Biomedical subjects

J L Jorcano

Publications and source records attributed to J L Jorcano.

At least 55 records · Page 3Linked to original sources

PubMed1994

Transcriptional activation of histone H1 zero during neuronal terminal differentiation.

We have examined the central nervous system (CNS) of developing and adult transgenic mice carrying sequences upstream of the histone H1 zero gene fused to the E. coli beta-galactosidase gene (lac Z). The transgene is induced in a subset of the neuronal population during postnatal development, coinciding with neuronal terminal differentiation. At postnatal day 9, the earliest time at which the transgene product can be detected, positive neurons are observed in the granular layer of the cerebellar cortex and in the pyramidal fields of the hippocampus. The transgene is then induced in other areas of the CNS, such as the neocortex, thalamus, hypothalamus, olfactory bulb, globus pallidus superior and inferior colliculus, substantia nigra, pontine nuclei and brain stem. Induction is unrelated with determination and quiescence, which are essentially prenatal. The overlapping of the temporal and regional patterns of transgene activity with those of the endogenous protein shows that the accumulation of H1 zero in differentiating neurons is at least in part under transcriptional control. In the light of these results, the H1 zero gene appears as the only mammalian histone gene that specifically responds to terminal differentiation. However, not all terminally differentiated neurons express H1 zero at detectable levels. For instance, Purkinje cells are negative. In neurons, terminal differentiation appears thus as a necessary, but not a sufficient condition for increased H1 zero expression.

Animals↗

PubMed1994

Augmented expression of cytokines in mouse epidermal tumor cells and its possible involvement in the induction of hematopoietic alterations.

Mice with skin tumors induced either by 7,12-dimethylbenz[a]anthracene complete carcinogenesis or subcutaneous injection of a carcinogenic keratinocyte cell line showed moderate to severe splenomegaly as a result of an increase in splenic granulocyte-macrophage and erythroid (erythroid burst-forming unit) progenitors. To test whether the observed alterations involve the release of soluble factors by the epidermal component of skin tumors, we used an in vitro approach. A series of mouse keratinocyte cell lines resembling progressive stages of skin carcinogenesis and carrying either normal or activated Ha-ras genes were assayed for their ability to produce the factors required for colony growth of hematopoietic-committed progenitors. Only the conditioned media of keratinocytes harboring activated Ha-ras genes were able to support the growth of granulocyte-macrophage colony-forming units. In addition, preincubation of normal bone-marrow cells with conditioned media from the transformed epidermal cell lines stimulated in vitro amplification of the hematopoietic granulocyte-macrophage progenitor compartment. To identify the possible factors responsible for the activities detected in the keratinocyte-conditioned media, we performed northern blot analysis using the cytokine probes granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, stem cell factor, interleukin-1 alpha, interleukin-3, and tumor necrosis factor-alpha. The cell lines expressed different cytokine mRNA combinations that positively correlated with the colony-stimulating activity detected in the corresponding conditioned medium. These results suggest that transformed epidermal tumor cells in vivo may alter normal hematopoiesis as a consequence of the production of cytokines that act in autocrine or paracrine loops probably related to tumor growth.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1994

Assembly dynamics of epidermal keratins K1 and K10 in transfected cells.

Keratins K1 and K10 are early markers of keratinization. Their expression starts when basal keratinocytes are committed to differentiate. To study keratin stability and intermediate filament (IF) assembly and dynamics, we have forced the expression of K1/K10 in epithelial and nonepithelial cell lines. It was observed that these keratins are unable to generate a normal cytoskeleton in fibroblasts, where they form, at most, abnormally short and twisted filaments. However, when transfected into epithelial cells they frequently cointegrate with the endogenous keratin into a well-developed cytoskeleton. These results suggest that the K1/K10 pair is unable to form a keratin network on its own and that, mimicking the in vivo situation, requires a preexisting cytoskeleton. Besides, filaments, transfected K1/K10 also form fairly stable, regularly sized round aggregates with no evidence of organization into IF. Transfections with single genes demonstrated that these structures were generated by K10 alone. These aggregates interacted with most components of the cellular cytoskeleton, altering the distribution of the endogenous keratins, actin, vimentin, and tubulin. Kinetic experiments in transfected PtK2 cells showed that, contrary to expectations, K10 does not integrate directly into the preexisting cytoskeleton, but assembles into the stable nonfilamentous aggregates. From these structures, K10 evolves toward the formation of IF together with the endogenous keratins through a complex and highly dynamic process, which involves substantial rearrangement of the endogenous keratin cytoskeleton. These results demonstrate that K1 and K10 have properties different from those described for other keratins and that epithelial IF are surprisingly dynamic structures.

Animals↗

PubMed1994

Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.

Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.

Aging↗

PubMed1993

Specific expression in adult mice and post-implantation embryos of a transgene carrying the histone H1(0) regulatory region.

Histone H1(0), a variant of the H1 group, has been found associated with the repressed state of chromatin and its content is increased in terminally differentiated cells. We have cloned a mouse H1(0) histone gene and introduced the promoter region, ligated to the beta-galactosidase reporter gene, into transgenic mice. By histochemistry we demonstrated a strong expression of the transgene in adult kidney, testis and brain. Intestine, uterus and ovarium were also positive. This expression followed the same pattern as that of the endogenous H1(0) gene, as demonstrated by in situ hybridization with a non-coding fragment of the mRNA, by Northern analysis, and by immunofluorescence with specific antibodies. In post-implantation embryos, the expression was very low up to day ten p.c. At this time, most of the X-Gal staining was found in the brain, retina and some of the large blood vessels. Hence, expression of the transgene as well as of the endogenous H1(0) gene is not exclusively linked to a differentiated phenotype or to a reduced cell proliferation capacity.

Animals↗

PubMed1992

Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.

Animals↗

PubMed1992

Aberrant expression of the simple epithelial type II keratin 8 by mouse skin carcinomas but not papillomas.

Keratins have been demonstrated to be suitable markers of changes taking place during epithelial neoplasia. Therefore, we analyzed 18 mouse skin tumors (nine papillomas and nine squamous cell carcinomas), induced either by two-stage carcinogenesis with 7,12-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetat e or complete carcinogenesis with DMBA, by immunofluorescence with a monoclonal antibody to keratin (K) 8 (TROMA-1). Immunoperoxidase staining and immunoblotting were also used on selected tumor samples to further explore for the presence of K8. All of the papillomas tested were negative for the presence of K8, whereas the carcinomas were positive. The level of K8 expression in carcinomas showed a positive correlation with the degree of malignancy. Northern blot analysis using a K8 cDNA probe suggested that control of K8 expression in mouse skin tumors occurs at the transcriptional level. Double-label immunofluorescence staining using TROMA-1 and RK13 antibodies demonstrated that K8 did not generally colocalize with K13, a keratin normally found in internal stratified epithelial but aberrantly expressed in mouse epidermal tumors. Furthermore, tumors expressing high levels of K8 showed a reduced expression of K13. Histological examination of immunoperoxidase-stained tumors demonstrated that K8-positive cells were mainly found in anaplastic areas, whereas K13 foci were restricted to well-differentiated regions. Our results demonstrate that K8 expression is a marker of late stages of carcinoma progression in the mouse skin carcinogenesis model.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Mapping of bovine cytokeratin sequences to four different sites on three chromosomes.

The chromosomal location of bovine class I and class II cytokeratin sequences was determined using in situ hybridization and Southern blot hybridization to DNA from hybrid somatic cells. The main signals were found over chromosome region 19q16----qter after in situ hybridization with two probes for the class I cytokeratin gene subfamily (KRT10 and KRT19) and over region 5q14----q23 after hybridization with probes for the class II gene subfamily (KRT1, KRT5, and KRT8). These regions most likely contain the loci of functional cytokeratin genes, with KRT10 and KRT19 mapping to 19q21 and KRT1, KRT5, and KRT8 to 5q21. The in situ hybridization data were corroborated by analysis of a somatic hybrid cell panel. The genes for the class I keratins segregated concordantly with each other and syntenic group U21 but were discordant with the class II keratin genes. The class II keratin genes segregated concordantly with each other and syntenic group U3. Two class II gene probes gave an additional minor signal above chromosome region 5q25----q33 after in situ hybridization, while another class II probe yielded a minor signal above chromosome region 10q31----qter. When the latter probe and an additional linked probe were hybridized to DNAs from a hybrid panel, two independently segregating loci were recognized, one of which cosegregated with the class II subfamily in syntenic group U3 and the other with syntenic group U5. These data confirm the chromosomal assignment of two syntenic groups and allow the assignment of a formerly unassigned syntenic group.

Animals↗

PubMed1989

Enhancer elements directing cell-type-specific expression of cytokeratin genes and changes of the epithelial cytoskeleton by transfections of hybrid cytokeratin genes.

The cytokeratins, which form the intermediate filaments (IFs) characteristic of epithelial cells, are encoded by a large family of genes whose members are differentially expressed in patterns different in the various kinds of epithelia. To identify possible cis-regulatory DNA elements involved in the cell-type-specific expression of these genes, we examined, in transfection assays, 5' upstream sequence intercepts of a certain cytokeratin gene, i.e. that for bovine cytokeratin IV* (CKIV*), in combination with the coding portions of either the chloramphenicol acetyltransferase (CAT) gene or other cytokeratin genes. A 5' upstream region located between the cap-site and nucleotide -605 was found to enhance the specific expression of these reporter genes in bovine mammary gland-derived BMGE + H cells, which express the endogenous gene, but not in bovine kidney epithelium-derived MDBK cells which synthesize cytokeratins other than IV*. This epithelium-type-specific expression was also observed in heterologous combinations, e.g. in murine keratinocytes, but not in other murine cell lines such as 3T3 fibroblasts. When a fragment located between -180 and -605 was coupled to the HSV-TK promoter it stimulated the expression of the reporter gene in a cell-type-specific manner. The enhancer character of this 425 nucleotide long region is also demonstrated. Moreover, the CKIV* promoter/enhancer complex was able to direct the expression of epidermal cytokeratins characteristic for suprabasal differentiation, i.e. bovine cytokeratins Ia and VIb, in cells that normally do not express these genes. We show that the newly synthesized cytokeratins integrate into the pre-existing cytokeratin IF system of the transfected cells and that the forced expression of one of these cytokeratins does not induce the endogenous gene encoding its normal pair partner.

Animals↗

PubMed1988

Synthesis and fate of keratins 8 and 18 in nonepithelial cells transfected with cDNA.

To study the assembly of intermediate filaments in vivo we have transfected fibroblast cell lines with the cDNAs coding for keratins 8 and 18 under the control of the promoter of the SV40 early region and followed keratin expression by RNA hybridization, two-dimensional gel electrophoresis, and immunofluorescence analysis. When expressed individually, keratins 8 and 18 failed to polymerize into intermediate filaments but formed granular aggregates of variable size distributed throughout the cytoplasm as seen by staining with specific antibodies. The expression of one of these two keratins did not induce the synthesis of its partner or of any other keratin. Coexpression of the two keratins produced filamentous structures, frequently perinuclear, indicating that the two types of polypeptides were able to assemble into intermediate filaments but could not form the cytoskeleton characteristic of epithelial cells. These results demonstrate that assembly in heterocomplexes stabilizes keratins against cellular degradation, helping to explain why excess pools of simple keratins have never been detected.

Animals↗

PubMed1988

Participation of cytoskeletal intermediate filaments in the infectious cycle of human respiratory syncytial virus (RSV).

RSV infection of Hep-2 or HeLa cells leads to biochemical and morphological changes of cytoskeletal intermediate filaments (IF). Thus, human cytokeratin 18 is modified to generate a more acidic polypeptide of slightly larger apparent molecular weight. In addition, the amounts of vimentin and other cytokeratins are reduced, probably as a consequence of proteolytic degradation. These changes are reflected in a decrease of immunofluorescence with specific antibodies in RSV-induced syncytia and a more disorganized arrangement of IF arrays. About 50% of virus nucleoprotein (NP) is extracted with the high salt and detergent-insoluble intermediate filament fraction. Pulse-chase experiments indicate that NP needs a maturation period after synthesis to associate with IF. It is suggested that RSV needs to interact with IF during its life cycle and that association of NP, and/or other viral components, with IF might then lead to cytoskeletal structures becoming unstable in RSV-infected cells.

Carcinoma, Hepatocellular↗

PubMed1987

Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5'-upstream sequences.

Cytokeratins are a family of approximately 20 polypeptides which form the intermediate-sized filaments (IFs) characteristic of epithelial cells. They are synthesized co-ordinately as 'pairs' consisting of one representative from each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the more basic (type II) polypeptides, in cell type-specific combinations. We have isolated and characterized the genes coding for four bovine cytokeratins of the basic (type II) subfamily, i.e. cytokeratins Ib, III, IV and 6*, by Southern blot hybridization, hybridization-selection-translation experiments, hetero-duplex mapping, and partial sequencing of the exons coding for the hypervariable carboxy-terminal 'tail' regions of the proteins and the 3'-non-translated ends of the mRNAs which are distinct for the individual cytokeratin polypeptides. Limited 'chromosomal walk' experiments demonstrated that the genes are organized into two tandems, i.e. 6*----Ib and III----IV, in which they are separated by approximately 11 kb. RNA analysis by Northern and dot blots show that both genes of the III----IV tandem are co-expressed in some bovine tissues (muzzle epidermis, hoof pad and tongue mucosa) and cultured cells (BMGE + H) but that in other tissues, cornea for example, only the gene encoding III is expressed. Unexpectedly, the genes linked in the tandem 6*----Ib are not co-expressed in any of the tissues examined. mRNA from gene 6* has been found in tongue mucosa but in none of the other cell lines and tissues examined, whereas mRNA for cytokeratin Ib is expressed in cornea and muzzle epidermis but not in, for example, tongue mucosa and in the epidermis of the heel pad.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Cytokeratin No. 9, an epidermal type I keratin characteristic of a special program of keratinocyte differentiation displaying body site specificity.

Plantar epidermis of the bovine heel pad as well as human plantar and palmar epidermis contain large amounts of an acidic (type I) keratin polypeptide (No. 9) of Mr 64,000 which so far has not been found in epidermis of other sites of the body. We present evidence for the keratinous nature of this protein, including its ability to form cytokeratin complexes and intermediate-sized filaments in vitro. We have isolated RNA from plantar epidermis of both species and show, using translation in vitro, that these polypeptides are genuine products of distinct mRNAs. Using immunofluorescence microscopy with specific antibodies against this protein, we demonstrate its location in most cells of suprabasal layers of plantar epidermis as well as in sparse keratinocytes which occur, individually or in small clusters, in upper layers of epidermis of other body locations. We conclude that cytokeratin No. 9 is characteristic of a special program of keratinocyte differentiation which during morphogenesis is expressed in most epidermal keratinocytes of soles and palms but only in a few keratinocytes at other body sites. This example of cell type-specific expression of a member of a multigene family in relation to a body site-related program of tissue differentiation raises important biological questions concerning the regulation of keratinocyte differentiation and morphogenesis as well as the function of such topological heterogeneity within a given type of tissue.

Animals↗

PubMed1986

Cytokeratin expression in simple epithelia. II. cDNA cloning and sequence characteristics of bovine cytokeratin A (no. 8).

Cytokeratin A (no. 8) is a cytoskeletal protein (Mr, approximately 53,000 in bovine cells) which is typical of all simple epithelia, is widespread in all cultured epithelial cells, and together with its partner cytokeratin D, is the first cytokeratin expressed during embryogenesis (synonyms for this protein are Endo A and TROMA-1 antigen). We isolated a clone (pKB8(1] from a pUC8 cDNA library prepared from poly(A)+-RNA of bovine bladder urothelium which contains the 3' nontranslated portion and the sequence coding for the carboxyterminal tail and almost the whole of the alpha-helical rod (369 amino acids). Northern-blot analysis showed that the mRNA coding for this cytokeratin is specifically synthesized in various epithelial tissues and in epithelial cell culture lines. The amino acid sequence of this cytokeratin, when compared with the sequences of other intermediate filament (IF) proteins, exhibits a high and specific homology with other cytokeratins of the basic (type II) subfamily; this homology is, however, restricted to the rod portion. The tail region, which is rich in hydroxy-amino acids (approximately 35%), is unique among the type-II cytokeratins in that it does not exhibit subdivision in three domains, specifically lacking the glycine-rich middle domain. Sequence comparison with a partial sequence of the corresponding cytokeratin of the amphibian species, Xenopus laevis, indicated high evolutionary conservation. The high sequence homology of bovine cytokeratin A with published sequences of human tissue polypeptide antigen (TPA), a soluble serum component used as tumor marker in clinical oncology, supports the view that TPA is a proteolytically solubilized fragment containing the rod portion of human cytokeratin no. 8. Our analysis of clone pKB8(1) made possible the first comparison of a simple epithelial cytokeratin with epidermal keratins and other IF proteins. This showed that, in some important molecular features, cytokeratin A (no. 8) differs drastically from the epidermal members of the same cytokeratin subfamily, probably reflecting different cellular functions of the tail region in stratified and simple epithelia.

Amino Acid Sequence↗

PubMed1985

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily.

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1985

Patterns of expression and organization of cytokeratin intermediate filaments.

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.

Amino Acid Sequence↗

PubMed1984

Drosophila melanogaster U1 snRNA genes.

We have isolated and characterized a recombinant which contains a Drosophila melanogaster U1 small nuclear RNA (snRNA) gene colinear with the published snRNA sequence. Southern hybridizations of the fly genomic DNA, using as probe a plasmid containing only the coding region of the gene, shows that the fly contains at most three or four genes and very few related sequences for the small nuclear U1 RNA. These genes were localized by in situ hybridization at different chromosomal loci and show no spatial relationship to the U2 snRNA genes.

Animals↗

PubMed1984

Divergence of U2 snRNA sequences in the genome of D. melanogaster.

Four different U2-snRNA genes/related sequences of D. melanogaster were cloned and characterized. The sequences of all four genes suggest that they were generated by a DNA-mediated mechanism. These genes/related sequences were found to be located in two loci, each locus containing two U2 snRNA sequences. Using coding sequences as well as flanking sequences as hybridization probes against polytene chromosomes of D. melanogaster Oregon R we were able to map these loci separately at positions 34BC and 84C. By Northern analysis we observed that the quantities of U2- and U1-snRNA are coordinated and change during the embryonic development of the fly.

Animals↗