Epithelial impedance analysis in experimentally induced colon cancer.

Epithelial impedance analysis was used to measure the alterations in resistance of the large bowel in a murine model of large bowel cancer. The technique was able to resolve the epithelial resistance from the total resistance of the bowel wall. A progressive decrease in resistance of the bowel epithelium occurs during carcinogenesis induced with dimethyhydrazine. About a 21% decrease in epithelial resistance from 22.0 +/- 1.3 omega.cm-2 to 17.5 +/- 1.1 omega cm-2 (p less than 0.025) was observed after 20 wk of carcinogen administration. The sensitivity of the technique in detecting altered epithelial resistance in premalignant bowel mucosa was improved by examining the impedance profile in a sodium-free Ringer's solution where the epithelium of control colons had a resistance of 24.4 +/- 1.8 omega.cm-2 compared with 19.0 +/- 1.1 omega.cm-2 (p less than 0.02) in colons from animals treated for only 4 wk with the carcinogen. Epithelial impedance analysis would seem to be a sensitive technique capable of identifying changes in the electrical properties or the large bowel early in disease states.     

Electrically evoked contractions of the triceps surae during and following 21 days of voluntary leg immobilization

The effects of 21 days voluntary leg (plaster) immobilization on the mechanical properties of the triceps surae have been studied in 11 young female subjects, mean age 19.4 years. The results show that during the period of immobilization the mean time to peak tension (TPT) and half relaxation time (1/2RT) and tension (Pt) of the maximal twitch increased significantly (p less than 0.001) but the effects were short lived. Maximal tension and contraction times of the twitch recovered within 2-14 days following the removal of the plaster cast. The electrically evoked tetanic tensions at 10 Hz and 20 Hz did not change significantly (P greater than 0.1) during immobilization, but the 50 Hz tetanic tension (Po50) and maximal voluntary contraction (MVC) were reduced (p less than 0.05). The fall in Po50 and MVC was associated with 10% decrease in the estimated muscle (plus bone) cross-sectional area. The relative (%) change in Po50 and MVC following immobilization was related to the initial physiological status (as indicated by the response of the triceps surae to a standard fatigue test prior to immobilization) of the muscle. The rate of rise and recovery fall of the tetanus were slightly but significantly (p less than 0.01) reduced on day 7 of immobilization, but thereafter remained constant. The isokinetic properties of the triceps surae as reflected in the measured torque/velocity relation of the muscle in 4 subjects did not change significantly if account was taken of the slight degree of atrophy present following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a conserved sequence deleted in Duchenne muscular dystrophy patients.

We have isolated a DNA sequence (HIP25) by subtraction- hybridisation which is deleted in a number of Duchenne muscular dystrophy (DMD) patients. HIP25 is conserved in evolution and hybridises to human fetal and adult muscle mRNA. HIP25 is absent in human fetal fibroblast mRNA. Physical mapping data localise this sequence within Xp21 between the breakpoints of X;autosome translocations found in two females suffering from the disease. HIP25 is a candidate exon sequence for the basic defect in DMD boys deleted at this locus.     

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.     

The evolutionary importance of mechanoreception in three erpobdellid leech species

Summary The ability of an erpobdellid leech, Erpobdella montezuma, to discriminate between two congeneric amphipod prey (Hyalella montezuma and Hyalella azteca) through mechanoreception was examined. Both E. montezuma and H. montezuma, a pelagic filter feeder, are endemic to the near thermally constant environment of Montezuma Well, Arizona, and form a highly specialized predator-prey relationship. In contrast, H. azteca, a benthic detritivore, is widely distributed throughout North America. Erpobdella montezuma was highly responsive to the prey signals of the endemic H. montezuma and showed only a limited response to H. azteca. An inverse relationship occurred between the frequency of attack and size of the leech. The abilities of Erpobdella punctata and Nephelopsis obscura, neither found in Montezuma Well, to detect the signals of both amphipod prey were also examined. These species with non-specialized diets showed a low response to the signals of both prey species. This study demonstrates that very close predator-prey relationships can develop over evolutionary time in isolated aquatic systems through the use of mechanoreception.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

E.s.r. spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect peroxyl, alkoxyl and carbon-centred radicals produced by reaction of t-butyl hydroperoxide (tBuOOH) with rat liver microsomal fraction. The similarity of the hyperfine coupling constants of the peroxyl and alkoxyl radical adducts to those obtained previously with isolated enzymes suggests that these species are the tBuOO. and tBuO. adducts. The effects of metal-ion chelators, heat denaturation, enzyme inhibitors and reducing equivalents demonstrate that these species arise from reaction of tBuOOH with a haem enzyme such as cytochrome P-450 or cytochrome b5. In the absence of NADPH or NADH the previously undetected peroxyl radical adduct is the major species observed. In the presence of these reducing equivalents the alkoxyl and carbon-centred radical adducts predominate, which is in accord with product studies on similar systems. These results demonstrate that both reductive and oxidative decomposition of tBuOOH can occur in rat liver microsomal fraction with the reductive pathway favoured in the presence of NADH or NADPH.