[Modelling the bronchial epithelium in chronic obstructive pulmonary disease using human induced pluripotential stem cells]. / Modélisation de l'épithélium bronchique dans la bronchopneumopathie chronique obstructive par les cellules souches pluripotentes induites humaines.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease leading to irreversible destruction of the terminal bronchioles. Although the precise patho-physiological mechanisms remain to be elucidated, the bronchial epithelium seems to play a pivotal role in the disease. Recent studies have highlighted a great heterogeneity among COPD patients, with various disease courses including, in about half the cases, an origin in childhood. Modelling of COPD is a major goal but currently available models are imperfect. Our work aims to create a new in vitro cellular model to study the pathology of the disease. The differentiation of human induced pluripotential stem cells (hiPSCs) in bronchial epithelium is a step towards a better understanding of the developmental origin and the identification of new therapeutic targets.

A set of BASIC programs to evaluate relationships among protein sequences by optimum alignment and distance matrix analysis.

These six programs evaluate relationships among a number (> 2) of protein (or DNA) sequences. Program 1 automatically computes optimum alignments and total distances for all pairwise sequence combinations over any user desired range of the two gap penalties. Programs 2, 3 and 4 generate a square, symmetrical distance matrix, which can be exported for cluster analysis, or further analysed with programs 5 and 6 to give specific distances between sequences and extract sequence relationships. Data and results are exchanged among these programs, which are written in BASIC and compiled to run on Macintosh (68020/030/040) type machines with coprocessor and at least one MB of RAM. BASIC graphics commands or those for the Macintosh interface are avoided to facilitate use on other machines. Two groups of sequences are used to demonstrate (a) alignment, inter sequence distance calculation and dendrogram generation and (b) specific distance calculation and its usage in detecting sub groups of related sequences in dendrograms.

DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax.

HPLC analysis was used to examine the cytosine methylation of total DNA extracted from four early-flowering lines that were induced by treating germinating seeds of flax (Linum usitatissimum) with the DNA demethylating agent 5-azacytidine. In the normal lines that gave rise to the induced early-flowering lines, flowering usually begins approximately 50 days after sowing. The early-flowering lines flower 7-13 days earlier than normal. The normal level of cytosine methylation was approximately 14% of the cytosines and 2.7% of the nucleosides. In the early-flowering lines, these levels were 6.2% lower than normal in DNA from the terminal leaf clusters of 14-day-old seedlings and 9.7% lower than normal in DNA from the cotyledons and immature shoot buds of 4-day-old seedlings. This hypomethylation was seen in lines that were five to nine generations beyond the treatment generation. The level of hypomethylation was similar in three of the four early-flowering lines, but was not as low in the fourth line, which flowers early but not quite as early as the other three lines. Unexpectedly, the degree of hypomethylation seen in segregant lines, derived by selecting for the early-flowering phenotype in the F(2) and F(3) generations of out-crosses, was similar to that seen in the early-flowering lines. Analysis of the methylation levels in segregating generations of out-crosses between early-flowering and normal lines demonstrated a decrease in methylation level during the selection of early-flowering segregants. The results suggest an association between hypomethylation and the early-flowering phenotype, and that the hypomethylated regions may not be randomly distributed throughout the genome of the early-flowering lines.

Relative mobility and activity of leaf malate dehydrogenase in flax (Linum usitatissimum) genotrophs and genotypes.

Malate dehydrogenase (MDH) band relative mobility (Rm) and activity were examined in leaf extracts of Durrant's flax genotrophs, L and S, and flax genotypes, R and M. MDH activity in leaves from just below the inflorescence was higher in the two smaller, sparsely branched plant types, S and M, than in the larger, more branched plant types, L and R. The MDH electrophoretic banding pattern in flax leaf extracts consisted of three major anionic bands, MDH-1, MDH-2, and MDH-3. No Rm differences were detected between corresponding isozymes of genotypes R and M. For the genotrophs, however, all three bands of S migrated faster than the corresponding bands of L. Codominance was absent in F1 hybrids; S Rm was dominant for MDH-2 and MDH-3 and L Rm was dominant for MDH-1. The observations suggest that MDH Rm in L and S may be controlled by a modifier locus (or loci). Previous studies indicate that a modifier locus may also control heritable genotrophic differences in peroxidase (PER) and acid phosphates (AP) Rm. The three enzyme systems are compared.

Heritable effects of 5-azacytidine treatments on the growth and development of flax (Linum usitatissimum) genotrophs and genotypes.

Seed of flax (Linum usitatissimum) were treated for short durations with 5-azacytidine and the direct and heritable effects of the treatments on plant growth and development in general and, more specifically, on the contrasting phenotypes of Durrant's large and small genotrophs were examined. 5-Azacytidine induced a reduction in the height of the plants grown from treated seed. Twenty-two percent of the first generation progeny of these plants also had short phenotypes and, in most cases, the short phenotype was stably and uniformly inherited by the second generation progeny of the short, first generation plants. Treatment also induced a marked decrease in the flowering age in a few of the first generation plants that was also transmitted to their second generation progenies. The effects seen in the progeny generations suggest that most, if not all, of the heritable changes induced by the treatment are epigenetic. Several differences were seen between the large and small genotrophs, which indicate that the genome of the small genotroph is less susceptible, than the genome of the large genotroph, to 5-azacytidine induced heritable alterations.

Using coomassie blue to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels.

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R-250 staining and destaining to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels, prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.

An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase.

When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADH-dependent formation of H2O2. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.

Malate dehydrogenase isozymes in flax genotroph leaves: differences in apparent molecular weight and charge between and within L and S.

Ferguson plots demonstrated that corresponding malate dehydrogenase (MDH) isozymes of Durrant's L and S flax genotrophs differ in apparent molecular weight (MW) and also in net negative charge. The MW differences explain heritable differences in electrophoretic relative mobility (Rm) between corresponding L and S isozymes. The MW for each MDH isozyme was higher for L than for S and resulted in a slower Rm for L. The net negative charge for each isozyme was higher for L than for S. MDH isozymes also differ in MW within L and S. MW was lower for isozymes in leaves from the bottom of the stem than in leaves from the top of the stem, particularly in L. Integration of information on the MDH isozyme system in the flax genotrophs and information on the peroxidase system suggests the possibility that common modifier loci may control Rm in both enzymes.

An examination of the beta-glucosidase (linamarase) banding pattern in flax seedlings using Ferguson plots and sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The reported polyacrylamide gel electrophoresis banding pattern for the main beta-glucosidase (linamarase) component from flax seed consists of five bands, made up of 62.5 and 65 kDa subunits; this component has an estimated molecular weight of 570-670 kDa. The present study used Ferguson plots to estimate the molecular weight of each electrophoretic band, plus two additional bands which were detected. From low to high relative mobility, the seven bands formed a linear series with estimated molecular weights from 1200 to 245 kDa. Each was 160 kDa smaller, and less charged, than the preceding band. This 160 kDa difference between bands did not appear to be consistent with the reported subunit size. Each band produced a corresponding band on sodium dodecyl sulfate (SDS)-gels. The decreases in molecular weight between the bands on nondenaturing gels and their corresponding bands on SDS-gels were multiples of the 62-65 kDa value. However, the estimated molecular weights of the SDS bands themselves and of the differences between the SDS bands were, again, not consistent with the proposed subunit size. The results suggest that active forms of this enzyme may contain a second minor component (possibly a 30-35 kDa component) in addition to the 62.5 and 65 kDa subunits.

Molecular weight and net charge of peroxidase isozymes in F1 hybrids between L and S flax genotrophs.

In the F1 hybrids between Durrant's L and Durrant's S flax genotrophs, the relative mobilities of the anionic peroxidase isozymes were essentially the same as those in the L parent. The isozymes in both parents and their F1's were compared over a range of acrylamide gel concentrations, with plots of log relative mobility against gel concentration. Plots of comparative mobility, relative to the internal standard hemoglobin, against concentration were also examined. Both approaches provided evidence that apparent molecular weight modifications underlay the shift in mobility between the parents and the resemblance of the F1's to L, the parent which was homozygous for the dominant alleles controlling the mobility shift for at least two of the isozymes.

Molecular weight differences in acid phosphatases of stem homogenates from L and S flax genotrophs.

Anionic acid phosphatase (AP) isozymes in stem homogenates of flax genotrophs L and S were examined electrophoretically. The banding pattern was similar in both genotrophs. Relative mobility (Rm) was faster in each band of L than in the corresponding band of S. These Rm shifts did not result from differences in the sample concentration or from differential rates of development. AP activity was the same in both genotrophs. The banding pattern of F1 hybrids resembled the parental pattern. No multiple banding was observed in any of the F1 bands. The Rm in the F1 hybrids showed a dominance of L. Plots of loge (Rm) against gel concentration were constructed. While the Y intercepts of the plots indicated some differences in free electrophoretic mobility, molecular conformation was the major factor underlying both the Rm differences between bands and the Rm shifts between L and S, for corresponding bands. Estimates of apparent molecular weight (MW) for the different bands ranged from 25,000 to 250,000, with a strong possibility that some bands were oligomeric forms of others. Apparent MW was, on average, 17% higher in S than L.

A BASIC program for orthogonal polynomials and retrieval of regression coefficients for original model.

A fast, short method for calculating orthogonal polynomials has been programmed in BASIC. This is combined with the retrieval of b coefficients for the original, non-orthogonal model in, for example, a curvilinear regression. The program may be used alone, or in conjunction with a package of BASIC programs for weighted least-squares solutions, to which it forms an update. Transformation of the columns of an X, or design matrix, to orthogonal vectors can detect linear dependence among the columns, as well as supplying orthogonal polynomials for regression situations with unequal increments in x values, the values of the independent variable. Retrieval of the b coefficients for the original X matrix prior to transformation allows the user to insert any x value to obtain the corresponding predicted y (dependent variable) values.

Possible posttranslational modification, and its genetic control, in flax genotroph isozymes.

Environmentally induced flax genotrophs L and S show heritable shifts in the relative mobilities of peroxidase, esterase, and acid phosphatase isozymes, plus a number of nonspecific glycoproteins. All L isozymes migrated faster than corresponding S isozymes in 10% acrylamide gels. Various aspects of these shifts are reviewed here; it is proposed that posttranslational modification, probably of the carbohydrate moieties of these glycoproteins, underlies the shifts. This proposal is discussed in relation to the switch model for genotroph induction.

Charge and molecular weight differences for fourteen guaiacol peroxidase isozymes in flax indicate that most are encoded by different structural genes.

Ferguson plots, linear regressions of log(relative mobility) against polyacrylamide gel concentration, provide an electrophoretic method of simultaneously obtaining estimates of net charge and molecular weight for nondenatured proteins, and are a powerful method of accurately comparing these characteristics among isozymes within an enzyme system. Here, Ferguson plots compare the characteristics of the nine isozymes (three cationic and six anionic) usually seen in soluble extracts of flax (Linum usitatissimum) seedling tissues, and of five anionic isozymes induced by heavy metal stress. Despite some differences in apparent molecular weight, all but one of the isozymes separated from each other on the basis of net charge and, except for a second isozyme with a comparatively high molecular weight, the net charges of the isozymes ranked in order of their relative mobilities on nondenaturing gels. Differences among the isozymes make it unlikely that any are related, which indicates that they are encoded by different structural genes.

Isozyme relative mobility (Rm) changes related to leaf position; apparently smooth Rm trends and some implications.

Relative mobilities (Rm's) of peroxidase and acid phosphatase isozymes were examined in leaves of flax (Linum usitatissimum L.). The leaves were sampled from four equidistantly spaced positions from main stem base to apex in various genotypes. Rm's for the two slowest-migrating isozymes of each enzyme system changed in a simple, coherent fashion in leaves from stem bases toward apices. The Rm trends up the stem seen in two highly branched flax types were somewhat different from those in two sparsely branched types. The coherent Rm trends in the four types, suggesting a smooth continuum and a potentially large number of slightly different forms of these isozymes, are discussed in relation to other data for such Rm trends. In the study reported here, both enzyme systems behaved similarly. This fact and the simple Mendelian genetical system with no codominance controlling Rm's in flax peroxidases and acid phosphatases suggest posttranscriptional or posttranslational modifications as plausible mechanisms underlying the numerous, presumably small molecular changes generating the small, consistent changes in Rm's.

Genetic control of acid phosphatase Rm and its relation to control of peroxidase Rm in flax (Linum) genotrophs.

Evidence from various workers has indicated that isozyme relative mobility (Rm) may not be defined solely by the corresponding structural gene but may also be modified by alleles at other loci. The instances of numerous, small ongoing temporal or tissue changes in Rm for certain enzyme systems in plants may be another aspect of this modification due to interactions between genes. A further possible example of Rm modification occurs in connection with environmentally (fertilizer treatment) induced heritable changes within particular completely inbred and genetically homogeneous plant genotypes. Fertilizer-induced, persistent relative mobility (Rm) shifts for peroxidases are controlled by two alleles at one locus, a dominant for faster Rm and a recessive for slower Rm; codominance is completely absent. There are similar Rm shifts in acid phosphatases, likewise stemming from molecular weight changes. This study examined genetic control of the acid phosphatase Rm shift and its relation to peroxidase Rm control. It showed that the environmentally induced heritable acid phosphatase Rm shift is controlled by an identical system of a dominant (faster) and recessive (slower) allele, closely linked to the locus controlling peroxidases. The Rm shifts for both these enzyme glycoproteins are unidirectional, with no codominance; at least 10 other nonidentified glycoproteins display the same unidirectional Rm shifts. The results suggest modification, possibly posttranslational or transcriptional, controlled by modifier loci. This supports indications in other organisms that small numbers of modifier loci may control widespread Rm changes in the protein products of a genome.

Visualization of both peroxidase and acid phosphatase isozymes from flax (Linum) on the same gels.

The staining of both peroxidases (PERs) and acid phosphatases (APs) following electrophoresis on acrylamide gels is described. Scanning doubly stained gels at appropriate wavelengths reveals individual isozyme bands for both enzyme systems, giving essentially identical results to those obtained from separately run and stained gels. Collection of relative mobility (Rm) data is unaffected by such tandem staining. Few complications arise from overlapping PER and AP bands, or interactions between the two staining methods and PER or AP isozymes. Even if repeat scans of a gel at different wavelengths are needed to retrieve all isozyme information, dual staining provides distinct savings in time and sample material.