Faculty Bibliography

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns

Lipids, in addition to being structural components of cell membranes, can act as signaling molecules. Bioactive lipids, such as sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), may act intracellularly as second messengers or be secreted and act as intercellular signaling molecules. Such molecules can affect a variety of cellular processes including apoptosis, proliferation, differentiation and motility. To investigate possible sources of bioactive lipids during development we have searched the Drosophila genome for homologs of genes involved in mammalian S1P and LPA metabolism. Here we report the developmental expression of 31 such genes by in situ hybridization to Drosophila embryos. Most show expression in specific tissues, with expression in the gut and nervous system being recurring patterns

Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol 151: 961-972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 +/- 122, knockout 2,322 +/- 114 Omega. cm(2); P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 +/- 0.06, knockout 1.83 +/- 0.14 cm/s x 10(-5); P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 +/- 0.24, knockout 2.93 +/- 0.31 cm/s x 10(-6); P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium

Mutations in the mouse p (pink-eyed dilution) and human P genes lead to melanosomal defects and ocular developmental abnormalities. Despite the critical role played by the p gene product in controlling tyrosinase processing and melanosome biogenesis, its precise biological function is still not defined. We have expressed p heterologously in the yeast Saccharomyces cerevisiae to study its function in greater detail. Immunofluorescence studies revealed that p reaches the yeast vacuolar membrane via the prevacuolar compartment. Yeast cells expressing p exhibited increased sensitivity to a number of toxic compounds, including arsenicals. Similarly, cultured murine melanocytes expressing a functional p gene were also found to be more sensitive to arsenical compounds compared with p-null cell lines. Intracellular glutathione, known to play a role in detoxification of arsenicals, was diminished by 50% in p-expressing yeast. By using the glutathione-conjugating dye monochlorobimane, in combination with acivicin, an inhibitor of vacuolar gamma-glutamyl cysteine transpeptidase, involved in the breakdown of glutathione, we found that p facilitates the vacuolar accumulation of glutathione. Our data demonstrate that the pink-eyed dilution protein increases cellular sensitivity to arsenicals and other metalloids and can modulate intracellular glutathione metabolism

The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs

BACKGROUND: The recent discovery of circulating endothelial progenitor cells (EPCs) has altered our understanding of new blood vessel growth such as occurs during collateral formation. Because diabetic complications occur in conditions in which EPC contributions have been demonstrated, EPC dysfunction may be important in their pathophysiology. METHODS AND RESULTS: EPCs were isolated from human type II diabetics (n=20) and age-matched control subjects (n=20). Proliferation of diabetic EPCs relative to control subjects was decreased by 48% (P<0.01) and inversely correlated with patient levels of hemoglobin A1C (P<0.05). Diabetic EPCs had normal adhesion to fibronectin, collagen, and quiescent endothelial cells but a decreased adherence to human umbilical vein endothelial cells activated by tumor necrosis factor-alpha (TNF-alpha) (P<0.05). In a Matrigel assay, diabetic EPCs were 2.5 times less likely to participate in tubule formation compared with controls (P<0.05). CONCLUSIONS: These findings suggest that type II diabetes may alter EPC biology in processes critical for new blood vessel growth and may identify a population at high risk for morbidity and mortality after vascular occlusive events

OBJECTIVE: To study the mechanism of intimal hyperplasia after coronary artery bypass grafting (CABG) and to find an effective way for preventing intimal hyperplasia. METHODS: Twenty-four male New Zealand rabbits were randomly divided into two groups of 12 rabbits: operation group and sham-operation (control) group. The external jugular vein was harvested and anastomosed end-to-side to the ipsilateral carotid artery in operation group or grafted in situ in the control group. Six rabbits in each group were killed and their grafted veins were taken 2 weeks and 4 weeks after operation respectively. The mRNA expressions of transforming growth factor beta (TGF-beta), collagen I, collagen III, and angiotension 1 receptor (AT1R) were measured by RT-PCR and electrophoresis. RESULTS: The intimal hyperplasia was much more remarkable in the operation group than in the control group either 2 weeks or 4 weeks after operation. The mRNA expressions of TGF-beta, AT1R, collagen I, and collagen III were significantly higher in the operation group than in the control group, especially 2 weeks after (P < 0.01). Four weeks after the operation, the expressions of TGF-beta, AT1R, collagen I and collagen III were 4.05 +/- 0.49 vs 2.05 +/- 0.26, 18.23 +/- 1.32 vs 4.61 +/- 0.53, 80 +/- 0.17 vs 0.90 +/- 0.18, and 7.05 +/- 0.68 vs 2.80 +/- 0.17 respectively (all P < 0.05). CONCLUSION: TGF-beta and AT1R may have an important role in the intimal hyperplasia of venous graft in CABG. Continuous arterial pressure may be the main factor of increased expression of TGF-beta and AT1R that cause the enormous synthesis and deposit of collagen

BACKGROUND: Interaction between hair cells and acellular gels of the mammalian inner ear, the tectorial and otoconial membranes, is crucial for mechanoreception. Recently, otoancorin was suggested to be a mediator of gel attachment to nonsensory cells, but the molecular components of the interface between gels and sensory cells remain to be identified. HYPOTHESIS: We report that the inner ear protein stereocilin is related in sequence to otoancorin and, based on its localisation and predicted GPI-anchoring, may mediate attachment of the tectorial and otoconial membranes to sensory hair bundles. TESTING: It is expected that antibodies directed against stereocilin would specifically label sites of contact between sensory hair cells and tectorial/otoconial membranes of the inner ear. IMPLICATIONS: Our findings support a unified molecular mechanism for mechanotransduction, with stereocilin and otoancorin defining a new protein family responsible for the attachment of acellular gels to both sensory and nonsensory cells of the inner ear.

The wiring of synaptic circuitry during development is remarkably precise, but the molecular interactions that enable such precision remain largely to be defined. Cadherins are cell adhesion molecules hypothesized to play roles in axon growth and synaptic targeting during development. We previously showed that N-cadherin localizes to ventrobasal (VB) thalamocortical synapses in rat somatosensory (barrel) cortex during formation of the whisker-map in layer IV (Huntley and Benson [1999] J. Comp. Neurol. 407:453-471). Such specific association of N-cadherin with one identified afferent pathway raises the prediction that other cadherins are expressed in barrel cortex and that these are, in some combination, also differentially associated with distinct inputs. Here, we first show that N-cadherin and three other classic cadherins (cadherin-6, -8, and -10) are expressed contemporaneously in barrel cortex with relative levels of postnatal expression that are highest during the first 2 weeks, when afferent and intrinsic circuitries are forming and synaptogenesis is maximal. Each displayed distinct, but partly overlapping laminar patterns of expression that changed over time. Cadherin-8 probe hybridization formed a particularly striking pattern of intermittent, columnar patches extending from layer V through layer III, which was first detectable at approximately postnatal day 3. The patches were centered precisely over regions of dysgranular layer IV and, in the whisker barrel field, over barrel septa. This pattern is similar to that formed by the terminal distribution of thalamocortical afferents arising from the posterior nucleus (POm), suggesting cadherin-8 association with the POm thalamocortical synaptic circuit. Consistent with this, cadherin-8 mRNAs were enriched in the POm nucleus, and cadherin-8 immunolabeling in layer IV was enriched in barrel septa and codistributed with labeled POm thalamocortical synaptic-like puncta. The striking molecular parcellation of at least two different cadherins to the two, converging thalamic pathways that terminated in non-overlapping barrel center and septal compartments in layer IV strongly suggested that cadherins provide requisite molecular recognition and targeting that enable precise construction of thalamocortical and other synaptic circuitry.

Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly