An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Classification of cultivated rices into indica and japonica types by the isozyme,RFLP and two milled-rice methods

Four methods for classifying cultivated rices (Oryza sativa L.) (including IR varieties) into indica and japonica types — waxy gene product in endosperm starch, glutelin ₃ molecular weight in milled rice, RFLP polymorphism at the Wx locus and Glaszmann's isozyme method — were compared. On the basis of the two endosperm traits and the RFLP method Glaszmann's group 1 (indica) was classified as mainly indica and intermediate groups 2, 3 and 4 as exclusively indica. However, the endosperm traits classified Glaszmann's group 5 as mainly indica, while the RFLP method classified it as japonica. The RFLP waxy gene probe was closest to the isozyme method in classifying group 6 as japonicas; the waxy gene product gave mainly indica reaction even in group 6, and the glutelin ₃ method was intermediate. All IR rices were classified as being indica on the basis of Wx gene product and by Glaszmann's method, but a few were classified as japonica by the glutelin ₃ method and by the RFLP waxy gene probe.     

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state

Abstract Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [³²P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with ³²P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.     

电脉冲穿孔法将苏云金杆菌δ-内毒素基因导入野生型芽孢杆菌

利用电脉冲穿孔法将带有苏云金杆菌毒蛋白基因的穿梭质粒导入几株野生型芽孢杆菌中。它们是野生型的蜡状芽孢杆菌、短芽孢杆菌和枯草芽孢杆菌。通过观察在新霉素和氨苄青霉素平板上长出的抗性菌落数,计算出转化效率为10~1—10~4转化子/μgDNA。从转化子中分离到的质粒DNA大小及其用HindⅢ酶切的片段与原始质粒DNA相同,毒性测试表明重组转化子对烟青虫六天的致死率达90—100％。     

Plant communities on anthropogenically disturbed sites on the Chukotka Peninsula,Russia

Abstract. The vegetation of anthropogenically disturbed sites on the Chukotka Peninsula was investigated in 1986 and 1990. 102 sites had been bulldozed free of vegetation and soil between 2 and 20 yr ago and revegetated. Anthropogenic vegetation differs considerably from natural tundra. The total plant cover decreases, lichens are absent, moss composition changes, and graminoids predominate. A floristic classification was developed in order to describe the diversity of anthropogenic communities formed on these sites. Four associations were distinguished, all belonging to the alliance Poion glaucomalacanthae. An additional alliance characterized by Alopecurus alpinus is suggested for colder sites, but it has not yet been described. There is a correspondence of associations to substrate types. The association Phippsietum algidae was found only on loam substrate. The Artemisietum glomeratae and Saxifragetum punctatae are connected with rubble or gravel. The graminoid association Arctagrostietum arundinaceae is generally widespread in disturbed habitats and occurs on a variety of substrate types.     

Multivariate analysis of the vegetation of the volcanoes Tláloc and Pelado,Mexico

Abstract. Multivariate analysis was used to describe the composition and distribution of vegetation types on the slopes of the volcanoes Tláloc and Pelado, Mexico. These volcanoes are situated in the transitional zone between the Holarctic and Neotropical floristic regions, which offers a partial explanation for the relatively high α and β diversities. Previous research argued that human activities, i.e. burning and grazing, rather than abiotic factors, play a major role in determining the distribution and floristic composition of the vegetation. TWINSPAN, Detrended Correspondence Analysis and Canonical Correspondence Analysis were used to test this hypothesis. Floristic and environmental data from 138 relevés and seven explanatory environmental variables were included: elevation, soil depth, soil moisture, percentage litter cover, percentage cover of bare ground, burning and grazing were included in the analysis. Soil moisture and elevation accounted for ca. 63% of the residual inertia and none of the remaining explanatory variables proved to be correlated significantly with the first two axes. The present results suggest that burning and grazing operate on a finer scale. In conclusion, soil moisture and elevation are the most relevant variables to explain the distribution of the vegetation under study.     

The fecundity patterns of S and L type rotifers of Brachionus plicatilis

Fecundity patterns of S and L type rotifers Brachionus plicatilis, which were previously found to have distinct growth ability, were analyzed using data of age-specific fecundity. The data were obtained by individual cultures of S and L type strains at 17, 24 and 34 °C. The pattern was analyzed by using normal probability functions. When the age was transformed into logarithmic value, the S and L types had an identical pattern of fecundity at every temperature. This fact indicates that the difference of the growth response to the temperature between S and L type strains stemmed only from the differences in net reproduction rate, not from the pattern.     

Axonal Glycoproteins with Immunoglobulin- and Fibronectin Type III-Related Domains in Vertebrates: Structural Features, Binding Activities, and Signal Transduction

Abstract: The L1- and F11-like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III-like domains. Their Ig-like and fibronectin type III-related domains are likely to be composed of seven β-strands arranged in two opposing β-sheets of highly similar topology. Whereas the F11-like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 -like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre-mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 -like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 -like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III-like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.