Identification of plectin in different human cell types and immunolocalization at epithelial basal cell surface membranes

The occurrence of plectin in various human tissues and cell lines was investigated using immunofluorescence microscopy and antibody gel overlay/immunoblotting techniques. Plectin was identified in all tissues and cell lines tested, namely placenta, kidney, cornea, foreskin and eyelid skin, skin fibroblasts, monocytes, keratinocytes and HeLa cells. In frozen sections of cornea and skin, plectin was found to be enriched at epithelial basal cell surface membranes. Consequently, antibodies to plectin could serve as a tool in the classification of mechanobullous diseases.     

Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase.

Human cytidine deaminase (HCD) catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. The genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals, but an alternative form of HCD has yet to be reported. Here we describe the cloning and characterization of a small form of HCD, HSCD, and it is likely to be a product of alternative splicing of HCD. The alignment of DNA sequences shows that the HSCD matches HCD in 2 parts, except for a deletion of 170 bp. Based on the HCD genome organization, exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of the reading frame from the point of splicing. The estimated molecular mass is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with glutathionine-S-transferase in the pGEX vector. The deletion and shift of the reading frame caused a loss of HCD activity, which was confirmed by enzyme assay and also with NIH3T3 cells modified to express HSCD and challenged against cytosine arabinoside. In this work we describe the identification and characterization of HSCD, which is the product of alternative splicing of the HCD gene.     

Galectin-1 receptors in different cell types

Summary Galectins are a family of animal lectins defined by two properties: shared amino acid sequences in their carbohydrate-recognizing domain, and -galactoside affinity. A wide variety of biological phenomena are related to galectins, i.e., development, differentiation, morphogenesis, tumor metastasis, apoptosis, RNA splicing, and immunoregulatory function. In this review, we will focus on galectin-1 receptors, and some of the mechanisms by which this lectin affects different cell types. Several galectin-1 receptors are discussed such as CD45, CD7, CD43, CD2, CD3, CD4, CD107, CEA, actin, extracellular matrix proteins such as laminin and fibronectin, glycosaminoglycans, integrins, a -lactosamine glycolipid, GM1 ganglioside, polypeptide HBGp82, glycoprotein 90 K/MAC-2BP, CA125 cancer antigen, and pre-B cell receptor.This revised version was published online in April 2005. In the previous version the name of the last author was missing.     

Zfa is an expressed retroposon derived from an alternative transcript of the Zfx gene.

ZFY, a gene on the Y chromosome encoding a zinc finger protein, has been proposed as a candidate for the human testis determining gene. Sequences related to ZFY, called ZFX, are present on the X chromosome of a wide range of placental mammals. Unlike most mammals the mouse has four genes homologous to ZFY; two on the Y chromosome, Zfy-1 and Zfy-2, an X-linked gene, Zfx, and an autosomal gene, Zfa. We show here that Zfa has arisen recently by retroposition of one of at least three alternatively spliced mRNAs transcribed from the Zfx gene. Zfa is an unusual retroposon in that it has retained an open reading frame and is expressed, although its function may be limited or altered by the presence of a potentially inactivating mutation in the third of its zinc fingers. This mutation must have occurred at the same time or soon after the retroposition event as it is also present in the Zfa gene of Mus spretus. Interestingly the third finger of the M. musculus musculus Zfy-2 gene has also sustained a mutation suggesting that this gene family may be rapidly evolving in mice.     

Identification of an S-adenosylhomocysteine hydrolase-like transcript induced during dendritic cell differentiation

Dendritic cells (DC) are the professional antigen-presenting cells that initiate immune responses. While DC take up antigen, migrate to lymph nodes and present processed antigen to T lymphocytes, little is known of the intracellular biochemical pathways controlling these events. Using the differential display technique, employing the activated blood DC-like cell line L428, we isolated a cDNA induced during DC differentiation likely to have a regulatory function. This cDNA encoded a putative 530-amino-acid (aa) protein consisting of a unique hydrophilic domain (106 aa) and a domain (424 aa) similar to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (AHCY). Therefore, this molecule was termed DC-expressed AHCY-like molecule (DCAL). DCAL mRNA was expressed moderately in fresh blood DC, but was not detectable in other peripheral blood mononuclear cells. DCAL mRNA increased markedly during activation of blood and skin DC (Langerhans cells), but was diminished in terminally differentiated tonsil DC. Cultured monocytes expressed little DCAL mRNA, but levels increased markedly when differentiated into DC by cytokines GM-CSF and IL-4. The DCAL gene [Chromosome (Chr) 1] and another previously identified DCAL-like molecule KIAA0828 (Chr 7) differed from the AHCY gene (Chr 20) in gene organization. Thus, DCAL may have a role in controlling critical events in DC differentiation and belong to a novel family of AHCY-like molecules.     

Identification of the alternative splice products encoded by the human protein phosphatase inhibitor-1 gene

We have isolated three major cDNA fragments of protein phosphatase inhibitor-1 from human brain and liver by RT-PCR. The 536 bp fragment encoded the wild-type of inhibitor-1 while two other fragments were alternative splice products of the inhibitor-1 gene, which was confirmed by partial genomic DNA sequencing. The 380 bp fragment encoded an in-frame 51-residue-deleted inhibitor-1, named inhibitor-1alpha, and the deletion occurred from residue 84 to 134 of inhibitor-1. The 316 bp fragment termed inhibitor-1beta was derived from an internal deletion of 536 bp fragment. This deletion resulted in an out of frame shift, allowing the 316 bp fragment that encoded the partial sequence of inhibitor-1. Based on the reported mRNA sequence of inhibitor-1 and evidence from our RT-PCR, we suggested that inhibitor-1beta consisted of 132 amino acids of which the N-terminal 61 amino acid sequences were identical to inhibitor-1 while the sequence after residue-61 was markedly different.