Human Leukocyte cDNA Research Articles

Gene expression profiling of human promyelocytic leukemia HL-60 cell treated by ajoene

Objective: Ajoene, a major compound extracted from crashed garlic, has been shown to have antitumor, antimycotic, antimicrobial, antimutagenic functions in vivo or in vitro and treated as a potential antitumor drug. However, the molecular mechanisms underlying the tumor cytotoxicity of ajoene and even garlic substances are poorly defined. In the present study, we aimed to generate gene expression profiles of HL-60 cell treated by ajoene. Methods: A cDNA microarray presenting 2400 of genes amplified from human leukocyte cDNA library was constructed and the gene expression profiles of HL-60 cell induced by ajoene were generated. Results: After data analysis, 28 differentially expressed genes were identified and sequenced. These genes include 21 known genes and 7 ESTs. Most of the known genes are related to cell apoptosis, such as secretory granule (PRG1), beta-2 microglobulin (B2M), 16S ribosomal RNA gene and ribosomal protein S12. Several genes are related to cell differentiation, including the genes similar to H3 histone and ribosomal protein L31. Northern blot analysis was used to verify and quantify the expression of selected genes. Conclusion: Ajoene can induce HL-60 cell apoptosis significantly and may play a role in differentiation. cDNA microarray technology can be a valuable tool to gain insight into molecular events of pharmacological mechanism of herbal medicine.

Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI)

The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67 RhoA-Arg68 RhoA-Leu69 RhoA as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.

Molecular Cloning and Characterization of a New Human Histamine Receptor, HH4R

A new histamine receptor, HH4R, was cloned from human leukocyte cDNA. The deduced amino acid sequence showed about 40% identity to that of the human histamine H3 receptor, HH3R. HH4R-expressing cells responded to histamine, inhibiting forskolin-induced cAMP accumulation. An H3 agonist, N-α-methylhistamine (NAMHA), bound specifically to HH4R, while another H3 agonist, R(−)-α-methylhistamine (RAMHA), and the H3 antagonist, thioperamide, competed with this binding. RAMHA, NAMHA, and imetit inhibited forskolin-induced cAMP accumulation in HH4R-expressing cells. However, the binding affinities and agonistic activities of H3 agonists to HH4R were weaker than those to HH3R. Low expression of HH4R was detected in a wide variety of peripheral tissues by RT-PCR; however, in contrast with HH3R, expression was not detected in the brain. These observations indicate that the clone is a distinct histamine receptor from HH3R, and thus is named HH4R.

Cloning and Characterization of cDNA Encoding a Novel Human Leukotriene B4 Receptor

By homology screening using BLAST searches of expressed sequence tags (ESTs), we have found a previously unidentified cDNA encoding a putative seven-transmembrane receptor with highest similarity to the leukotriene B4 receptor, BLTR. Analysis of calcium flow in transfected cells, along with sequence analysis, revealed that the EST encoded a functionally inactive protein, lacking the segment corresponding to the C-terminal part of the putative receptor protein. The missing segment was obtained by PCR amplification of a human leukocyte cDNA library and ligated to the truncated EST cDNA. The novel cDNA encodes a full-length receptor with 39% identity to the previously cloned BLTR. Studies of intracellular calcium flow of transfected HeLa cells exposed to various leukotrienes showed that also the novel BLTR-like receptor can be activated by leukotriene B4, and it is therefore tentatively named BLTR2.

Molecular Cloning and Characterization of a Novel Human Gene Containing Ankyrin Repeat and Double BTB/POZ Domain

A novel human gene containing an ankyrin repeat and BTB/POZ domains (BPOZ) was isolated from a human leukocyte cDNA library. The cDNA sequence contains an open reading frame of 1434 bp that encodes 478 amino acid residues with a predicted molecular mass of 53.9 kDa. Sequence pattern analysis shows that BPOZ contains an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Using semiquantitative RT-PCR, the BPOZ transcript was found to be ubiquitously expressed in all fetal tissues examined (heart, brain, liver, and kidney) suggesting that BPOZ is involved in basic cellular function. Low expression of BPOZ in adult tissues (normal and hypertrophic heart) suggests that BPOZ mRNA is developmentally regulated and may play a role in developmental processes. Chromosomal localization by radiation hybrid mapping revealed that this gene is localized between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a region frequently associated with leukemia. It is thus suggested that BPOZ may be functionally involved in protein-protein interaction, perhaps in forming protein complexes, and may have an important role in normal development and in the development of leukemia.

Molecular cloning and characterization of a cDNA encoding the human leucocyte vacuolar protein sorting (hlVps45)

We have isolated a novel cDNA clone from human leucocyte cDNA library, encoding a Sec1p-like vacuolar protein sorting (hl Vps45) which is believed to be implicated in vesicular transportation. Although the deduced amino acid (AA) sequence of this cDNA has revealed 97% identity to other known mammalian vacuolar protein sorting, there is an extensive variation in nucleotide sequence in comparison to that of three previously reported human (h Vps45), rat (r Vps45) and mouse (m Vps45) vacuolar protein sorting ( Vps45) cDNAs [ 1–3]. At the nucleotide sequence level hl Vps45 demonstrated 90% homology to the h Vps45 and r Vps45 and 89% identity to m Vps45 with no significant homology in their noncoding regions. The 2.4 Kb mRNA corresponding to the hl Vps45 clone is widely distributed in a variety of human tissues expressing highest levels in peripheral blood mononuclear cells (PBMC), neutrophils, heart, spleen, and testis. The chromosomal mapping studies have demonstrated that the hl Vps45 is localized to long arm of human chromosome 1 at q21–q22. Our data indicates that we have isolated, characterized and mapped a novel cDNA encoding hl Vps45, which may play an important role in protein trafficking as well as have clinical significance in the release of inflammatory mediators e.g. histamine, bradykinin and cytokine release.

Isolation and mapping of a putative b subunit of human ATP synthase (ATP-BL) from human leukocytes.

From a human-leukocyte cDNA library, we cloned cDNA encoding a novel protein, which has a significant homology with the b subunit of ATP synthase (proton-transporting ATPase, F1F0-ATPase; EC3.6.1.34) derived from Anabaena sp. strain PCC 7120. The cDNA has an open reading frame of 1314 nucleotides corresponding to 438 amino acids. The coding sequence was 37.9% identical over 57 amino acid with b subunit of ATP synthase. The 34-amino-acid region of the predicted peptide sequence displays a coiled-coil motif that could form a complex with some other protein(s). We designated this novel gene as ATP-BL because of its homology to the b subunit of ATP synthase. The ATP-BL locus was mapped by fluorescence in situ hybridization (FISH) and radiation hybrid mapping to the q24 region of chromosome 16.

A novel form of cytochrome P-450 family 4 in human polymorphonuclear leukocytes. cDNA cloning and expression of leukotriene B4 omega-hydroxylase

Isolation of cDNA clones for human leukotriene B4 (LTB4) omega-hydroxylase clearly demonstrates that the hydroxylase is a member of the cytochrome P-450 (CYP) superfamily. cDNA clones isolated from a human leukocyte cDNA library with CYP4A4 cDNA as a probe encode a protein of 520 amino acids with a molecular weight of 59,805. The deduced amino acid sequence contains an invariant cysteine in the conserved heme-binding domain near the C terminus, characteristic of the P-450 superfamily. The microsomes from yeast cells transfected with an expression vector pAAH5 carrying isolated cDNA catalyzed the omega-hydroxylation of LTB4 with a Km value of 0.71 microM, and its activity was significantly inhibited by carbon monoxide and by antisera against CYP4A4, consistent with the properties previously reported with LTB4 omega-hydroxylase in human polymorphonuclear leukocytes. The amino acid sequence of LTB4 omega-hydroxylase (P-450LTB omega) shows 31-44% similarity to those of CYP4A, CYP4B, and CYP4C, whereas less than 25% similarity was observed with any of the other P-450 families. According to the systematic classification of the P-450 superfamily, P-450LTB omega is classified into the CYP4 family but does not belong to any of the known CYP4 subfamilies. This P-450 composes a new subfamily of CYP4. RNA blot analysis indicated that mRNA hybridized to the cDNA was expressed in the polymorphonuclear leukocytes as well as leukocytes from four individuals. Isolation of the cDNA opens the way to investigate the physiological role and to regulation of the omega-hydroxylase in the inflammation process.

Molecular cloning and expression of platelet-activating factor receptor from human leukocytes.

The cDNA for a platelet-activating factor (PAF) receptor was cloned from a human leukocyte cDNA library using a 0.8-kilobase pair fragment of the guinea pig lung PAF receptor cDNA (Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T. (1991) Nature 349, 342-346). The cDNA (1.8-kilobase pairs) had an open reading frame encoding 342 amino acid residues with a calculated Mr of 39,203. The clone was shown to code for a PAF receptor based on the following criteria: 1) the amino acid sequence possesses seven putative membrane spanning domains with 83% identity to the guinea pig lung PAF receptor, 2) Xenopus laevis oocytes injected with the transcript of the clone showed an electrophysiological response to PAF, and 3) COS-7 cells expressing the encoded receptor showed ligand binding with the pharmacological properties of the PAF receptor. Activation of the PAF receptor yielded inositol 1,4,5-trisphosphate production in both COS-7 cells and oocytes, and guanosine 5'-O-(2-thio)bisphosphate injection into the oocytes inhibited PAF-induced Cl- current, providing an evidence that PAF stimulates phosphoinositide turnover via G-protein(s). PAF receptor mRNA was abundant in leukocytes and less so in an undifferentiated human eosinophilic cell line (EoL-1 cells) or human erythroleukemia cells (HEL cells). The production of the mRNA was prominently increased when EoL-1 cells were treated with granulocyte macrophage colony stimulating factor, interleukin-5, and n-butyrate.

Three novel molecular forms of biliary glycoprotein deduced from cDNA clones from a human leukocyte library

Three cDNA clones that encode three novel variants of biliary glycoprotein a (BGPa), a glycoprotein belonging to the CEA gene family, were identified in a human leukocyte cDNA library. The domain structures of the predicted proteins of the three clones W211, W233 and W239 are N-A1-B1-A2, N-A1-B1 and N-A1-B1-C, respectively; they lack the transmembrane and cytoplasmic domains that exist in the four BGP species (BGPa, BGPb, BGPc and BGPd) previously reported. Their sequences from N to B1 or to A2 are virtually identical to those of BGPa-d. Comparison with the genomic sequence for BGPa-d suggested that these three new BGP variants as well as BGPa-d are generated from the same single gene by alternative splicing of RNA.

Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocyte

Human leukocyte cDNA library was screened to isolate cDNA clones coding for hepatocyte growth factor using cDNA from human liver as a probe. Nucleotide and deduced amino acid sequences were analyzed for two of four clones obtained. One of them contained an open reading frame coding for a polypeptide chain of 728 amino acid residues like that of cDNA clone derived from human liver. In another clone a spontaneous deletion of 15 base pairs was found within the coding sequence. When expressed transiently using COS-1 cells both clones produced protein with similar biological activity against rat hepatocyte in vitro.

A human immunoglobulin G receptor exists in both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms.

Several cDNA clones encoding the human immunoglobulin G receptor CD16 were isolated from human lung or peripheral blood leukocyte cDNA libraries. Nucleotide sequence comparisons revealed that the cDNAs could be divided into two groups. cDNA clones in one group encode a protein that terminates 4 amino acids after the putative transmembrane domain. Clones in the second group encode a protein with an extra 21 amino acids that could comprise a cytoplasmic domain. Direct peptide sequencing was used to determine the N terminus of the mature CD16 receptor protein and supported the existence of the two forms of the receptor. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C resulted in the release of a large percentage of the CD16 molecules from the cell surface. In contrast, treatment of natural killer cells with phosphatidylinositol-specific phospholipase C did not release any CD16 from the cell surface. These data demonstrate that both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms of the CD16 molecule exist and that they are differentially expressed on neutrophils and natural killer cells.