A case of multifocal Langerhans cell granulomatosis: a BAL follow up study

UDP-glucuronosyltransferase (UGT) 2B9, isolated from a cynomolgus monkey liver cDNA library, is 89% identical to human UGT2B7 in primary amino acid sequence, and the two expressed enzymes were previously shown to catalyze the glucuronidation of many common endogenous substrates. The purpose of the present study was to characterize the reactivity of expressed UGT2B9 with important therapeutic agents and other xenobiotics. UGT2B9, stably expressed in human embryonic kidney 293 cells, catalyzes the 3-O- and 6-O-glucuronidation of morphine and the 6-O-glucuronidation of codeine. A number of other morphinan (e.g. naloxone, naltrexone, and nalorphine) and oripavine (e.g. buprenorphine) derivatives are substrates for this enzyme. In general, morphinan derivatives are glucuronidated at higher rates, compared with oripavines; however, glucuronidation efficiency values (Vmax/KM) for the compounds are similar. Stably expressed UGT2B9 also catalyzes the glucuronidation of profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and straight-chain fatty acids at the carboxylic acid moiety. Monoterpenoid alcohols and propanolol are glucuronidated at aliphatic hydroxyl positions. Expressed UGT2B9 exhibits enantioselective glucuronidation for (R/S)-ibuprofen, (R/S)-propanolol, and (+)/(-)-menthol. The data suggest that monkey UGT2B9 and human UGT2B7 are functionally similar.     

Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.     

Craf-1 protein kinase is essential for mouse development

The complete cDNA of the mouse integral membrane protein 2B gene (Itm2b) was determined by sequence analysis of expressed sequence tag (EST) clone L26775 and a clone isolated from a cDNA library of the osteogenic stromal cell line MN7 (Mathieu et al., 1992. Calcif. Tissue Int. 50, 362-371) and by 5' rapid amplification of cDNA ends (RACE). Alignment of different mouse ESTs confirmed the entire sequence. Northern blot analysis of different neonatal and adult mouse tissues showed that Itm2b is ubiquitously expressed. There are three mRNAs with different lengths in neonatal as well as in adult tissues, originating from alternative polyadenylation by usage of one consensus and two additional variant polyadenylation signals. The cDNA sequence of the human Itm2b homolog (ITM2B) was assembled using data from available human ESTs. Both the mouse and the human gene code for a protein of 266 amino acids (aa) that is homologous to a previously described integral membrane protein, Itm2A, of which the expression is restricted to osteo- and chondrogenic tissues. Itm2A and Itm2B belong to a family of type II integral membrane proteins, which contains a third member, Itm2C (Deleersnijder et al., 1996. J. Biol. Chem. 271, 19475-19482). The human ITM2B and mouse Itm2b genes were previously mapped as unknown ESTs to conserved syntenic regions Homo sapiens 13q12-13 and Mus musculus 14.     

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.     

Functional magnetic resonance imaging of median nerve stimulation in rats at 2.0 T

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C.     

Suppression of caveolin expression induces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancer cells

Although prostate cancer cells are often initially sensitive to androgen ablation, they eventually lose this response and continue to survive, grow and spread in the absence of androgenic steroids. The mechanism(s) that underlie resistance to androgen ablation therapy remain mostly unknown. We have demonstrated that elevated caveolin protein levels are associated with human prostate cancer progression in pathological specimens. Here we show that suppression of caveolin expression by a stably transfected antisense caveolin-1 cDNA vector converted androgen-insensitive metastatic mouse prostate cancer cells to an androgen-sensitive phenotype. Orthotopically grown tumors and low-density cell cultures derived from antisense caveolin clones had increased apoptosis in the absence of androgenic steroids, whereas similarly grown tumors and cells from vector (control) clones and parental cells were not sensitive to androgens. Studies using a representative antisense caveolin clone showed that selection for androgen resistance in vivo correlated with increased caveolin levels, and that adenovirus-mediated caveolin expression blocked androgen sensitivity. Our results identify a new candidate gene for hormone-resistant prostate cancer in man and indicate that androgen insensitivity can be an inherent property of metastatic prostate cancer.     

Sex hormones differentially regulate isoforms of UDP-glucuronosyltransferase

PURPOSE: To investigate the role of sex hormones in the regulation of UDP-glucuronosyltransferase (UGT). METHODS: We examined liver from adult, prepubertal, gonadectomised and gonadectomised plus hormone replaced rats of both sexes. Immunohistochemistry and immunoblots were performed using a polyclonal UGT antibody to a number of family 1 and family 2 UGT isoforms. Northern blot analysis was performed utilising cDNA probes to family 1 and family 2 isoforms. RESULTS: Immunohistochemistry demonstrated variations in intensity and distribution of staining in the hormonally manipulated rats. Immunoblots showed variations in individual band intensity between rat groups. Immunoblots using a more specific antibody (anti-17 beta-hydroxysteroid UGT, which recognises UGT2B3 and UGT2B2) demonstrated marked differences between male and female rats and significant alterations after gonadectomy and testosterone replacement in the male rats. In northern analysis, UGT2B3 and 2B1 mRNA were significantly higher in adult males than females, and in prepubertal males compared to prepubertal females. In male rats, gonadectomy resulted in a 45-53% reduction in UGT2B3 and 2B1 levels respectively, which increased significantly with testosterone treatment to greater than normal adult levels. No change in UGT2B3 or 2B1 occurred after gonadectomy in females. In contrast, UGT1*1 mRNA tended to be higher in adult female and prepubertal female rats than in their male counterparts. In females, gonadectomy resulted in significant up-regulation of UGT1*1, while gonadectomy plus oestradiol treatment resulted in markedly reduced levels. UGT1*1 mRNA was not significantly altered by gonadectomy in males. CONCLUSIONS: This study demonstrates the differential effects of sex hormones on the expression of isoforms from the two phylogenetically distinct UGT families.     

Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme

An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purified C. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of approximately 40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectularius cDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with native C. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized both C. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum. C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegans and in mouse and human expressed sequence tags from fetal and tumor EST libraries.     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.     

Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen

Shrimp is a common cause of seafood hypersensitivity. To study the mechanism of seafood hypersensitivity at the molecular level, we have determined the primary structure of the major heat-stable allergen of shrimp by cloning, expression, nucleotide sequencing, and amino acid sequence determination of an IgE-reactive cDNA clone, Met e I, isolated from a Metapenaeus ensis expression library in lambda gt 11. We first constructed a cDNA library from the shrimp M. ensis in lambda gt 11. We then screened the library with sera from patients with hypersensitivity reactions to shrimp and identified a positive IgE-reactive clone, designated as Met e I. This cDNA was purified to homogeneity and subsequently expressed in the plasmid pGEX. Serum antibodies from patients with shrimp allergy demonstrated positive IgE reactivity by immunoblotting to a protein encoded by the clone Met e I; sera from nonallergic control subjects were not reactive. The nucleotide sequence of this cDNA clone revealed an open reading frame of 281 amino acid residues, coding for a protein of 34 kd. Comparison of the Met e I amino acid sequence with the Genbank database showed that Met e I is highly homologous to multiple isoforms of tropomyosin.