Faculty Bibliography

The removal of introns from pre-mRNA requires as an initial event the accurate molecular recognition of the proper exon-intron borders. It is now evident that RNA sequence elements in addition to the consensus splice site sequences themselves are required for this recognition. Genomic analyses have contributed to the definition of these elements as exonic and intronic splicing enhancers and silencers, comprising what has been called the "splicing code." Many computational methods have been brought to bear in such studies. We describe here some of the methods we have used to discover functional splicing signals. What these methods have in common is a comparison of sequences in and around exons to sequences found elsewhere in the genome. We have especially made use of comparisons to "pseudo exons," intronic sequences resembling exons by virtue of being bounded by sequences indistinguishable from splice sites. Two computational strategies are emphasized: (1) the use of a machine learning technique in which a computational algorithm, a support vector machine, is first trained on known examples and then used to predict sequences associated with splicing; and (2) straight statistical analysis of differences between regions associated with exons and other regions in the genome. In most cases, the predictions made using these methods have been validated by subsequent empirical tests. An attempt has been made to make this description understandable by researchers unfamiliar with computational practice and to include practical references to specific databases and programs.

As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long-term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged-triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC50 values in the range of 10 microM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with L-3,4-dihydroxyphenylalanine (L-DOPA) for binding to the DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 muM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage-gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan-3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan-3. We have examined the molecular composition of the PNS nodes in syndecan-3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan-3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of cartilage and bone. The destructive lesions result from both immune responses and non-antigen-specific inflammatory processes. Little is known about the primary cause of RA. Although the primacy of T-cell-related events early in the disease remains debated, strong evidence indicates that autoantigen recognition by specific T cells is crucial to the pathophysiology of rheumatoid synovitis. We will discuss evolving concepts about T-cell involvement in RA and the roles for various T cell subsets in the development of joint abnormalities. The hypothesis that RA is a T-cell driven disease was put forward when studies of RA synovium showed numerous T cells carrying activation markers. These T cells were found to participate in the complex network of cell- and mediator-driven events leading to joint destruction. Conceivably, these T cells may be stimulated by an autoantigen (whether specific to the joints or ubiquitous), a highly conserved foreign protein cross-reacting with its human homolog, or a neo-antigen expressed as a result of posttranslational events. For many years, animal models have provided valuable evidence supporting a role for T cells in RA. We will review three murine models of arthritis caused by different mechanisms. In collagen-induced arthritis, the immune response to a joint antigen is mediated by pathogenic Th1 cells that elicit severe inflammatory synovitis. Spontaneous arthritis in K/BxN T-cell-receptor transgenic mice is related to an adaptive immune response against a ubiquitous protein whose end-stage effector mechanisms are heavily dependent on the innate immune system. In the SKG model of autoimmune inflammatory arthritis, a point mutation in the gene encoding a key signal-transduction molecule in T cells causes defective T cell selection in the thymus, which releases polyclonal autoreactive T cells. Studies in these and other animal models have established that a variety of T-cell subsets whose roles vary with cell location and disease stage can contribute to synovitis. Finally, in addition to direct autoimmune attack by effector T cells, arthritis may result from defective homeostatic control of immunity by regulatory T cells.

Boxing hundreds of thousands of particles in low-dose electron micrographs is one of the major bottle-necks in advancing toward achieving atomic resolution reconstructions of biological macromolecules. We have shown that a combination of pre-processing operations and segmentation can be used as an effective, automatic tool for identifying and boxing single-particle images. This paper provides a brief description of how this method has been applied to a large data set of micrographs of ice-embedded ribosomes, including a comparative analysis of the efficiency of the method. Some results on processing micrographs of tripeptidyl peptidase II particles are also shown. In both cases, we have achieved our goal of selecting at least 80% of the particles that an expert would select with less than 10% false positives

Cardiolipin is a unique mitochondrial phospholipid with an atypical fatty acid profile, but the significance of its acyl specificity has not been understood. We explored the enormous combinatorial diversity among cardiolipin species, which results from the presence of four fatty acids in each molecule, by integrated use of high-performance liquid chromatography, mass spectrometry, diacylglycerol species analysis, fatty acid analysis, and selective cleavage of fatty acids by phospholipase A2. The most abundant cardiolipin species from various organisms and tissues (human heart, human lymphoblasts, rat liver, Drosophila, sea urchin sperm, yeast, mung bean hypocotyls) contained only one or two types of fatty acids, which generated a high degree of structural uniformity and molecular symmetry. However, an exception was found in patients with Barth syndrome, in whom an acyltransferase deficiency led to loss of acyl selectivity and formation of multiple molecular species. These results suggest that restriction of the number of fatty acid species, rather than the selection of a particular kind of fatty acid, is the common theme of eukaryotic cardiolipins. This limits the structural diversity of the cardiolipin species and creates molecular symmetry with implications for the stereochemistry of cardiolipin

Obesity and overweight, well known risk factors for cardiovascular disease and type 2 diabetes, are now associated with Alzheimer's disease (AD). It remains to be determined if obesity and overweight contribute to the risk of developing AD through modulating levels of amyloid-beta (Abeta), a key molecule in AD pathogenesis. Thus, we investigated whether there were any associations between plasma Abeta levels and body mass index (BMI) or fat mass (FM) in a group of 18 healthy adults. A statistically significant correlation was found between BMI, FM, and plasma levels of Abeta42 (BMI r = 0.602, P = 0.008; FM r = 0.547, P = 0.019), the longer, more pathogenic form of Abeta, but not with plasma levels of the shorter, less pathogenic Abeta40. Although not significant, positive correlations between plasma levels of Abeta42 and levels of insulin and the inflammatory marker C-reactive protein (CRP), along with an inverse trend between plasma Abeta42 levels and levels of high density lipoprotein (HDL) were answered. These results suggest that proteins implicated in inflammation, cardiovascular disease and type 2 diabetes, which in turn are risk factors for AD, may contribute to the associations between BMI/FM and plasma Abeta42 levels. Longitudinal studies involving larger cohorts are required to determine if elevated body fat may predispose individuals to AD through increasing Abeta42 levels throughout early to late adulthood.

BACKGROUND: To survey arboviruses in Yunnan province. METHODS: Mosquitoes were collected from Yunnan Province in 2002 and 2004. Virus strains were isolated by the inoculation of homogenates of the mosquitoes onto BHK cell line. The isolated strains and their molecular biological characteristics were identified by real-time PCR, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent antibody technique. RESULTS: Twelve strains of viruses producing CPE in BHK cells were isolated from 4810 mosquitoes. All the 12 isolates were identified to be Japanese encephalitis viruses. Genotype analysis showed the new virus (DL-0437 strain) belonged to genotype III. CONCLUSION: Twelve strains of Japanese encephalitis viruses were isolated from mosquito pools collected in Yunnan. It was the first isolation of genotype III Japanese encephalitis viruses in Yunnan Province in recent years.

The discovery of long-lived epithelial stem cells in the bulge region of the hair follicle led to the hypothesis that epidermal renewal and epidermal repair after wounding both depend on these cells. To determine whether bulge cells are necessary for epidermal renewal, here we have ablated these cells by targeting them with a suicide gene encoding herpes simplex virus thymidine kinase (HSV-TK) using a Keratin 1-15 (Krt1-15) promoter. We show that ablation leads to complete loss of hair follicles but survival of the epidermis. Through fate-mapping experiments, we find that stem cells in the hair follicle bulge do not normally contribute cells to the epidermis which is organized into epidermal proliferative units, as previously predicted. After epidermal injury, however, cells from the bulge are recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a marked radial pattern. Notably, although the bulge-derived cells acquire an epidermal phenotype, most are eliminated from the epidermis over several weeks, indicating that bulge stem cells respond rapidly to epidermal wounding by generating short-lived 'transient amplifying' cells responsible for acute wound repair. Our findings have implications for both gene therapy and developing treatments for wounds because it will be necessary to consider epidermal and hair follicle stem cells as distinct populations