Human rhinovirus VPg uridylylation AlphaScreen for high-throughput screening.

As an obligate step for picornaviruses to replicate their genome, the small viral peptide VPg must first be specifically conjugated with uridine nucleotides at a conserved tyrosine hydroxyl group. The resulting VPg-pUpU serves as the primer for genome replication. The uridylylation reaction requires the coordinated activity of many components, including the viral polymerase, a conserved internal RNA stem loop structure, and additional viral proteins. Formation of this complex and the resulting conjugation reaction catalyzed by the polymerase, offers a number of biochemical targets for inhibition of an essential process in the viral life cycle. Therefore, an assay recapitulating uridylylation would provide multiple opportunities for discovering potential antiviral agents. Our goal was to identify inhibitors of human rhinovirus (HRV) VPg uridylylation, which might ultimately be useful to reduce or prevent HRV-induced lower airway immunologic inflammatory responses, a major cause of asthma and chronic obstructive pulmonary disease exacerbations. We have reconstituted the complex uridylylation reaction in an AlphaScreen suitable for high-throughput screening, in which a rabbit polyclonal antiserum specific for uridylylated VPg serves as a key reagent. Assay results were validated by quantitative mass spectrometric detection of uridylylation.

Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(Phenylthio)acyclouridine, a uridine phosphorylase inhibitor.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in reducing 5-fluorouracil (FUra) host-toxicity and enhancing its chemotherapeutic efficacy against human colon tumors. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. METHODS: SCID mice bearing human colon DLD-1 or HCT-15 tumors were injected intraperitoneally with FUra (50, 200 or 300 mg/kg) on days 17, 24 and 31 after tumor cell inoculation. PTAU (120 mg/kg), uridine (1,320 mg/kg) or their combination was administered orally 2 or 4 h after FUra injection. Another four administrations of PTAU+uridine were given every 8 h after the first treatment with PTAU plus uridine. Survival and body weight were used to evaluate host toxicity. Tumor weight was used to evaluate the efficacy of the drugs on tumor growth. The mice were monitored for 38 days. RESULTS: Administration of the maximum tolerated dose (50 mg/kg) of FUra reduced DLD-1 and HCT-15 tumor weights by 48 and 59%, respectively, at day 38 post implantation. Administration of 200 mg/kg FUra resulted in 100% mortality. Oral administration of uridine (1,320 mg/kg) alone, 2 h following the administration of 200 mg/kg FUra, did not alleviate FUra host-toxicity as all the mice died. Administration of 120 mg/kg PTAUresulted in partial rescue from this lethal dose of FUra as 63% of mice survived and tumor weights were reduced by approximately 60%. Coadministration of PTAU plus uridine resulted in complete rescue from the toxicity of FUra as 100% of the mice survived and tumor weights were reduced by 81-82%. Delaying the administration of the combination of PTAU plus uridine to 4 h post FUra treatment was less effective in rescuing from FUra toxicity as only 88% of the mice survived and tumor weights were reduced by only 62%. Administration of PTAU alone, under the same conditions, resulted in a 38% survival rate while the tumor weights were reduced by 47%. Treatment with uridine alone did not protect from FUra toxicity at the dose of 200 mg/kg as all mice died. At the higher dose of 300 mg/kg FUra, neither uridine nor PTAU alone, administered 2 h following the treatment with FUra, had any rescuing effect. On the other hand, the use of the PTAU plus uridine combination reduced the tumor weight by 79%, although this reduction in the tumor weight was accompanied by 37% mortality. There was no significant difference between DLD-1 and HCT-15 in their response to the different regimens employed in this study despite the fact that the tumors have different levels of UrdPase. CONCLUSIONS: The present results demonstrate that the combination of PTAU plus uridine represents an exceptionally efficient method in increasing FUra chemotherapeutic efficacy while minimizing its host-toxicity. The efficiency of the PTAU plus uridine combination can be attributed to the extraordinary effectiveness of this combinationin raising and maintaining higher levels of uridine in vivo (Al Safarjalani et al., Cancer Chemo Pharmacol 55:541-551, 2005). Therefore, the combination of PTAU plus uridine can provide a better substitute for the large doses of uridine necessary to rescue or protect from FUra host-toxicities, without the toxic side-effects associated with such doses of uridine. This combination may also allow for the escalation of FUra doses for better chemotherapeutic efficacy against human colon carcinoma while avoiding FUra host-toxicities. Alternatively, the combination of PTAU and uridine may be useful as an antidote in the few cases when cancer patients receive a lethal overdose of FUra.

Fine tuning of rabbit equilibrative nucleoside transporter activity by an alternatively spliced variant.

The full-length cDNA encoding an equilibrative nucleoside transporter (rbENT2) and its novel C-terminal variant, rbENT2A, were isolated from rabbit trachea. Rabbit ENT2 protein consists of 456 amino acid residues; rbENT2A is shorter by 41 residues. Both rbENT2 and rbENT2A transcripts are found in rabbit tissues including intestine, kidney cortex, kidney, and trachea, at varying levels of expression. When transfected in a heterologous expression system-Madin Darby canine kidney (MDCK) epithelial cell line-both rbENT2 and rbENT2A were expressed. rbENT2 had a molecular mass of 49 kDa; rbENT2A had a molecular mass of 44 kDa. Clones of both transporters yielded functional proteins that were capable of mediating uridine uptake and efflux without the needing to be coupled to a secondary ion (e.g. Na(+)). Remarkably, rbENT2A displayed a higher affinity (K(m) = 41 microM) and a lower capacity (V(max) = 0.6 nmol/mg protein/5 min) towards substrates than rbENT2 (K(m) = 272.8 microM, V(max) = 1.26 nmol/mg protein/5 min). Pharmacological profiles showed that nitro-benzyl-mercapto-purine-ribose (NBMPR) potently inhibited (3)H-uridine uptake mediated by rbENT2A, but not uptake mediated by rbENT2. The constitutive splicing, broad expression, markedly different kinetics, and distinct pharmacological characteristics of rbENT2A appear to act in conjunction with the wild type, rbENT2, to fine-tune basolateral nucleoside transport function in rabbit trachea.

Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans.

BACKGROUND: Adenosine receptor stimulation induces several effects that could limit ischemia-reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia-reperfusion injury in humans. METHODS: Ischemia-reperfusion injury was studied in forearm skeletal muscle by technetium Tc 99m-labeled annexin A5 scintigraphy. Ischemia-reperfusion injury was induced by unilateral forearm ischemic exercise. Immediately on reperfusion, annexin A5 labeled with technetium Tc 99m was administered intravenously, and ischemia-reperfusion injury was expressed as the percentage difference in radioactivity between the experimental arm and the control arm 1 and 4 hours after reperfusion. Targeting was quantified in the region of the thenar muscle and forearm flexor muscles. This approach was used in 9 healthy male volunteers after a 1-week treatment with dipyridamole (200 mg, slow release, twice daily) and in 23 control subjects. RESULTS: Dipyridamole treatment significantly reduced annexin A5 targeting in skeletal muscle compared with the control group (thenar region, 13% +/- 7% versus 22% +/- 15% at 1 hour after reperfusion and 9% +/- 6% versus 27% +/- 13% at 4 hours for dipyridamole and control groups, respectively [P = .01]; flexor region, 4% +/- 8% versus 7% +/- 6% at 1 hour after reperfusion and 1% +/- 4% versus 10% +/- 9% at 4 hours for dipyridamole and control groups, respectively [P = .01]). CONCLUSIONS: One week of oral treatment with the nucleoside uptake inhibitor dipyridamole (200 mg, slow release, twice daily) significantly limits ischemia-reperfusion injury in humans in vivo, as assessed by technetium Tc 99m-labeled annexin A5 scintigraphy of forearm skeletal muscle.

Inhibition of nucleoside transport by protein kinase inhibitors.

Recently we reported that the pyridinylimidazole class of p38 mitogen-activated protein (MAP) kinase inhibitors potently inhibited the facilitated transport of nucleosides and nucleoside analogs in K562 cells. These compounds competed with the binding of nitrobenzylthioinosine (NBMPR) to K562 cells, consistent with inhibition of the NBMPR-sensitive equilibrative transporter (ENT1). In this study we examined a large number of additional protein kinase inhibitors for their effects on nucleoside transport. We find that incubation of K562 cells with tyrosine kinase inhibitors (AG825, AG1517, AG1478, STI-571), protein kinase C (PKC) inhibitors (staurosporine, GF 109203X, R0 31-8220, arcyriarubin A), cyclin-dependent kinase inhibitors (roscovitine, olomoucine, indirubin-3'-monoxime), or rapamycin resulted in a dose-dependent reduction of intracellular uptake of [3H]uridine. In contrast, neither the MAP kinase kinase inhibitors (U0126, PD 98059) nor the phosphatidyl inositol-3 kinase inhibitors (wortmannin, LY 294002) affected this process. Furthermore, both transient uptake and prolonged [3H]thymidine incorporation in K562 cells were inhibited by protein kinase inhibitors, inactive analogs of kinase inhibitors (R0 31-6045, SB202474), and NBMPR, independently of effects on cell proliferation as determined by MTT assay. These studies demonstrate that a wide variety of protein kinase inhibitors affect nucleoside uptake through selective inhibition of nucleoside transporters, independently of kinase inhibition.

Uridine uptake inhibition assay: an automated micromethod for the screening of cytotoxicity.

The uridine uptake inhibition assay is a sensitive microassay for measuring cytotoxicity. This assay is normally performed with Hela S3 cells, which lack metabolic activity. In an earlier study, we adapted the test to HepG2 cells, a human hepatoma cell line that retains many hepatocyte characteristics, such as functional metabolic enzymes. This study describes a new automated protocol for the assay that makes it much more rapid. In the previous protocol, after the cells were treated with the test compounds and allowed to take up uridine for 30 min, samples were taken manually one by one and spotted onto 3MM Whatman paper. After drying, the paper sheet was then chromatographed in 5% (P/V) TCA for 2 h in order to precipitate and measure the total amount of RNA. In the new method, instead of paper chromatography, samples are transferred onto a 96-well microplate equipped with GF/C glass filters. Then, RNA precipitation by TCA is carried out with a manifold system, and the amount of radiolabeled uridine taken up by the cells is counted directly with a radioactivity microplate reader. This method makes it possible to screen many compounds simultaneously for cytotoxicity. To evaluate its sensitivity, we compared the IC(50) values obtained with new and original protocol for each eight toxic compounds. We found an excellent correlation between the two methods (r(2)=0.99). With the automated protocol, the uridine uptake inhibition assay is both sensitive and rapid enough for high-throughput daily screening.

Transient expression of a purine-selective nucleoside transporter (SPNTint) in a human cell line (HeLa).

PURPOSE: The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na(+)-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of nucleosides and nucleoside analogs with this transporter. METHODS: Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using [3H]inosine, [3H]uridine, [3H]-dideoxyinosine (ddI), and [3H]-2-chloro-2'-deoxyadenosine (2CdA) as model substrates. RESULTS: Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na(+)-stimulated uptake of [3H]inosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na(+)-stimulated uptake of both inosine and uridine was saturable (K(m) = 28.1 +/- 7.1 microM and 20.6 +/- 5.6 microM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddI (IC50 = 46 microM) and 2CdA (IC50 = 13 microM) also significantly inhibited the Na(+)-stimulated uptake of [3H]inosine. A Na(+)-stimulated uptake of [3H]2CdA was observed suggesting that 2CdA is also a permeant of SPNTint. CONCLUSIONS: HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.

Characterization of nucleoside transport activity in rabbit cortical synaptosomes.

Rabbit central nervous system (CNS) preparations have been used to study the central effects of adenosine, but little is known about the specific uptake mechanisms in rabbit brain involved in the regulation of extracellular adenosine concentrations. The present study assessed the kinetic and pharmacological characteristics of the uptake of [3H]uridine (a poorly metabolized substrate for adenosine transporters) by rabbit cortical synaptosomes, to define the transporter subtypes involved and to evaluate species variability in transporter characteristics. [3H]Uridine transport into rabbit cortical synaptosomes was mediated by two saturable, facilitated diffusion systems with characteristics compatible with the es and ei transporter subtypes identified in other mammalian species. About 65% of the total transport was mediated by the es system, and Km estimates of 320 and 94 microM were determined for [3H]uridine uptake by the es and ei transporter, respectively. These results differ significantly from the subtype ratio and kinetic characteristics reported for rat and guinea pig cortical synaptosomes, where most of the transport was mediated by an ei subtype. Dipyridamole, dilazep, nitrobenzylthioinosine, R75231, soluflazine, and mioflazine were relatively more effective as inhibitors of es-mediated uptake (compared with ei), while the substrates adenosine, cytidine, and guanosine did not distinguish between the es and ei transporters in rabbit cortical synaptosomes. These results highlight the significant species-tissue variability in nucleoside transporter characteristics and subtype expression, and emphasize the need to characterize the transporters in human CNS tissue to allow the rational development of CNS-active therapeutics based on inhibition of nucleoside transport.

Uridine transport in basolateral plasma membrane vesicles from rat liver.

The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurements conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were KM 8.9 microM and Vmax 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rats and, thirdly, the permeant nature of the Na+ counterions determined uridine, transport rates (SCN- greater than NO3- greater than Cl- greater than SO4(2-)). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.

Facilitated diffusion and sodium-dependent transport of purine and pyrimidine nucleosides in rat liver.

In mammalian cells, nucleoside transport usually is mediated by facilitated diffusion. In addition, a Na(+)-dependent, concentrative nucleoside transport system has been detected in several tissues but not the liver. To further clarify hepatic nucleoside transport mechanisms, we measured the uptake of [2-14C]uridine (2 to 100 mumol/L) and of [8-14C]adenosine (10 to 75 mumol/L) by the isolated perfused rat liver in the presence or absence of extracellular sodium or specific inhibitors of facilitated nucleoside diffusion. Uridine transport and metabolism were monitored by the release of labeled catabolites including 14CO2, which indicated complete degradation of the pyrimidine. Adenosine, uridine and uridine catabolites were measured in the effluent perfusate by reversed-phase high-performance liquid chromatography and a radioactivity flow monitor. The existence of a Na(+)-dependent nucleoside transport system could be inferred from the following observations: (a) Sodium depletion caused a strong inhibition of nucleoside transport reflected by an up to threefold and 15-fold increase in extracellular uridine and adenosine, respectively. The sodium-dependent transport of uridine was saturated when the influent uridine concentration was raised beyond 20 mumol/L. No such saturation was observed for much higher concentrations of adenosine used (10 to 75 mumol/L). (b) Na(+)-free perfusion resulted in a strong suppression of the release of uridine catabolites by the liver. Complete uridine breakdown was depressed to 7% of the amount of 14CO2 released in the presence of sodium and at influent uridine concentrations below 20 mumol/L. (c) Inhibition of uridine (10 mumol/L) transport and degradation was observed after coperfusion with adenosine, deoxyadenosine, guanosine and deoxyguanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin.

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.

Transport characteristics of renal brush border Na(+)- and K(+)-dependent uridine carriers.

The uptake of uridine into rat renal brush-border membrane vesicles is mediated by Na(+)- and K(+)-dependent concentrative transport processes. At a 100 mM extravesicular cation concentration the apparent Km values were 9.7 +/- 4.2 and 28 +/- 5 microM, and Vmax values were 28 +/- 4 and 7 +/- 1 pmol.mg protein-1.s-1 for the Na(+)- and K(+)-dependent systems, respectively. Uracil, D-ribose, and D-glucose failed to inhibit the uptake processes, indicating that these carriers are specific for nucleosides. Other purines and pyrimidines inhibited uridine uptake competitively, although these two transport systems seem to favor adenosine and pyrimidines as permeants. Evidence is also given that transport is rheogenic, involving a net transfer of positive charge. The Na+:uridine and K+:uridine coupling stoichiometry was found to be 1:1 and 3:2, respectively. Both systems can also be driven by an anion gradient with apparent NO3- affinity (KNO3-) values of 42 +/- 13 and 163 +/- 54 mM for Na(+)- and K(+)-dependent systems, respectively.

Pyridoxal phosphate annuls acetaldehyde inhibition of uridine uptake by hepatocytes.

Methods were evaluated for depressing the acetaldehyde (AcH) inhibition of uridine uptake by Chang liver cells and isolated autologous liver cells, obtained by percutaneous liver biopsy from cases of alcoholic hepatitis. Hepatocytes so obtained were significantly more susceptible to AcH-induced inhibition of uridine uptake than hepatocytes from normal liver, alcoholic fatty liver, stable alcoholic cirrhosis and acute viral hepatitis. Benzylamine (an aldehyde buffer) and pyridoxal-5' phosphate (PLP) counteracted the inhibition of uridine uptake by AcH in vitro. We suggest that benzylamine neutralizes AcH toxicity through a Schiff-base condensation with AcH thus taking AcH out of the field of action. PLP protects against AcH-induced inhibition of uridine uptake by probably forming a Schiff base with cellular amino acids thus blocking further condensation of these amino groups with AcH. In this manner, uridine uptake by liver cells for RNA synthesis can proceed without interference by AcH.

RNA polymerase II transcripts as targets for 5-fluorouridine cytotoxicity: antagonism of 5-fluorouridine actions by alpha-amanitin.

The cytotoxicity of 5-fluorouridine (FUrd) results from actions directed at the synthesis of both DNA and RNA. The role of mRNA as a target for FUrd was investigated by selectively decreasing the incorporation of FUrd into RNA polymerase II transcripts of K-562 erythroleukemia cells, which was accomplished by the addition of alpha-amanitin to cultures of K-562 cells permeabilized with lysolecithin. In these cells alpha-amanitin at concentrations of 1-5 micrograms/ml inhibited the incorporation of [3H]-uridine into polyadenylated RNA by up to 45% and decreased the steady-state levels of two specific mRNAs but had no effect on poly A- RNA synthesis. alpha-Amanitin decreased the incorporation of FUrd into poly A+ RNA by up to 60%. The decrease in FUrd incorporation produced by alpha-amanitin was accompanied by an antagonism of the growth inhibitory effects of the fluorinated pyrimidine nucleoside by the mycotoxin, as measured by both growth in suspension culture and colony formation in 0.12% agar. Antagonism between these agents increased as the concentration of alpha-amanitin was elevated; furthermore, it was sequence-dependent, occurring only when alpha-amanitin preceded FUrd. These findings provide evidence that the actions of FUrd directed against mRNA are antagonized when FUrd incorporation into mRNA transcripts is decreased and that the effects of FUrd on mRNA produce cytotoxic consequences.

Isolation and characterization of a mutant of L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nucleoside transport.

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.

Mechanism of the inhibitory effect of 11ß hydroxypregna 1,4 diene 3,20 dione (delta HOP) at high concentrations in RNA synthesis / 303-12

La potente inhibición de la síntesis de ARN en timocitos de rata por la 11ß -hidroxipregna-1,4-dien-3,20-diona (DeltaHOP) demostrado recientemente, cumple las tres condiciones requeridas para un efecto no-genómico: no perdurabilidad del efecto después de ser retirado el esteroide por lavado, acción instantánea y efecto en presencia de inhibidores de la síntesis de ARN. La inyección intraperitoneal de DeltaHOP en ratones (2 mg/100 gm) provoca un 32% de inhibición de la síntesis de ARN en los timocitos; queda por aclarar si esta inhibición es debida también a un efecto no-genómico

Mechanism of the inhibitory effect of 11ß hydroxypregna 1,4 diene 3,20 dione (delta HOP) at high concentrations in RNA synthesis / 303-12

La potente inhibición de la síntesis de ARN en timocitos de rata por la 11ß -hidroxipregna-1,4-dien-3,20-diona (DeltaHOP) demostrado recientemente, cumple las tres condiciones requeridas para un efecto no-genómico: no perdurabilidad del efecto después de ser retirado el esteroide por lavado, acción instantánea y efecto en presencia de inhibidores de la síntesis de ARN. La inyección intraperitoneal de DeltaHOP en ratones (2 mg/100 gm) provoca un 32% de inhibición de la síntesis de ARN en los timocitos; queda por aclarar si esta inhibición es debida también a un efecto no-genómico (AU)

[Clinical significance of the short-term incubation test for the therapy of metastatic breast cancer]. / Klinische Bedeutung des Kurzzeitinkubationstests für die Therapie des metastasierten Mammakarzinoms.

Between January 1978 and October 1980 97 tissue samples of histologically verified carcinoma of the breast were received for performance of the short-term incubation test. 25 tumour samples could not be prepared as cell suspension sufficient for testing. Among the 72 performed tests there were 9 with stimulation of 3H-uridine uptake by doxorubicin. Thus only 63 tests could be evaluated. Results were correlated with clinical data of the patients. No significant correlations could be established between tumour stage at time of diagnosis, age, menopausal state, receptor state, and the test result. There was also no differentiation between favourable and unfavourable prognoses as regards free interval and rate of survival. A correlation between results of tests and success or failure of cytostatic treatment could not be ascertained.

Vasoconstrictor effects of uridine and its nucleotides and their inhibition by adenosine.

Uridine, uridine monophosphate (UMP) and uridine diphosphate (UDP) increased blood pressure when infused into intact anaesthetized rats and had similar effects on the perfusion pressure in the rat isolated perfused kidney. In an isolated vascular preparation, the everted rat portal vein, uridine was without effect while UMP and UDP caused log dose-related increases in contractile work. Adenosine infused at a dose of 200 nmol/kg per min blocked the response to uridine in the intact rat, converting it to a depressor response at higher doses, and reduced the response to UMP. Uridine may need to be phosphorylated to UMP to act on blood vessels. The two compounds are effective at similar dose ranges and suppress renin secretion in the isolated kidney, while UDP, which is effective at lower doses and stimulates renin secretion, may act by a different mechanism. Adenosine competes for membrane transport with uridine but its inhibition of the effects of UMP is consistent with activity at intracellular sites as well.