Radiosensitivity of mouse skin epithelial cell line established in serum-free culture: an alternative to animal use.

A cultured line of murine skin epithelial cells was established to investigate the potential use of cultured cells as an alternative to animal use in radiation research. C3Hf/Sed newborn mouse skin cells have been successfully cultured in serum- and Ca(2+)-free medium with no terminal differentiation to keratinized cells. Presently, more than 25 passages have passed with no loss of stem cell capability. The radiosensitivity and repair of sublethal and potentially lethal radiation damages were investigated in this epithelial cell line. The population cell doubling time was 25 +/- 2.9 hr at 37 degrees C. The clonal growth of epithelial cells after irradiation was performed in the serum-free medium in the presence of lethally irradiated skin fibroblasts. Single dose survival curves of exponentially growing epithelial cells were investigated from the seventh to the twenty-third passages, and no significant changes in radiosensitivity and doubling time were found. The confluent epithelial cells also showed an identical sensitivity to radiation. The alpha/beta ratios of survival curves fitted by the linear quadratic model were 6.1 +/- 1.0 and 5.9 +/- 1.3 Gy for cells in exponential and confluent phases, respectively. The survival curve of epithelial cells left in confluence for 8 hr after irradiation showed a smaller beta value than that of cells plated immediately after irradiation with a resultant alpha/beta ratio of 9.5 +/- 3.8 Gy. This alpha/beta ratio was identical to those found in many animal experiments, suggesting a potential use of this cell line as an alternative to animal use. The magnitude of repair of sublethal damage following 6 Gy was greater than that following 3.9 Gy. Survival curves were also obtained following twice-a-day irradiations with no sign of rapid repopulation. These results are discussed by comparing with published in vivo and in vitro data.     

Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae

Summary The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.     

APOE epsilon3 gene transfer attenuates brain damage after experimental stroke.

Apolipoprotein E (apoE, protein; APOE, gene) is the major lipid-transport protein in the brain and plays an important role in modulating the outcome and regenerative processes after acute brain injury. The aim of the present study was to determine if gene transfer of the epsilon3 form of APOE improves outcome in a murine model of transient focal cerebral ischaemia. Mice received an intrastriatal injection of vehicle, a second-generation adenoviral vector containing the green fluorescent protein gene (Ad-GFP) or a vector containing the APOE epsilon3 gene (Ad-APOE) 3 days before 60 mins focal ischaemia. Green fluorescent protein expression was observed in cells throughout the striatum and subcortical white matter indicating successful gene transfer and expression. ApoE levels in the brain were significantly increased after Ad-APOE compared with Ad-GFP or vehicle treatment. Ad-APOE treatment reduced the volume of ischaemic damage by 50% compared with Ad-GFP or vehicle treatment (13+/-3 versus 29+/-4 versus 27+/-5 mm(3)). The extent of postischaemic apoE immunoreactivity was enhanced in Ad-APOE compared with Ad-GFP or vehicle treated mice. These results show the ability of APOE gene transfer to markedly improve outcome after cerebral ischaemia and suggest that modulating apoE levels may be a potential strategy in human stroke therapy.     

Comparing the validity of clinician-generated diagnosis of conduct disorder to the diagnostic interview schedule for children.

Clinician diagnoses of conduct disorder (CD) were compared to the diagnoses of CD generated by a structured interview against an observed criterion. Participants were 534 youth from a large residential program in the Midwest for delinquent youth. Rates of in-program CD behaviors were gathered from staff observations of the youth over a 9-month time period. Youth diagnosed with CD by the Diagnostic Interview Schedule for Children (DISC) displayed significantly more CD behaviors in the first 6 months of treatment compared to both youth without an externalizing disorder and youth diagnosed with CD by a clinician. Youth diagnosed with CD by a clinician had rates of CD identical to youth without an externalizing disorder. Clinicians may have weighted contextual information more heavily, as this group was significantly more likely to have an arrest record. Results support the use of structured interviews and provide evidence that typical clinician diagnoses may lack adequate validity.     

Improved bioavailability and clinical response in patients with chronic liver disease following the administration of a spironolactone: β-cyclodextrin complex

Aims To compare the absorption and clinical effect of spironolactone from an inclusion complex with β-cyclodextrin (SP-COMP) to Aldactone tablets (ALD) in chronic liver disease.
Methods Patients, admitted with chronic liver disease, completed a randomized crossover steady state study. They received their spironolactone dose as either daily SP-COMP or ALD for 7 days. Serial blood samples were drawn over a 24  h period from day 7 of each therapy. Accurate fluid balance was recorded on days 5–7 and 12–14. Thirteen (six females) whose mean (s.d.) age and weight was 58.4(9.3) years and 74.3(19.0)  kg completed the study.
Results The mean (95% confidence limits) relative bioavailability for SP-COMP (compared with ALD) from steady state serum concentrations of canrenone, 6β-hydroxyl 7α-thiomethyl spironolactone and 7α-thiomethyl spironolactone was 310.0 (265.4, 336.7), 233.4(212.9, 250.8) and 254.8(230.8, 279.0)%, respectively. Improvements in clinical status and fluid balance occurred over the last 3 days of SP-COMP with a mean (s.d.) net loss, in fluid balance, of 1370(860)ml compared with a gain of 228(936)ml during ALD.
Conclusions Better absorption of spironolactone from the spironolactone: β-cyclodextrin complex formulation should lead to a reduction in dosage and perhaps a more consistent effect in patients with chronic liver disease.     

A 94-kDa protein, identified as neutral endopeptidase-24.11, can inactivate atrial natriuretic peptide in the vascular endothelium.

Neutral endopeptidase 24.11 (EC 3.4.24.11) inactivates atrial natriuretic peptide by cleaving the hormone between Cys7 and Phe8, and inhibitors of the enzyme have consequent natriuretic and diuretic properties. The in vivo sites of degradation of this peptide by the zinc-metallopeptidase, however, remain to be established. Because an endopeptidase-24.11-like activity has recently been reported in the rat mesenteric artery, we have further investigated the degradation of atrial natriuretic peptide in vascular tissue. Endopeptidase-24.11 activity was detected in solubilized membrane preparations from rat and rabbit vascular tissue, using [3H]D-Ala2-leucine enkephalin as substrate, and both rabbit and rat aorta preparations were also found to cleave atrial natriuretic peptide between Cys7 and Phe8. In both cases, hydrolysis was inhibited by neutral endopeptidase inhibitors, with Ki values close to their Ki values for the pure enzyme. In preparations of rabbit aorta denuded of endothelium by saponin treatment, the hydrolysis of the Gly3-Phe4 bond of [3H]D-Ala2-leucine enkephalin and the Cys7-Phe8 bond of atrial natriuretic peptide was reduced by greater than 90%. The high performance liquid chromatography method used to follow the degradation of atrial natriuretic peptide differed from previously published procedures, in that samples to be injected were first treated with excess dithiothreitol to reduce the Cys7-Cys23 disulfide bridge. This facilitated the separation of the intact peptide and its metabolites. The presence of the 94-kDa neutral endopeptidase in rabbit aortic tissue was definitively established using a new potent 125I-labeled inhibitor, [125I]RB104 [2-[(3-[125I]iodo-4-hydroxy)phenylmethyl]-4-N-[3- hydroxyamino-3-oxo-1-phenylmethyl propyl]amino-4-oxobutanoic acid] (Ki, 30 pM), which selectively labeled the enzyme after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membrane preparations. Therefore, despite its low concentrations in the vasculature, the presence of endopeptidase-24.11 almost exclusively in endothelial tissue suggests that the enzyme is ideally localized to inactivate circulating atrial natriuretic peptide.     

Prospective Evaluation of the Chemiluminescence Test for the Detection of Granulocyte Antibodies: Comparison with the Granulocyte Immunofluorescence Test

A series of 213 neutropenic patients were tested for the presence of granulocyte antibodies using the granulocyte chemiluminescence test (GCLT) and the granulocyte immunofluorescence test (GIFT). Sera containing lymphocyte (HLA) antibodies were excluded from the study. A direct GIFT was performed on granulocytes from 56 patients. Samples were obtained from patients with a range of clinical conditions including primary adult autoimmune neutropenia, autoimmune neutropenia of infancy, autoimmune neutropenia secondary to Felty's syndrome, rheumatoid arthritis, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, proliferative disorders, bone marrow transplantation and patients with documented febrile or pulmonary transfusion reactions. Overall, granulocyte antibodies were detected in 52.1% of patient sera. Results for the GCLT and GIFT (IgG) were strongly correlated (plt;0.001) for both primary and secondary immune neutropenias. The results confirm the applicability of the GCLT in the granulocyte serology laboratory.     

Allergic contact dermatitis from grasses

This study attempts to demonstrate the existence of allergic contact dermatitis from grass, and to develop a patch test series to screen patients with grass intolerance. 6 common grass species from lawns and military training areas were collected. Solvent extracts of polar. non-polar and volatile fractions were prepared and used for patch testing in 20 control subjects and 46 patients with a history of grass intolerance. The 20 control had negative responses to patch testing. 5 out of 46 patients had positive patch tests to Axonopus compressus (carpet grass). Ischaenmum muticum (sea-shore centipede grass). Imperata evlindrica (lalang). Panicum maximum (Guinea grass) and Pennisetum purpureum (elephant grass). Reactions to the non-polar fraction for all 5 species were noted. This study demonstrates the existence of allergic contact dermatitis from various common species of grass. In our series, this is seen in 11% of those with a history of grass intolerance.