Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.

Dendritic cells are potent antigen-presenting cells that initiate primary immune responses. Although dendritic cells derive from bone marrow stem cells, the intermediate stages in their development remain unknown. In this study, plastic-adherent blood monocytes (CD14+, CD1a-) cultured for 7 days with granulocyte-monocyte colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha were shown to differentiate into CD1a+ CD83+ dendritic cells. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were the most potent stimulatory cells in allogeneic mixed leukocyte reactions. The identification of specific culture conditions that generate large numbers of dendritic cells from purified monocytes uncovers an important step in dendritic cell maturation that will allow the further characterization of their role in autoimmune diseases, graft rejection, and human immunodeficiency virus infection.

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.

Telomere length and replicative aging in human vascular tissues.

Because repeated injury of the endothelium and subsequent turnover of intimal and medial cells have been implicated in atherosclerosis, we examined telomere length, a marker of somatic cell turnover, in cells from these tissues. Telomere lengths were assessed by Southern analysis of terminal restriction fragments (TRFs) generated by HinfI/Rsa I digestion of human genomic DNA. Mean TRF length decreased as a function of population doublings in human endothelial cell cultures from umbilical veins, iliac arteries, and iliac veins. When endothelial cells were examined for mean TRF length as a function of donor age, there was a significantly greater rate of decrease for cells from iliac arteries than from iliac veins (102 bp/yr vs. 47 bp/yr, respectively, P < 0.05), consistent with higher hemodynamic stress and increased cell turnover in arteries. Moreover, the rate of telomere loss as a function of donor age was greater in the intimal DNA of iliac arteries compared to that of the internal thoracic arteries (147 bp/yr vs. 87 bp/yr, respectively, P < 0.05), a region of the arterial tree subject to less hemodynamic stress. This indicates that the effect is not tissue specific. DNA from the medial tissue of the iliac and internal thoracic arteries showed no significant difference in the rates of decrease, suggesting that chronic stress leading to cellular senescence is more pronounced in the intima than in the media. These observations extend the use of telomere size as a marker for the replicative history of cells and are consistent with a role for focal replicative senescence in cardiovascular diseases.

Selenophosphate synthetase: detection in extracts of rat tissues by immunoblot assay and partial purification of the enzyme from the archaean Methanococcus vannielii.

In Escherichia coli and Salmonella typhimurium it has been shown that selenophosphate serves as the selenium donor for the conversion of seryl-tRNA to selenocysteyl-tRNA and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs. Although selenocysteyl-tRNA also is formed in eukaryotes and is used for the specific insertion of selenocysteine into proteins, the precise mechanism of its biosynthesis from seryl-tRNA in these systems is not known. Because selenophosphate is extremely oxygen labile and difficult to identify in biological systems, we used an immunological approach to detect the possible presence of selenophosphate synthetase in mammalian tissues. With antibodies elicited to E. coli selenophosphate synthetase the enzyme was detected in extracts of rat brain, liver, kidney, and lung by immunoblotting. Especially high levels were detected in Methanococcus vannielii, a member of the domain Archaea, and the enzyme was partially purified from this source. It seems likely that the use of selenophosphate as a selenium donor is widespread in biological systems.

Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis.

Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.

bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death.

Previous studies have implicated the bcl-2 protooncogene as a potential regulator of neuronal survival. However, mice lacking functional bcl-2 exhibited normal development and maintenance of the central nervous system (CNS). Since bcl-2 appears dispensable for neuronal survival, we have examined the expression and function of bcl-x, another member of the bcl-2 family of death regulatory genes. Bcl-2 is expressed in neuronal tissues during embryonic development but is down-regulated in the adult CNS. In contrast, Bcl-xL expression is retained in neurons of the adult CNS. Two different forms of bcl-x mRNA and their corresponding products, Bcl-xL and Bcl-x beta, were expressed in embryonic and adult neurons of the CNS. Microinjection of bcl-xL and bcl-x beta cDNAs into primary sympathetic neurons inhibited their death induced by nerve growth factor withdrawal. Thus, Bcl-x proteins appear to play an important role in the regulation of neuronal survival in the adult CNS.

Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages.

Human macrophages are believed to damage host tissues in chronic inflammatory disease states, but these cells have been reported to express only modest degradative activity in vitro. However, while examining the ability of human monocytes to degrade the extracellular matrix component elastin, we identified culture conditions under which the cells matured into a macrophage population that displayed a degradative phenotype hundreds of times more destructive than that previously ascribed to any other cell population. The monocyte-derived macrophages synthesized elastinolytic matrix metalloproteinases (i.e., gelatinase B and matrilysin) as well as cysteine proteinases (i.e., cathepsins B, L, and S), but only the cathepsins were detected in the extracellular milieu as fully processed, mature enzymes by either vital fluorescence or active-site labeling. Consistent with these observations, macrophage-mediated elastinolytic activity was not affected by matrix metalloproteinase inhibitors but could be almost completely abrogated by inhibiting cathepsins L and S. These data demonstrate that human macrophages mobilize cysteine proteinases to arm themselves with a powerful effector mechanism that can participate in the pathophysiologic remodeling of the extracellular matrix.

Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.