U.S. Food Aid and Agricultural Cargo Preference Policy

Replaced with revised version of paper 07/20/10.Food Aid Policy, Cargo Preference, Agricultural and Food Policy, Food Security and Poverty,

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes

A dimerization model for the concentration dependent photophysical properties of diphenylhexatriene and its phospholipid derivatives. DPHpPC and DPHpPA

We have investigated the reason for the sensitivity of the fluorescence excited-state lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its phospholipid derivatives, 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl)carbonyl)-3-sn-phosphatidylcholine (DPHpPC) and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl)carbonyl)-3-sn-phosphatidic acid (DPHpPA), to the concentration of these probes in dipalmitoylphosphatidylcholine (DPPC) multilamellar membranes (Barrow, D. A., and B. R. Lentz, 1985. Biophys. J. 48:221-234; Parente, R. A., and B. R. Lentz. 1985. Biochemistry. 24:6178-6185). We have interpreted self-quenching data, excitation and emission spectra, and phase and modulation lifetime data in terms of a model that envisions dimerization of these probes in a membrane bilayer. It is proposed that dimerization alters the symmetry of the DPH excited state so as to allow more rapid decay via the normally symmetry-disallowed route from the 1Ag* state. Global analysis of fluorescence phase shift and modulation ratio data for DPHpPC in terms of the dimerization model provided a good fit of these data as a function of both modulation frequency and probe concentration. Global analysis of a similar set of data for the charged phosphatide DPHpPA predicted that this probe was much less prone to dimerize than was the uncharged DPHpPC. This physically reasonable result provides support for the assumptions made in the development of our model. We conclude that the dimerization model allows rationalization of many of the anomalous photophysical properties of DPH and its derivatives in membranes

Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex

ABSTRACT Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae . The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. IMPORTANCE Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR

Specific contribution of different phospholipid surfaces to the activation of prothrombin by the fully assembled prothrombinase

This paper addresses, in thermodynamic and kinetic terms, the reasons for the acidic lipid specificity of the human prothrombinase complex. We obtained, from the measured lipid titrations of the initial rates of prothrombin activation, the empirical binding constants for prothrombinase assembly on different membranes. These favored assembly on phosphatidylserine (PS)- as opposed to phosphatidylglycerol (PG)-containing membranes. In addition, we have used full time courses of prothrombin activation, in conjunction with a calculation of the equilibrium distribution of factor Xa between four enzymatic forms, to obtain the intrinsic kinetic constants of the prothrombinase assembled on PS- or PG-containing membranes. The resulting values of kcat, Km, and kcat/Km increased as acidic lipid content increased, and kcat/Km reached a plateau at 12 mol % PS and 50 mol % PG. Using the measured assembly and kinetic constants, the observed shapes of the phospholipid titration curves of human prothrombin activation were interpreted. We conclude that the difference in activity of prothrombinase assembled on PS- versus PG-containing membranes results both from the different binding properties of factors Xa and Va to these surfaces and from the different intrinsic activities of the prothrombinase when assembled on different membranes

Analysis of the Type IIn Supernova 1998S: Effects of Circumstellar Interaction on Observed Spectra

We present spectral analysis of early observations of the Type IIn supernova 1998S using the general non-local thermodynamic equilibrium atmosphere code \tt PHOENIX}. We model both the underlying supernova spectrum and the overlying circumstellar interaction region and produce spectra in good agreement with observations. The early spectra are well fit by lines produced primarily in the circumstellar region itself, and later spectra are due primarily to the supernova ejecta. Intermediate spectra are affected by both regions. A mass-loss rate of order $\dot M \sim 0.0001-0.001$\msol yr$^{-1}$ is inferred for a wind speed of 100-1000 \kmps. We discuss how future self-consistent models will better clarify the underlying progenitor structure.Comment: to appear in ApJ, 2001, 54

Viral vectors for gene delivery to the central nervous system

The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features

Promoting sustainability in quality improvement: an evaluation of a web-based continuing education program in blood pressure measurement.

BACKGROUND: The accuracy of blood pressure measurement is variable in office-based settings. Even when staff training programs are effective, knowledge and skills decay over time, supporting the need for ongoing staff training. We evaluated whether a web-based continuing education program in blood pressure measurement reinforced knowledge and skills among clinical staff and promoted sustainability of an existing quality improvement program. METHODS: Medical assistants and nurses at six primary care clinics within a health system enrolled in a 30-min online educational program designed to refresh their knowledge of blood pressure measurement. A 20-question pre- and post-intervention survey addressed learners\u27 knowledge and attitudes. Direct observation of blood pressure measurement technique before and after the intervention was performed. Differences in responses to pre- and post-module knowledge and attitudes questions and in observation data were analyzed using chi-square tests and simple logistic regression. RESULTS: All 88 clinical staff members participated in the program and completed the evaluation survey. Participants answered 80.6% of questions correctly before the module and 93.4% afterwards (p \u3c 0.01). Scores improved significantly among staff from all job types. Licensed practical nurses and staff who had been in their current job at least a year were more likely to answer questions correctly than registered nurses and those in their current job less than a year. Attitudes toward correct blood pressure measurement were high at baseline and did not improve significantly. Prior to the intervention, staff adhered to 9 of 18 elements of the recommended technique during at least 90% of observations. Following the program, staff was more likely to explain the protocol, provide a rest period, measure an average blood pressure, and record the average blood pressure, but less likely to measure blood pressure with the arm at heart level and use the right arm. CONCLUSIONS: We designed, implemented, and evaluated a web-based educational program to improve knowledge, skills, and attitudes in blood pressure measurement and use of an automated device among nurses and medical assistants in ambulatory care. The program reinforced knowledge related to recommended blood pressure measurement technique. TRIAL REGISTRATION: Retrospectively registered with ClincalTrials.gov on March 22, 2012; registration number NCT01566864