Adenovirus-mediated gene transfer of human lipoprotein lipase ameliorates the hyperlipidemias associated with apolipoprotein E and LDL receptor deficiencies in mice.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglyceride-rich lipoproteins. We tested the efficacy of adenovirus-mediated gene transfer of LPL as treatment of experimental hyperlipidemias associated with apolipoprotein (apoE) deficiency (apoE-/-) and low-density lipoprotein receptor (LDLr) deficiency (LDLr-/-) in mice. Replication-defective adenovirus containing the human LPL cDNA driven by a cytomegalovirus promoter (Ad.hLPL) efficiently transduced CHO-ldlA7 cells in vitro, inducing in these cells the production of bioactive LPL (73 mU/ml). Intravenous injection of Ad.hLPL (2 x 10(9) pfu) led to high-level expression of hLPL mRNA and LPL activity in the liver (88.3 mU/ml) and in post-heparin plasma (116.1 mU/ml). Overexpression of LPL resulted in marked reductions in total plasma cholesterol (TC; 48%, 43%, 25%) and triglycerides (TTg; 63%, 40%, 70%, p < 0.01) in apoE-/-, LDLr-/-, and wild-type (WT) mice, respectively. Fast protein liquid chromatography (FPLC) fractionation of plasma lipoproteins showed a marked decrease in very-low-density lipoprotein (VLDL)/chylomicron remnant cholesterol (V/CR-C) in apoE-/- (83%), LDLr-/- (84%), and WT mice (58%, p < 0.01). VLDL/chylomicron remnant triglycerides (V/CR-Tg) were virtually eliminated in apoE-/- (92%), LDLr-/- (86%), and WT mice (84%, p < 0.05). No significant changes were detected in LPL activities, plasma lipids, or lipoproteins of mice injected with a control virus, Ad.Luc, containing the luciferase instead of the LPL cDNA. In summary, infusion of Ad.hLPL leads to increased liver and post-heparin plasma LPL activities, significantly reduced TC, TTg, V/CR-C, and V/CR-Tg in WT mice, as well as in mice with apoE and LDLr deficiencies. Adenovirus-mediated LPL gene transfer to the liver is an effective means of reversing many of the lipoprotein abnormalities in apoE- and LDLr-deficient mice.

Human very-low-density lipoprotein receptor complementary DNA and deduced amino acid sequence and localization of its gene (VLDLR) to chromosome band 9p24 by fluorescence in situ hybridization.

A complementary DNA for the very-low-density lipoprotein receptor (VLDLR) that codes for a protein of 873 amino acids was cloned from a human heart cDNA library. The mature protein of 846 amino acids, preceded by a 27-residue signal peptide, shares 97% amino acid sequence identity with the rabbit VLDLR. Like the low-density lipoprotein receptor, the VLDLR contains five different domains, all of which are highly conserved between human and rabbit. A tetrapeptide NPVY that potentially serves as a signal for clustering of the VLDLR on coated pits is present in the cytoplasmic domain, which is 100% conserved between human and rabbit. We localized the VLDLR gene to chromosome 9p24 by fluorescence in situ hybridization using the cloned cDNA as hybridization probe. The high amino acid sequence homology of the VLDLR between two mammalian species suggests that the receptor plays a fundamental role in lipoprotein metabolism and that energy metabolism mediated by triglyceride utilization may be an evolutionarily highly conserved mechanism.

Structure of the mouse cholesterol 7 alpha-hydroxylase gene.

Three overlapping cholesterol 7 alpha-hydroxylasecDNAs were cloned from total mouse liver RNA by a combination of the polymerase chain reaction and mouse liver cDNA library screening. One of the cDNA clones was used to screen a mouse genomic library; three genomic clones were isolated and one of them was fully characterized. The mouse cholesterol 7 alpha-hydroxylase gene spans approximately 10 kb. The gene contains six exons including a 1509-bp open reading frame encoding 503 amino acid residues. The predicted amino acid sequence of mouse cholesterol 7 alpha-hydroxylase showed 93 and 82% identity to the rat and human enzymes, respectively. The transcription initiation site is mapped to 64 bases 5' of the translation initiation codon. Several transcription factor binding sequences are identified in the 5' flanking region of the mouse cholesterol 7 alpha-hydroxylase gene.

Detection of low level of human papilloma virus type 16 DNA sequences in cancer cell lines derived from two well-differentiated nasopharyngeal cancers.

Human papilloma virus type 16 (HPV16) related sequences were detected in two EBV-negative nasopharyngeal carcinoma (NPC) cell lines derived from two well-differentiated NPC by polymerase chain reaction. E2 and E6 related sequences of HPV16 were demonstrated using two pairs of primers derived from these two regions. DNA sequence analysis of amplified products excluded the possibility of laboratory viral DNA contamination.

Establishment and characterization of two nasopharyngeal carcinoma cell lines.

Two human nasopharyngeal carcinoma (NPC) cell lines have been established. One derived from a 64-year-old male, and the other from a 36-year-old female Chinese patient living in Taiwan. Both were keratinizing squamous cell carcinoma in nature and designated as NPC-TW039 and NPC-TW076. Both have been grown in culture system for more than 100 passages. Single cells from both cell lines could form colonies in 0.3% soft agar. In the nude mouse transplantation experiment, both cell lines could produce tumor mass with metastasis. The karyotypic analysis showed multiple chromosomal abnormality. The number of chromosomes ranged between 76 to 109 and 80 to 105 with an average of 98 and 95, respectively. The doubling time was 10.5 hours and 10.8 hours, respectively. The NPC-TW039 cell line has been subcloned and three subclones have been obtained. Ultrastructural studies from those two cell line, three subcloned cell lines and two transplanted tumor masses, all showed two types of morphology: the dark and light cells. This morphologic difference is probably derived from the different metabolic state, but not due to an artifact. Three oncogene probes have been used to check the oncogene expression; none of those five cell lines is positive. Similarly, six Epstein-Barr virus fragments have been labeled to hybridize with NPC cellular DNA preparations, results from the Southern blotting showed no detectable Epstein-Barr virus DNA sequence.

Genomic evidence for a complete sexual cycle in Candida albicans.

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.