Cecilia Montañez

Dystrophins in developing retina: Dp260 expression correlates with synaptic maturation.

Dystrophin, the protein altered in Duchenne muscular dystrophy (DMD), is necessary for normal retinal function and exists in several isoforms. We examined the expression of dystrophin and utrophin proteins and transcripts in the rat retina at different developmental stages using Western blots and semi-quantitative RTPCR. Our results revealed the presence of utrophin (DRP1), G-utrophin and/or DRP2 and four dystrophin isoforms (Dp427, Dp260, Dp140, Dp71) in the normal adult rat retina. Only Dp260 showed a marked progressive increase with age at both protein and mRNA levels. This variation is consistent with the establishment of synaptic functions in the developing retina and suggests a key role for this apo-dystrophin in synaptogenesis.

Sp1 and AP2 transcription factors are required for the human fragile mental retardation promoter activity in SK-N-SH neuronal cells

The promoter of the human fragile mental retardation gene (FMR1) was functionally analyzed in order to identify elements responsible for its regulation. Plasmids carrying the wild type or different deleted-promoter sequences driving the chloramphenicol acetyl transferase gene (CAT) were transiently transfected into the SK-N-SH cells and the CAT activity was assessed. Deletion studies suggested that major regulatory elements are present in a DNA region between positions -123 and -51. Gel mobility shift and footprinting assays using a DNA fragment encompassing that promoter region showed that SP1 and AP2 transcription factors could be involved in the functioning of the FMR1 promoter.

Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

Giardia lamblia: Isolation of RNA

In this study we report the isolation of RNA from two strains of the parasitic protozoan Giardia lamblia: Pl and WB. In order to detect all rRNA populations present in this parasite, we used three different methods to isolate total RNA. These included phenolchloroform extraction (Brawerman 1974), guanidine isothiocyanate extraction (Chirgwin et al. 1979), and a modified method of hydroxylapatite chromatography (Markow and Ivanov 1974). This latter one consisted of the use of 0.19 M sodium phosphate solution, pH 6.8, to elute total RNA. The yields of total RNA by the three methods used were 143, 21, and 51 kg/l X IO8 trophozoites, respectively. Among the methods used, phenol chloroform extraction was the most efficient procedure to obtain G. lamblia RNA. Gel electrophorcsis analysis, under nondenaturing conditions, of total RNA obtained from the Pl and WB strains by the three different methods, revealed the presence of two prominent rRNA bands which correspond to the large (LSrRNA) and small (SSrRNA) rRNA species of G. lamblia, as has also been reported by Boothroyd et al. (1987) and Edlind and Chakraborty (1987). The electrophoretic mobility of these species is shown in Fig. 1. A difference was observed in the pattern of total RNA extracted by guanidine isothiocyanate in that low molecular weight RNAs were hardly detected (lane 2). The RNA samples from Pl and WB strains were also analyzed under strong denaturing conditions. In this, RNA preparations were heated in a solution of formamideformaldehyde at 60°C followed by electrophoresis in agarose gels containing formaldehyde. This analysis revealed only two populations of 2390 and 1420 nucleotides. According to their intensity after staining with ethidium bromide, these RNA populations may be present in similar amounts in this parasite (rat brain and Escherichia coli rRNAs were used as standards). None of the methods used revealed a large labile rRNA as has been described for some protozoan (Albath et al. 1984; Castro er al. 1981). RNA purified by phenol extraction or by hydroxylapatite chromatography was centrifuged through sucrose gradients under nondenaturing conditions. The profiles of the optical density measurements from the collected fractions revealed the presence of two peaks which corresponded to the two rRNA species of G. lamb& (Pl strain) (Fig. 2). Sedimentation values of large and small rRNAs calculated using rat brain rRNAs as standards were 21s and 15S, respectively. The same sedimentation values were obtained for rRNAs from WB strain (data not shown). In order to determine the abundance of low molecular weight RNA species, RNA preparations from Pl and WB strains obtained by the three methods were fractionated by denaturing polyacrylamide gel electrophoresis. Two major RNA populations and three less abundant species of lower molecular weight were observed after isolation by phenol extraction and hydroxylapatite chromatography (Pl strain) (Fig. 3). The sizes of the two prominent RNA molecules were determined relative to denatured rat brain and yeast 5.8s and 5s RNA standards, and corresponded to approximately 130 and 118 bases. RNA isolated by guanidine isothiocyanate extraction contains only one band which corresponds to the 130 bases specie; other populations were not observed. This may be due, in part, to the low recovery of smaller RNA populations by this method. Integrity of the isolated RNA by phenolchloroform extraction, guanidine isothiocyanate extraction, and hydroxylapatite chromatography was confirmed by in vitro translation experiments in which a broad spectrum of polypeptides were obtained. Interestingly, all RNA species identified from both strains of G. fumblia are smaller than most of the prokaryotic and eukaryotic rRNAs described (Noller 1984). These results, together with the data obtained for other protozoa, show a considerable diversity in eukaryotic rRNAs, their genes, and the processing mechanisms in these organisms. The complete characterization of rRNA as well as their genes will contrib-

Expression and localization of utrophin in differentiating PC12 cells

To ascertain the role of utrophin in cultured neuronal cells, we investigated its expression and distribution along the NGF-induced differentiation of PC12 cells grown on different substrata. Utrophin mRNA was measured by RT-PCR assay and utrophin protein was quantified by immunoblot analysis. The distribution of utrophin and β-dystroglycan was analyzed by confocal microscopy. We demonstrate that utrophin protein was increased 4-fold during differentiation of cells grown on laminin. Concomitant with this up-regulation, utrophin was enriched at the growth cones in differentiating cells, where it co-localizes with β-dystroglycan. These data suggest the presence of a utrophin–β-dystroglycan complex in PC12 cells that participates in the formation and/or stabilization of the growth cone-extracellular matrix adhesion.

Dp71, utrophin and beta-dystroglycan expression and distribution in PC12/L6 cell cocultures.

Function of dystrophin Dp71 isoforms is unknown but seems related to neurite outgrowth and synapse formation. To evaluate Dp71 role in myoneural synapses, we established a coculture model using PC12 cells and L6 myotubes and analyzed expression and localization of Dp71 and related proteins, utrophin and &bgr;-dystroglycan, in PC12 cells. Confocal microscopy showed Dp71d isoform in PC12 nuclei, golgi-complex-like and endoplasmic reticulum-like structures, whereas Dp71ab concentrates at neurite tips and cytoplasm, colocalizing with &bgr;-dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest that Dp71ab isoform, unlike Dp71d, may take part in neurite-related processes. This is the first work on Dp and members of Dp-associated protein complex roles in a cell-line based coculturing system, which may be useful in determining Dp71 isoforms associations.

Myotonic Dystrophy Protein Kinase (DMPK) Gene Expression in Lymphocytes of Patients with Myotonic Dystrophy

BACKGROUNDnMyotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder with defects in many tissues, including skeletal muscle myotonia, progressive myopathy, and abnormalities in heart, brain, and endocrine systems. It is associated with a trinucleotide repeat occurring in the 3 (UTR) untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Several studies have been carried out to determine DMPK gene expression in muscle and non-muscle tissues.nnnMETHODSnDMPK gene expression was determined in lymphocytes of adult-onset patients with DM and normal controls. To quantitate total locus expression as well as allele-specific mRNA levels, semiquantitative RT-PCR assay was used. Allele-specific expression was analyzed using a Bpm1 polymorphism located at exon 10 of the DMPK gene.nnnRESULTSnIn heterozygous patients with DM, we observed a fourfold difference between mRNA levels produced by the Bpm1-undigested allele (187 bp) compared to the Bpm1-digested allele (136 bp). By using (CTG) trinucleotide (with cytosine, thymine, and guanine) expansion polymorphism, it was shown that the down-regulated allele corresponds to the mutant allele. Interestingly, the reduction in the mutant allele-transcript levels is compensated by an increase of the wild-type allele, yielding no significant differences in total locus mRNA amount between patients and normal individuals.nnnCONCLUSIONSnThese results suggest that the expression of the two alleles at the DMPK locus in lymphocytes is coordinated. The reduction in mutant-allele transcript levels is compensated by an increase in wild-type allele mRNA levels.

Altered astrocyte morphology and vascular development in dystrophin-Dp71-null mice

Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti‐GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild‐type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71‐null mice, a reduction in GFAP‐immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real‐time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post‐natal vascular development. Regarding morphology in Dp71‐null and WT mice, a significant decrease in overall astrocyte process number in Dp71‐null retinas at P6 to adult age was found. Using fluorescence‐conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71‐null mice was found. An evidence that the Dystrophin Dp71, a membrane‐associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided. GLIA 2016;64:716–729

Molecular cloning of cellulolytic enzyme genes from Cellulomonas flavigena in E. coli

Abstract A genomic bank of Cellulomonas flavigena was constructed in E. coli using the pUC18 vector, and over 14000 clones screened for cellulolytic activity. Three different cellulolytic enzyme genes were cloned, one coding for an endo-β-glucanase (pJS10, CMC activity) and two coding for β-glucosidases, each with a distinct substrate specificity (pJS3, X-glu, and pJS4, X-glu and MUC activities). These three inserts have different restriction patterns to each other and the previously isolated cellulolytic enzyme genes from C. fimi and C. uda .

Dp71Δ78-79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1.

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.