Single-Spin Magnetomechanics with Levitated Micromagnets.

We demonstrate a new mechanical transduction platform for individual spin qubits. In our approach, single micromagnets are trapped using a type-II superconductor in proximity of spin qubits, enabling direct magnetic coupling between the two systems. Controlling the distance between the magnet and the superconductor during cooldown, we demonstrate three-dimensional trapping with quality factors around 1×10^{6} and kHz trapping frequencies. We further exploit the large magnetic moment to mass ratio of this mechanical oscillator to couple its motion to the spin degrees of freedom of an individual nitrogen vacancy center in diamond. Our approach provides a new path towards interfacing individual spin qubits with mechanical motion for testing quantum mechanics with mesoscopic objects, realization of quantum networks, and ultrasensitive metrology.

New classes of systematic effects in gas spin comagnetometers.

Atomic comagnetometers are widely used in precision measurements searching for spin interactions beyond the standard model. We describe a new (3)He-(129)Xe comagnetometer probed by Rb atoms and use it to identify two general classes of systematic effects in gas comagnetometers, one associated with diffusion in second-order magnetic-field gradients and another due to temperature gradients. We also develop and confirm experimentally a general and practical approach for calculating spin relaxation and frequency shifts due to arbitrary magnetic-field gradients.

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant.

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.

Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11.

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.

A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae.

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.

Correlation between activation of quiescent 3T3 cells by retinoic acid and increases in uridine phosphorylation and cellular RNA synthesis.

Short exposure of cultured quiescent cells to micromolar quantities of beta-all-trans-retinoic acid (RA) has been reported to potentiate the effects of phorbol myristate acetate in promoting the transition from the resting to growing states of these cells. Longer periods of exposure to RA result in substantial inhibition of cellular proliferation. We now show that short-term treatment of quiescent Swiss 3T3 cells with RA yields marked increases in uridine phosphorylation and total cellular RNA synthesis as well as 2-deoxyglucose uptake. Upon subsequent treatment of the cells with phorbol myristate acetate, a direct correlation between the comitogenic activity of RA and its stimulation of uridine phosphorylation and RNA synthesis is apparent. The increases in 2-deoxyglucose uptake persist after long-term exposure of the cells to RA when the growth-inhibitory effects of this agent are observed.

Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity.

Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae. We report that Rab GDI releases GDP-bound Sec4 from yeast membranes. dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes. An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Permeability measurement of macromolecules and assessment of mucosal antigen sampling using in vitro converted M cells.

INTRODUCTION: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells. METHODS: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production. RESULTS: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes. DISCUSSION: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

Organized practice and the quality of medical care.

Larger, more complex medical care organizations have the inherent capability to improve the quality of the care they deliver because of the improved competency that follows higher volumes of service, the interdependence of staff, and the emergence of responsible leadership in large organizations. The potential for slackened physician-patient relationships, however, could jeopardize that quality. We suggest that professional associations can counterbalance the negative influences of large organizations. We envision that the changing political and economic environment of medical practice, along with the greater professional and public scrutiny of care in highly visible large organizations, will act together to exert pressure on organized practices to examine and demonstrate quality clinical practice.

Lessons in cooperation: an update on improving the quality of hospital care.

BACKGROUND: Between 1989 and 1997 the Robert Wood Johnson Foundation (Princeton, NJ) launched a demonstration project, the Improving the Quality of Hospital Care Program, to test a consortium approach to quality improvement. As part of the project, four hospital consortia in various parts of the United States shared quality resources (for example, training) and collaborated on improvement efforts. Although cooperation was not a natural approach for enhancing quality in hospitals, the consortia mounted improvements in multiple clinical areas, such as diabetes care, the intensive care unit (ICU), prevention of wound infections, and care in rural areas. WHERE ARE THEY NOW? Of the four consortia that received implementation funding, all are continuing some explicit focus on improving quality, but only two have retained the organizational form of a consortium. Based at the University of Iowa (Iowa City), the Institute for Quality Healthcare continues to operate as a free-standing consortium with more than 40 hospital members. The Vermont Program for Quality in Health Care (Montpelier) provides information and education to improve quality of care statewide. LESSONS FROM THE PROGRAM: The program taught valuable lessons about what hospitals can do together and what they can achieve when they cooperate around quality of care issues. Sharing resources for education, providing a forum for quality improvement professionals to work together on specific issues, and identifying means of improving specific aspects of care in the group are all feasible in the consortium model. Even a chaotic environment can support cooperation.

Beyond survival: toward continuous improvement in medical care.

Using the scientific method, continuous improvement strives to attain unprecedented levels of performance - improved patient outcomes while maintaining or reducing costs. The needs for, and benefits of, continuous improvement are discussed along with a description of its basic elements. The approaches outlined can serve to greatly increase the pace of improvement in health care.

Reducing cesarean section rates safely: lessons from a "breakthrough series" collaborative.

BACKGROUND: Almost one million cesarean operations are performed each year in the United States. The objective of this project was to test the hypothesis that a structured collaborative effort can help participating health care organizations to reduce their cesarean delivery rates safely. METHODS: Experts associated with the collaborative helped participant organizations to explore several categories of change concepts and to develop action plans for safely reducing their cesarean delivery rates. Over the course of one year participants attended three two-day learning sessions. In the interval between these sessions, collaborative participants communicated by weekly conference calls and a dedicated Internet site. RESULTS: Of 28 participating organizations, 15 percent achieved cesarean delivery rate reductions of 30 percent or more during the 12-month period of active collaborative work. An additional 50 percent achieved reductions between 10 and 30 percent. CONCLUSIONS: The Healthy People 2000 goal of a cesarean delivery rate below 15 percent by the year 2000 is attainable. Clinical leadership from doctors and nurses toward the achievement of that goal is timely, ethical, and in the best interests of childbearing women in the United States.

Reconstitution of transport from the ER to the Golgi complex in yeast using microsomes and permeabilized yeast cells.

We have developed a highly efficient in vitro-transport assay that couples translocation across the ER membrane and transport to the Golgi complex using the secreted pheromone alpha-factor as a marker protein. Radiolabeled prepro-alpha-factor of high specific radioactivity is obtained by in vitro-translating this protein in a yeast lysate. Prepro-alpha-factor synthesized in vitro is then translocated directly into microsomes or the ER of permeabilized yeast cells. Conversion of the 26-kDa ER form of pro-alpha-factor to the high molecular weight Golgi form is dependent on the presence of ATP and soluble and membrane-bound factors. Differential centrifugation and fractionation on a sucrose gradient have shown that the ER and Golgi forms of alpha-factor are enriched in separate compartments after the transport reaction. These and other findings (see Ruohola et al., 1988, for a more complete discussion) indicate that conversion to the high molecular weight form of alpha-factor is the result of authentic intercompartmental transport. Permeabilized mammalian cells have been used to reconstitute transport from the ER to the Golgi complex. In these systems (Becker et al., 1987; Simons and Virta, 1987), a viral membrane glycoprotein protein (vesicular stomatitis virus G protein) is used as the marker protein. This protein is radiolabeled with [35S]methionine during virus infection, either before or after the cells are permeabilized. Radiolabeled G protein, residing in the ER, is then transported to the Golgi complex in the presence of an ATP-regenerating system. In the mammalian system the donor and acceptor compartments are retained within the permeabilized cells (Simons and Virta, 1987); however, on occasion the addition of an exogenous acceptor compartment is required (Beckers et al., 1987). The assay we developed (Ruohola et al., 1988) differs from the mammalian assay (Beckers et al., 1987) in that we introduce radiolabeled marker protein into the ER in vitro during translocation rather than during virus infection. In addition, in our assay the acceptor Golgi compartment is always provided exogenously to the permeabilized cells. Therefore, if acceptor membranes are present in the PYC, they are not utilized. Because the permeabilized cells and the S3 fraction are prepared differently, the conditions used to prepare the cells may lead to inactivation or loss of the acceptor compartment. The in vitro assay will enable us to purify components involved in transporting proteins from the lumen of the ER to the Golgi complex. Antibody prepared to purified components can be used to clone the genes that code for these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic.

Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non-functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H-ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild-type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild-type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.

Growth inhibitory and stimulatory effects of retinoic acid on murine 3T3 cells.

All-trans-beta-retinoic acid (RA) has both comitogenic and antiproliferative effects on murine Swiss 3T3 cells. Treatment of quiescent 3T3 cells for less than 24 hr with micromolar concentrations of RA potentiates subsequent mitogenic response of those cells to phorbol 12-myristate 13-acetate. Longer exposures of 3T3 cells to RA result in inhibition of DNA replication as measured by [3H]thymidine incorporation and decreased growth rates and saturation densities for cells grown in either 2% or 10% serum. Both the comitogenic and antiproliferative activities of RA for 3T3 cells are RA-dose dependent. RA-induced decreases in the 3T3 cell saturation density are reversible only after resuspension of cells by trypsinization and replating. Treatment of 3T3 cells for 48 hr with RA inhibits the rate of [3H]thymidine incorporation by 35--50%, while autoradiographic data show that labeling indices are similar to control values. Equal percentages of control and 48-hr RA-treated quiescent 3T3 cells respond to subsequent stimulation with 10% serum as determined by autoradiographic and flow cytometric analyses. However, the progression of RA-treated cells through the S phase of the cell cycle is slowed. These data suggest that inhibition of 3T3 cell proliferation by RA is established after a minimum 24-hr treatment and that this inhibition is the result of a decreased rate of DNA replication in S-phase cells.