Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

The dependence of the frequency of recessive lethal and vital mutations inArabidopsis thaliana (L.)Heynh. on the concentration of N-nitroso-N-methylurea

The dependence of the frequency of recessive lethal (two groups), chlorophyll and morphological mutations on the mutagen concentration was determined in M₂ after subjection to N-nitroso-N-methylurea applied to seeds ofArabidopsis in three concentrations (0·05, 0·10 and 0·20mm). The observed frequencies were compared with the theoretically expected ones for the linear and for two exponential types of dependence, by using the t-test, according to the formulas m=k. C, m=k. C^3/2, m=k. C². No satisfactory agreement with any expected type of dependence was found when directly observed frequencies were used. Since a considerable deficit of mutation frequency was observed in high concentrations, the correction of frequency values was done with respect to the probability of occurence of double mutations. After such a correction, a clear exponential relation was found in both types of lethals and a linear one in chlorophyll and morphological mutations. The probable occurence of multiple mutations should be, therefore, taken into account if the dependence of mutation frequency on the concentration of mutagen is discussed.     

Changes in the radioresistance of bacteria after the inhibition of proteosynthesis in the preirradiation phase

The strain ofEscherichia coli WP₂ (try_v) was irradiated with UV light, at a dosage of 240 erg/mm. Proteosynthesis was inhibited by the elimination of the essential amino acid from the cultivation medium. Changes in radioresistance were followed during 45 minutes of starvation and during the subsequent 45 minutes of restitution after the addition of the essential amino acid. The radioresistance of the cells showed a linear increase immediately after the removal of the essential amino acids, proportional to the duration of the inhibition of proteosynthesis. The increase in radioresistance was shown to be reversible. After the addition of the essential amino acid there was an immediate decrease in radioresistance which was most marked in the first 15 minutes.     

Enhanced tumor cell adhesion to the subendothelial matrix resulting from 12(S)-HETE-induced endothelial cell retraction

A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.     

Effect of Glutathione and N-Acetylcysteine on in Vitro and in VIVO Cardiac Toxicity of Doxorubicin

The effects of two sulfhydryl compounds, glutathione (GSH) and N-acetylcysteine (NAC), on the cardiotoxicity of doxorubicin (DXR) were tested on in vitro and in vivo models. DXR was administered to rats as 4 weekly i.v. doses of 3mg/kg. GSH (1.5 mmoles/kg), given i.v. 10 min before and 1 hr after DXR, was found to prevent the development of the delayed cardiotoxic effects of DXR, as assessed by electrocardiographic and mechanical parameters, as well as by histological examination of left ventricular preparations. In contrast, equimolar oral doses of NAC (1 hr before and 2hrs after DXR) were found to be ineffective. Both GSH and NAC prevented the negative inotropic effect produced by DXR on isolated rat atria. A good correlation exists between the cardioprotective effects of the two agents and their ability to enhance the non-protein sulfhydryl group content of the myocardium. Differences observed in vivo between GSH and NAC might be accounted for by pharmacokinetic factors.     

Alternating bolus and continuous infusion 5-fluorouracil: a strategy to overcome resistance to this fluoropyrimidine in advanced colorectal cancer patients

Focusing our effort on the importance of FUra scheduling we have tested the hypothesis that pulse and continuous infusion (CI) of the fluoropyrimidine have different mechanisms of cytotoxicity. Our initial approach was to compare the mechanism of resistance of a cell line resistant to a short term exposure to FUra (HCT-8/FU4hR) to that of a cell line resistant to a prolonged exposure to the fluoropyrimidine (HCT-8/FU7dR). Cytotoxicity studies showed that HCT-8/FU4hR cells were still sensitive to FUra given as a 7-d exposure, suggesting different mechanisms of resistance. Indeed, rapid recovery of TS activity after drug removal was evident in the HTC-8/FU7dR cell line while HCT-8/FU4hR cells were similar to the parental cell line with regard to both the degree of in situ TS inhibition by FUra and duration of inhibition after FUra removal. In contrast, labelling studies with [³H-6] FUra (4 h exposure, 100 M) showed that the incorporation of the fluoropyrimidine into RNA is significantly decreased in HCT-8/FU4hR cells as compared to parental HCT-8 cells.Given the lack of cross resistance between the two schedulesin vitro, a pilot trial was done on patients with colorectal cancer refractory to bolus FUra. On 15 patients failing after FUra+LV or FUra alone 1 PR, 3 MR, 3 SD and 8 P were observed, confirmng a certain degree of activity of CI FUra in patients clinically resistant to bolus FUra.Based on this rationale, a phase II trial of schedule-oriented biochemical modulation of FUra in advanced colorectal cancer patients was conducted, employing a hybrid regimen of 2 biweekly cycles of FUra bolus (600 mg/sqm), preceeded by (24 h interval) methotrexate, 200 mg/sqm (in order to maximize the RNA effect of the drug) alternating with FUra continuous infusion, 200 mg/sqm daily for 3 weeks, modulated by leucovorin, 20 mg/sqm weekly bolus (in order to maximize the DNA effect).Thirty-three consecutive patients (median ECOG PS 1) with advanced measurable colorectal cancer and no prior therapy for metastatic disease entered the study, from February 1992 to August 1993. Three complete and 13 partial responses were obtained among these 33 patients (RR=48%, 95% confidence limis, 31–66%). After a median follow-up time of 23 months, 16 patients are still alive. The median progression free survival and overall survival were 9.6 and 20.8 months, respectively. No toxic deaths or grade 4 toxicity occurred. The incidence of grade 3 toxicity per patient in any cycle was: mucositis 6%, diarrhea 3% and vomiting 3% for the bolus part and 21%, 3% and 6% respectively, for the continuous infusion part of the regimen. Hand-foot syndrome occurred in 27% of the patients treated with the continuous infusion regimen.In conclusion, this experimental and clinical project has generated a novel regimen of schedule oriented biochemical modulation that is twice as active and half as toxic compared to bolus FU+LV given with either the daily x 5 or the weekly schedule. This high clinical activity is very encouraging, especially considering that 1) consecutive patients were entered, 2) the responses were independently reviewed, 3) the progression free survival and survival were much longer than those actually reported for this disease, 4) the toxicity of the program, in particular the bolus regimen, was relatively low allowing further intensification.     

Study of the reversibility of the water saturation deficit as one of the methods of causal phytogeography

Jedním z kritérií pro posuzování odolnosti rostiin v??i suchu je jejich schopnost sná?et vodní deficit ani? nastanou irreversibilní změny v jejich pletivech. Tato schopnost byla zkoumána metodikou, popsanou v p?edlo?ené práci. Listy některých xerothermních trav (druhy roduStipa, Melica atd.) vysýchaly za p?esně definovaných pokusných podmínek tak, ?e dosáhly r?zně odstupňovaného vodního deficitu. Potom byly roz?ezány na segmenty a ve speciálním ráme?ku dosycovány vodou. P?vodní deficit i jeho vyronání bylo sledvváno váhově. Výsledné hodnoty byly znázorněny graficky. Zatímco ztráta vody z list? během vysýchání probíhala v některých p?ípadech lineárně, k?ivka dosycování ukazovala charakteristický zlom, který indikoval, jak dalece byla ztráta vody nahraditelná, a kdy do?lo ji? k irreversibilním změnám. Ukázalo se, ?e ze studované série rostlin druhy typicky kontinentální mají schopnost doplňovat svou zásobu vody ad integrum i p?i zna?ném vodním deficitu. Rostliny s areálem spí? oceánického charakteru tuto schopnost nemají. Je pravděpodobné, ?e i tato vlastnost bude směrodatná p?i výkladu kausální fytogeografie.