Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276 -335. This region, which contains consensus clathrin-and AP2-binding sites, functions in conjunction with the coiledcoil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrinmediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP6O) genes using a set of universal degenerate HSP6O PCR primers. The cloned partial HSP60 DNA sequences f rom nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98O/0), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. A t the subspecies level, DNA sequence similarity among members of 5. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. A t the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93 O/ O (mean 82 YO). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59 %, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 165 rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP6O gene offer a convenient and accurate tool for species-specif ic identification and phylogenetic analysis of staphylococci.