Faculty Bibliography

Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.

The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

The lifetime risks for both breast and ovarian cancer for BRCA mutation carriers far exceeds the general population risk of 13% for breast cancer and 1.4% for ovarian cancer. BRCA carriers have unique and medically complicated decisions to make regarding their cancer treatment or risk reduction. As BRCA testing becomes increasingly common among unaffected individuals in families with a previously documented BRCA mutation, there are a growing number of individuals with unique psychosocial needs and concerns. This review paper describes the BRCA 1/2 population, discusses preimplantation genetic diagnosis (PGD), and describes the decisions and ethical issues related to PGD among the BRCA 1/ 2 population.

The extracellular matrix plays a key role in organ formation and tissue homeostasis. Recent studies have revealed that fibrillin assemblies (microfibrils) confer both tissue integrity and regulate signaling events that instruct cell performance and that perturbation of either function manifests in disease. These analyses have also indicated that fibrillin assemblies impart contextual specificity to TGFbeta and BMP signaling. Moreover, correlative evidence suggests functional coupling between cell-directed assembly of microfibrils and targeting of TGFbeta and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals with TGFbetas and BMPs as nodal points that convert extracellular inputs into discrete and context-dependent cellular responses

It is well accepted that complex biological processes such as angiogenesis are not controlled by a single family of molecules or individually isolated signaling pathways. In this regard, new insight into the interconnected mechanisms that regulate angiogenesis might be gained by examining this process from a more global network perspective. The coordination of signaling cues from both outside and inside many different cell types is required for the successful completion of angiogenesis. Evidence is accumulating that the multifunctional integrin family of cell adhesion receptors represent an important group of molecules that play active roles in sensing, integrating, and distributing a diverse set of signals that regulate many cellular events required for angiogenesis. Given the ability of integrins to bind numerous extracellular ligands and transmit signals in a bi-directional fashion, we will discuss the multiple ways by which integrins may serve as a functional hub during pathological angiogenesis. In addition, we will highlight potential imaging and therapeutic strategies based on the expanding new insight into integrin function.

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

An early step in mammalian fertilization is species-restricted binding of sperm to the egg's zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1-3, constitute the mouse egg's ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 from fertilized eggs has no effect on sperm binding and is unable to induce the acrosome reaction. These and other properties of mZP3 suggest that it is a receptor for sperm and inducer of the acrosome reaction. Mapping of the mZP3 combining-site for sperm suggests that it is located near the C-terminus of the polypeptide, just downstream of the ZP domain, in a region encoded by exon-7 of the mZP3 gene. This region of mZP3 is a site of positive Darwinian selection. When mZP3 exon-7 is fused to the Fc fragment of human IgG and sperm exposed to the chimeric protein, sperm are inhibited from binding to eggs. However, the chimeric protein does not induce the acrosome reaction. Therefore, polypeptide encoded by mZP3 exon-7 is necessary and sufficient for binding of mouse sperm.

An epidemiologic study of childhood leukemia in Denmark (2,400 cases; 6,697 controls) from 1968 to 1994 suggested a weak, but statistically significant, association of residential radon exposure and acute childhood lymphoblastic leukemia (ALL). The Danish study estimated a relative risk (RR) = 1.56 (95% CI, 1.05-2.30) for a cumulative exposure of 1,000 Bq m-3 y. For an exposure duration of 10 y their RR corresponds to a radon concentration of 100 Bq m-3. There are two dose pathways of interest where alpha particles could damage potential stem cells for ALL. One is the alpha dose to bone marrow, and two is the dose to bronchial mucosa where an abundance of circulating lymphocytes is found. Compared with an exposure of about 1 mSv y-1 from natural external background, radon and decay products contribute an additional 10 to 60% to the bone marrow equivalent dose. The other pathway for exposure of T (or B) lymphocytes is within the tracheobronchial epithelium (BE). Inhaled radon decay products deposit on the relatively small area of airway surfaces and deliver a significant dose to the nearby basal or mucous cells implicated in human lung cancer. Lymphocytes are co-located with basal cells and are half as abundant. Using a 10-y exposure to 100 Bq m-3, our dose estimates suggest that the equivalent dose to these lymphocytes could approach 1 Sv. The relatively high dose estimate to lymphocytes circulating through the BE, potential precursor cells for ALL, provides a dose pathway for an association