Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

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.     

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.     

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.