TH. The dorsal CMZ cells can be induced to proliferate if deiodinase activity is inhibited. D3 or dominant-negative thyroid hormone receptor transgenes inhibit both TH-induced proliferation of the ventral CMZ cells and the formation of the ipsilateral projection. Thus, the localized expression of D3 in the dorsal CMZ cells accounts for the asymmetric growth of the frog retina. (+info)