The critical role of alpha‐folate receptor in the resistance of melanoma to methotrexate

Although methotrexate (MTX) is an effective drug for several types of cancer, it is not active against melanoma. Experiments following methotrexate treatment indicated a reduced accumulation of the drug in the cytosolic compartment in melanoma cells, suggesting that the mechanisms that control the transport and retention of this drug could be altered in melanoma. For this reason, we analyzed the presence and function of folate receptor‐α (FRα) in melanoma cells. In this study, we have identified the presence of FRα in normal and pathological melanocytes and demonstrated that MTX is preferentially transported through this receptor in melanoma cells. FRα‐induced endocytic transport of MTX, together with drug melanosomal sequestration and cellular exportation, ensures reduced accumulation of this cytotoxic compound in intracellular compartments. The critical role of FRα in this mechanism of resistance and the therapeutic consequences of these findings are also discussed.     

Interactions among unit-price, fixed-ratio value, and dosing regimen in determining effects of repeated cocaine administration

Previous research has shown tolerance to cocaine that was dependent on fixed-ratio (FR) parameter size in the context of a multiple FR schedule of food reinforcement. Completion of the FR requirement in these studies resulted in the same magnitude of reinforcement, regardless of ratio size. The cost-to-benefits ratio (unit-price) was therefore not equated across the different FR components. The current study examined the effects of repeated administration of cocaine to pigeons when unit-price under FR schedules was either the same or different. Additionally, the role of a chronic variable-dosing versus chronic fixed-dosing procedure was examined when unit-price was equated across different ratio values. Pigeons were trained to key peck in daily sessions under a three-component multiple FR schedule of food presentation, according to which either 10, 30, or 100 pecks were required for each delivery of food. In Experiment 1, completion of the FR 10, FR 30, and FR 100 resulted in 1.5, 4.5, and 15.0 s access to food, respectively. That is, the response requirement was correlated with access to food time so that unit-price (pecks per second of access to food) was equated across components. After assessing acute effects of a range of doses of cocaine, drug administration occurred daily before each session, with the dose varying from day to day. Tolerance, the magnitude of which was unrelated to the peck requirement, developed under the repeated-dosing regimen. In order to assess whether having equal unit-price was responsible for producing similar levels of tolerance across components, daily drug administration continued in Experiment 2 using the variable-dose regimen, but the amount of food presented each time was fixed at 4.5 s access to food, yielding different unit-prices under the three pecking requirements. Subsequently, the conditions of Experiment 1 were reinstated, i.e., unit-price was equated. Making unit-price different or the same had little influence on effects of cocaine. In Experiment 3, a fixed dose of cocaine was administered before each session while programmed unit-price remained the same across components. Under these conditions, tolerance became peck-requirement related. Specifically, tolerance was most prevalent under the smaller requirements and less robust or absent when the largest requirement was in effect. Differences in unit-price, therefore, were not related to degree of tolerance, but work requirement was. Differences in effects of cocaine across responses requirements, however, were observed only when each session was preceded by the same dose, not when dose varied from session to session.     

Identification of a selective ERK inhibitor and structural determination of the inhibitor-ERK2 complex

Selective inhibition of extracellular signal-regulated kinase (ERK) represents a potential approach for the treatment of cancer and other diseases; however, no selective inhibitors are currently available. Here, we describe an ERK-selective inhibitor, FR180204, and determine the structural basis of its selectivity. FR180204 inhibited the kinase activity of ERK1 and ERK2, with K(i) values 0.31 and 0.14microM, respectively. Lineweaver-Burk analysis of the binding interaction revealed that FR180204 acted as competitive inhibitor of ATP. In mink lung epithelial Mv1Lu cells, FR180204 inhibited TGFbeta-induced luciferase-expression. X-ray crystal structure analysis of the human ERK2/FR180204 complex revealed that Q105, D106, L156, and C166, which form the ATP-binding pocket on ERK, play important roles in the drug/protein interaction. These results suggest that FR180204 is an ERK-selective and cell-permeable inhibitor, and could be useful for elucidating the roles of ERK as well as for drug development.     

Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways

Background

Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii.

Methodology/Principal Findings

Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite.

Conclusions/Significance

Our findings provide an explanation for the observed differences in activity of fosmidomycin and {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.     

Synthesis and evaluation of folate-based chlorambucil delivery systems for tumor-targeted chemotherapy

The development of tumor-targeting drug delivery systems, able to selectively transport cytotoxic agents into the tumor site by exploiting subtle morphological and physiological differences between healthy and malignant cells, currently stands as one of the most attractive anticancer strategies used to overcome the selectivity problems of conventional chemotherapy. Owing to frequent overexpression of folate receptors (FRs) on the surface of malignant cells, conjugation of cytotoxic agents to folic acid (FA) via suitable linkers have demonstrated to enhance selective drug delivery to the tumor site. Herein, the chemical synthesis and biological evaluation of two novel folate-conjugates bearing the anticancer agent chlorambucil (CLB) tethered to either an aminoether (4,7,10-trioxa-1,13-tridecanediamine) or a pseudo-β-dipeptide (β-Ala-ED-β-Ala) linker is reported. The two drug delivery systems have been prepared in high overall yields (54% and 34%) through straightforward and versatile synthetic routes. Evaluation of cell specificity was examined using three leukemic cell lines, undifferentiated U937 (not overexpressing FRs, FR(-)), TPA-differentiated U937 (overexpressing FRs, FR(+)), and TK6 (FR(+)) cells. Both conjugates exhibited high specificity only to FR(+) cells (particularly TK6), demonstrating comparable antitumor activity to CLB in its free form. These data confirm the reliability of folate-based drug delivery systems for targeted antitumor therapy; likewise, they lay the foundations for the development of other folate-conjugates with antitumor potential.     

Behavioral economic analysis of water intake in a laboratory rhesus macaque

Behavioral economics is useful for understanding the influence of environmental manipulation on a variety of behaviors, including drug self-administration, food intake, and stock behavior. The present study employed behavioral economics to investigate the psychologically satisfying amount of water intake in a laboratory rhesus macaque. Our institutional guidelines set a minimum amount of daily water intake. However, no study to date has determined whether that minimum amount is psychologically sufficient. In the present experiment, a monkey lived in an individual cage in which the only water available was delivered by chain pulling. A fixed number of responses was required for water delivery. This fixed ratio (FR) of responses per water delivery was progressively increased from FR 2 to FR 10. The study findings showed that during the FR 2 condition, demand for water was saturated at 131.3 ml/kg body weight (BW) (ranging from 95.1 to 211.2). The monkeys daily intake of water decreased as FR size incrementally increased, approaching an asymptote under the FR 8 and FR 10 conditions. During the FR 8 and FR 10 conditions, responding ceased when this monkey earned 53.5 ml/kg-BW (ranging from 32.7 to 74.9) of water. Therefore, the amount of water obtained under these conditions might provide a psychologically satisfying amount. Although these values were obtained from the behavioral study of one monkey, they were almost equivalent to values in our institutional guidelines that were determined by veterinary observations. These findings imply that behavioral economics is useful for studying the welfare of laboratory animals.     

Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.     

Exploration of robustness indicators using adaptive responses to short-term feed restriction in suckling primiparous beef cows

Animal robustness is a complex trait of importance for livestock production systems and genetic selection. Phenotyping is essential for evaluation of the adaptation of different genotypes to changing environments. This study tested an experimental framework to induce marked deviations in the adaptive responses of suckling beef cows and to identify relevant indicators of responses to characterise individual differences in the robustness of cows. The production and metabolic responses of primiparous suckling Charolais cows to two periods of feed restriction (FR, 50% of their net energy requirements) of different durations were monitored. After calving, 13 cows (aged 39 ± 2 months, BW of 680 ± 42 kg at calving) had ad libitum access to a diet composed of hay and supplemented with concentrate to meet their energy and protein requirements. Starting at 54 ± 6 days postcalving, the cows underwent two periods of FR: 4 days of FR (FR4), which was followed by 17 days of ad libitum intake to study the recovery from FR4, and 10 days of FR (FR10), which was followed by 18 days of ad libitum intake to study the recovery from FR10. The milk yield (MY), BW, body condition score and plasma non-esterified fatty acid (NEFA), β-hydroxybutyrate, glucose and urea concentrations were measured before, during and after each FR. Among all measured variables, the MY and NEFA concentrations showed the most significant changes in response to FR. A functional data analysis approach was applied to the MY and NEFA data to model the adaptive responses and extract quantifiable indicators of deviation and recovery. Linear correlations (P < 0.03–0.07) between FR4 and FR10 were found for some indicators describing MY and NEFA levels before and after FR. The overall repeatability of MY and NEFA responses between both FR accounted for 46% based on quartile analysis performed on average responses. Moreover, the variance in both the MY and NEFA variables did not differ significantly between FR4 and FR10, despite a trend for higher variances in FR10. Altogether, (1) the calculated variables derived from the functional data analysis of the time patterns of the MY and NEFA accounted for the differences in the cow responses to FR, and (2) the animal responses appeared to show concordance between FR4 and FR10. In conclusion, short-term FR is a relevant framework for studying productive and metabolic adaptive responses in suckling cows and allows the identification of potential robustness indicators.     

Physiologic responses of grizzly bears to different methods of capture

The physiologic effects of two methods of capture, chemical immobilization of free-ranging (FR) bears by remote injection from a helicopter and physical restraint (PR) by leg-hold snare prior to chemical immobilization, were compared in 46 grizzly bears (Ursus arctos) handled during 90 captures between 1999 and 2001. Induction dosages and times were greater for FR bears than PR bears, a finding consistent with depletion of, or decreased sensitivity to, catecholamines. Free-ranging bears also had higher rectal temperatures 15 min following immobilization and temperatures throughout handling that correlated positively with induction time. Physically restrained bears had higher white blood cell counts, with more neutrophils and fewer lymphocytes and eosinophils, than did FR bears. This white blood cell profile was consistent with a stress leukogram, possibly affected by elevated levels of serum cortisol. Serum concentrations of alanine aminotransferase, aspartate aminotransferase, and creatine kinase were higher in PR bears that suggested muscle injury. Serum concentrations of sodium and chloride also were higher in PR bears and attributed to reduced body water volume through water deprivation and increased insensible water loss. Overall, different methods of capture resulted in different patterns of physiologic disturbance. Reducing pursuit and drug induction times should help to minimize increase in body temperature and alteration of acid-base balance in bears immobilized by remote injection. Minimizing restraint time and ensuring snare-anchoring cables are short should help to minimize loss of body water and prevent serious muscle injury in bears captured by leg-hold snare.     

A general model for ontogenetic growth under food restriction

Food restriction (FR) retards animals' growth. Understanding the underlying mechanisms of this phenomenon is important to conceptual problems in life-history theory, as well as to applied problems in animal husbandry and biomedicine. Despite a considerable amount of empirical data published since the 1930s, there is no relevant general theoretical framework that predicts how animals vary their energy budgets and life-history traits under FR. In this paper, we develop such a general quantitative model based on fundamental principles of metabolic energy allocation during ontogeny. This model predicts growth curves under varying conditions of FR, such as the compensatory growth, different age at which FR begins, its degree and its duration. Our model gives a quantitative explanation for the counterintuitive phenomenon that under FR, lower body temperature and lower metabolism lead to faster growth and larger adult size. This model also predicts that the animals experiencing FR reach the same fraction of their adult mass at the same age as their ad libitum counterparts. All predictions are well supported by empirical data from mammals and birds of varying body size, under different conditions of FR.     

New Arabidopsis recombinant inbred lines (Landsberg erecta x Nossen) reveal natural variation in phytochrome-mediated responses

We used 52 Arabidopsis (Arabidopsis thaliana) accessions and developed a new set of 137 recombinant inbred lines between Landsberg erecta (Ler) and Nossen (No-0) to explore the genetic basis of phytochrome-mediated responses during deetiolation. Unexpectedly, most accessions showed weak or moderate hypocotyl growth and cotyledon unfolding responses to pulses of far-red light (FR). Crosses between Columbia and No-0, two accessions with poor response, segregated seedlings with unfolded cotyledons under pulsed FR, suggesting the occurrence of accession-specific loci in the repression of morphological responses to weak light signals. Confirming the latter expectation, mapping of responses to pulsed FR in the Ler x No-0 lines identified novel loci. Despite its weak response to pulsed FR, No-0 showed a response to continuous FR stronger than that observed in Ler. By mapping the differential effect of pulsed versus continuous FR, we identified two high-irradiance response loci that account for the steeper response to continuous FR in No-0. This underscores the potential of the methodology to identify loci involved in the regulation of the shape of signal input-output relationships. Loci specific for a given phytochrome-mediated response were more frequent than pleiotropic loci. Segregation of these specific loci is predicted to yield different combinations of seedling responsivity to light. Such flexibility in combination of responses is observed among accessions and could aid in the adjustment to different microenvironments.     

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea ( Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA₁ varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA₁ between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA₁ and GA₂₀ (0.01–1 µg explant⁻¹), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA₁ was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA₁ was negated by colchicine, indicating that microtubules may be involved in the response to both factors.     

Phenotypic plasticity of sun and shade ecotypes of Stellaria longipes in response to light quality signaling: Cytokinins

From two distinct ecotypes of Stellaria longipes, one genotype was chosen from each of two very different locations, an alpine (sun) and a prairie (shade) habitat. Plants were clonally propagated and grown in controlled environment chambers under low and moderate red to far-red (R/FR) ratios. The prairie ecotype plants exhibited increased stem elongation, leaf expansion and flowering (6-fold) in response to a low R/FR ratio, relative to plants grown under the moderate R/FR ratio. In contrast, plants of the alpine ecotype showed no increased growth in response to a low R/FR ratio and their flowering was reduced, all relative to the plants grown under the moderate R/FR ratio. These different phenotypic responses to the reduction in R/FR ratio were associated with very different profiles and concentrations of endogenous cytokinins (CKs) assessed in growing tissues of the upper shoots. Specifically, increased total CKs were associated with the rapid growth of plants of the prairie ecotype under a low R/FR ratio. In particular, concentrations of bioactive trans-zeatin and dihydrozeatin, were increased during the period of most rapid shoot growth by 2- to 4- fold for these prairie ecotype plants grown under the low R/FR ratio treatment. In contrast, changes in CK levels for the alpine ecotype plants grown under low R/FR ratios were muted. Of especial interest, plants of the alpine ecotype had a predominance of cis-pathway CKs, whereas the low elevation, prairie ecotype plants accumulated predominantly trans-pathway CKs. Speculatively, the pattern emphasizing trans-pathway CKs may be explained by increased LONELY GUY enzyme activity. This enzyme converts and activates nucleotide CKs to free base CKs (bypassing riboside CKs). It could thus explain, in part, the prairie ecotype's ability to respond to shade light with such a high degree of plasticity if one assumes that high trans-CKs levels are causal for the increased shoot growth seen under a low R/FR ratio.     

Effects of red, far-red and blue light in maintaining growth in latitudinal populations of Norway spruce (Picea abies)

Seedlings of trees with a free growth pattern cease growth when night-lengths become shorter than a critical value, and this critical night-length (CNL) decreases with increasing latitude of origin. In northern populations, the light quality also appears to play an important role and a clinal variation in requirement for far-red (FR) light has been documented. In this study we dissected the light quality requirements for maintaining growth in different latitudinal populations of Norway spruce (Picea abies (L.) H. Karst.) using light emitting diodes for red (R), FR and blue (B) light, as 12 h day extension to provide 24 h photoperiod. At equal spectral photon flux, FR light was more effective than R light in maintaining growth, and the requirement of both R and FR increased with northern latitude of origin. One-to-one mixtures of R and FR light were more effective in maintaining growth than either FR or R light alone, indicating a possible interaction between R and FR light maintaining growth. Using the blue light as day extension could not prevent growth cessation in any of the populations, but delayed the bud set slightly in all populations. Our results suggest that phytochrome(s) are the primary photoreceptors in high irradiance responses maintaining growth in Norway spruce seedlings.     

The spinal antinociceptive effect of FR140423 is mediated through kyotorphin receptors

We investigated the antinociceptive effect of a novel anti-inflammatory and analgesic drug, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyraz ole (FR140423), in the tail-pinch test in mice, and evaluated the mechanism of action of FR140423 using L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist, L-NG-nitroarginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, and methylene blue (MB), an inhibitor of activation of guanylate cyclase. Oral administration of FR140423, at doses of 5-80 mg/kg, produced a dose-dependent antinociceptive effect with an ED50 value of 18 mg/kg. This antinociception was reversed by intrathecal (i.t.) (10 microg/mouse), but not by intracerebroventricular (i.c.v.) (100 microg/mouse), injection of Leu-Arg. Moreover, the antinociceptive effect of i.t. injection of FR140423 with an ED50 value of 3.7 microg/mouse was completely antagonized by co-administered Leu-Arg 10 microg/mouse. However, L-NAME (2000 mg/kg s.c.) and MB (200 mg/kg s.c.) did not antagonize the antinociception of FR140423. These findings suggest that FR140423 plays a role in nociceptive modulation in the spinal cord, being antinociceptive via the kyotorphin-Met-enkephalin pathway but not via the peripheral NO-cyclic GMP pathway.     

From natural products to clinically useful antifungals

In our search for natural products with a broad spectrum of antifungal activity as lead compounds for novel treatments for mycoses, we have isolated echinocandin-type lipopeptide FR901379 and lipopeptidolactone FR901469, as novel water-soluble antifungal agents that inhibit the synthesis of 1,3-beta-glucan, a key component of the fungal cell wall. Since the cell wall is a feature unique to fungi and is not present in nonfungal eukaryotic cells, inhibitors of the synthesis of fungal cell wall components such as 1,3-beta-glucan have potential for selective toxicity to fungi and not to the host. In this short review, we describe efforts directed at synthetic modification of FR901469 and FR901379 with the ultimate goal of identifying new entities with suitable profiles as development candidate compounds. The main thrust of our work to date has been replacement of the highly flexible lipophilic side chains of the natural products with a view to reducing the hemolytic potential associated with these compounds, and to enhance chemical stability and/or in vivo antifungal efficacy. As a result of these efforts, we recently discovered a novel analog, FK463 (micafungin). Micafungin is currently in phase III clinical trials worldwide as a parenteral agent for various mycoses, and a new drug application (NDA) was recently filed in Japan.     

Diagnosis of B-cell lymphoma. Utility of the polymerase chain reaction for detecting clonality from archival cytologic smears

OBJECTIVE: To apply the polymerase chain reaction (PCR) to detect clonality for potentially helping to establish a definitive diagnosis of lymphoma in cytologic material. STUDY DESIGN: In this retrospective study, Papanicolaou-stained cytologic smears and formalin-fixed, paraffin-embedded tissues from 17 cases of B-cell lymphoma were examined to investigate their clonality by a PCR technique using three different approaches (FR3, FR3A and FR2) for amplification of immunoglobulin heavy chain genes. Cytologic smears from 10 cases of nonneoplastic lymphoid tissues and T-cell lymphomas served as negative controls. RESULTS: Monoclonality was detected in 9 of 17 cases (53%) of B-cell lymphoma in cytologic smears as compared with 8 of 16 cases (50%) in tissue sections. Semi-nested PCRs (FR3A/FR2) were superior to the single PCR (FR3) in the detection rate (41% vs. 18%). Five of seven cases (71%) of marginal zone B-cell lymphomas showed monoclonality, whereas only 4 of 10 cases (40%) of diffuse large B-cell lymphomas did so. Monoclonality was demonstrated in none of the negative controls. CONCLUSIONS: Clonality detection in B-cell lymphomas by PCR using cytologic smears is specific and equal in sensitivity to that using formalin-fixed, paraffin-embedded tissues. The detection rate is especially excellent in marginal zone B-cell lymphoma, in which the cytologic diagnosis is particularly challenging. Combined seminested PCRs for FR3A and FR2 are advocated for a reliable assessment of clonality.     

Morphological plasticity ofFestuca rubra clones from three neighbouring communities in response to red: Far-red levels

Morphological parameters ofFestuca rubra clones and their response to differences in the red/far red ratio (R : FR) were investigated in a growth chamber experiment. The twenty-four clones tested came from 15 micro-sites in three neighbouring meadow communities in the Krkono?e Mts. (Czech Republic). The community types were characterized by different canopy height and structure, and by different radiation conditions (relative R : FR) within the canopies. Low R : FR treatment resulted in reduced tillering and stimulated shoot elongation in most of the clones. In some clones, low R : FR ratios stimulated extravaginal tillering and rhizome system formation. The origin of the clones (i.e., community type) explained a part of variation in morphological parameters, but did not explain variation in the response to R : FR levels. Evidence for a consistent relationship between clone performance and radiation environment in the micro-sites from where the clones originated was not found. It seems that there is only limited selection ofF. rubra clones (ecotypes) specialized for particular community types and no selection of clones specialized for particular micro-sites within communities.     

Long-term ozone exposure of potato: free radical content and leaf injury analysed by Q-band ESR spectroscopy and image analysis

This paper presents Q-band electron spin resonance (ESR) studies on free radicals (FR) generated in potato leaves exposed to different O₃ levels in open-top chambers (OTC), together with a quantitative study of the relationship between FR signal intensity and area of potato leaf damage. The advantages of Q-band when compared to X-band ESR spectroscopy are analysed, the main advantage being an absence of overlapping between Mn(II) and FR signals, allowing a quantitative study of FR signal intensity. This study also reports on a graphical method developed to quantitatively measure the damaged area on leaves caused by ozone exposure. Results indicate a direct relationship between FR signal intensity (measured as area under the signal) and percentage of O₃ damage and clearly demonstrate a close relationship between visible ozone-induced symptoms and permanent FR concentration in potato leaves.     

Diaryl ester prodrugs of FR900098 with improved in vivo antimalarial activity

The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.