Small Angle Scattering Of Large Protein Units Under Osmotic Stress

Indiana University-Purdue University Indianapolis (IUPUI)Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt% and 36 wt%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography

ANALYSIS OF MULTICHANNEL SIGNALS USING A CHANNEL SIMULATOR

poster abstractsIn biological systems ion channels can be thought of as opening and clos-ing pores that allow the flow of ion through a membrane. Ion channels are responsible for intracellular communication and keeping an osmotic equilib-rium across cell membranes. Measurements of ion channel activities that generate multichannel events (the opening of more than one channel at a time) are important to understand, but difficult to analyze. In many cases the multichannel events are discarded instead of used in the analysis. The availability of a channel signal simulator offers an excellent opportunity to develop and test statistical models for analysis of multichannel signals. We have generated single channel traces for various open probabilities and then digitally superimposed these signals to obtain multichannel events. We then applied our simplified analysis to these multichannel traces to calculate sin-gle channel parameters such as the average ON and OFF times and their statistical distribution of the ON times. The average ON time as well as the ON time distribution matched the single channel input mean open time. The importance of our findings is that our proposed analysis will be able to use the statistical distribution of ON times, in addition to the traditional “dwell time” (or average ON time) parameter, to characterize ion channels

Structural Changes of Alpha 1-Antitrypsin under Osmotic Pressure and in the Presence of Lipid Membranes

poster abstractAlpha 1-Antitrypsin (A1AT) is a glycoprotein that has been shown to have protective roles of lung cells against emphysema, a disease characterized by lung tissue destruction. Most known glycoproteins have been shown to play a role in cellular interactions but the exact role of the glycan chains is still under investigation. Previous electrophysiological measurements show that A1AT has a strong affinity to lipid bilayers perturbing the function of ion channels present in the membrane. We have performed contrastmatching small-angle neutron scattering (SANS) experiments to study the conformational changes of the glycosylated form of A1AT for different concentrations of the osmolyte poly(ethelene glycol) (PEG) and in the presence of two different lipid membranes: POPC and POPS. We also monitor the structural changes of the lipid vesicles in the presence of A1AT by SANS. Guinier fits were used as a first approximation to obtain the radius of gyration (Rg) of A1AT. Bragg peaks were used to study structural changes of lipid vesicles. We observed that the Rg of A1AT changes as a function of PEG concentration in solution and when in the presence of lipid vesicles. The deformations monitored through changes in A1AT’s Rg in the presence of lipid vesicles are compared to the deformations of the glycoprotein observed under osmotic pressure and to the structural changes observed in the lipid vesicles

Cardiopatías congénitas en el perro : conducto arterioso persistente, estenosis pulmonar y estenosis aórtica

En el presente artículo se describen los hallazgos clínicos, radiológicos, electrocardiográficos y ecográficos de las principales cardiopatías congénitas en el perro de acuerdo con una serie de casos clínicosClinical signs and radiological, electro and echocardiographic findings of the main congenital heart diseases in the dog are described in this paper based on c1inicalcases

Finding Active Ingredients in Pharmaceuticals by UV Spectrophotometry

The active ingredient in any pharmaceutical is the chemical that will ultimately deliver the desired effect on a patient. Knowing the quality and the quantity of the active ingredient in a pill right before ingestion is of paramount importance for the patient’s health and the desired results. Unfortunately drugs only undergo quality control testing at the manufacturing plant but not at the point of sale. Moreover, to an untrained eye, one pill may not appear different from another and if the wrong pill or the wrong dose is taken, adverse health effects may arise. Indeed, manufacturers of counterfeit drugs rely on these two points of appearance and testing. In this study we examine whether ultra violet (UV) spectrophotometry absorbance can be used to separate an active ingredient’s UV peaks from the combination of peaks generated by the inactive ingredients of the tablet. A well-understood active ingredient, acetaminophen, was used for this study. Samples were prepared by crushing Tylenol tablets, dissolving the powder in different solvents (0.1M HCl, 0.1M NaOH, and H2O) at various concentrations and mixed by vortex. After preparation, the samples were measured by UV Spectroscopy. Experimental results were compared to standard UV curves for the pure active ingredient to correlate the observed changes in absorbance within the relevant UV wavelength range. We observe that more than one solvent is needed to identify the active ingredient. Development of a simple method to accurately identify the quality of the active ingredient will provide an additional safeguard to consumers, particularly in regions where counterfeit drugs are prevalent

Detecting Counterfeit Pharmaceuticals through UV Spectrophotometry

poster abstractAccording to the World Health Organization between 10%-30% of medicines, in Africa, Asia and South America, are counterfeit or sub-standard, affecting the health of millions of people. Currently, there is no effective way to check the quality of a medicine at the point of care, leaving many with treatable diseases at risk. The goal of this study is to identify UV-Vis (240nm - 500nm) absorbance patterns that would indicate if a drug is sub-standard or counterfeit. UV-Vis spectroscopy was selected as the method for testing due to the maturity and availability of the technology. Pure Acetaminophen and Tylenol were used as controls for proof of concept. Samples were prepared by dissolving different combinations of the pure active ingredient and adulterants such as cement, rice flour, vitamin C and lactose in three different types of solvents (H2O, 0.1 M HCl, 0.1 NaOH). Various concentrations (ranging from 0.01mg/ml to 0.04mg/ml) and mixing ratios were analyzed using a UV-Vis Spectrophotometer. It was found that adulterants significantly decrease the absorption of acetaminophen at 245nm by interacting with its benzene ring, while showing a slight increase in other parts of the spectrum. UV-Vis scans show that the amount of change in absorbance at specific wavelengths, coupled with characteristic wavelength shifts produced by different solvents, can be used for detection of counterfeit drugs. The methods presented here could be used for quality control of medicines at or near the point of care in parts of the world at higher risk of encountering defective pharmaceuticals

The separate effects of self-estimated and actual alcohol intoxication on risk-taking: a field experiment

First
 Version
(“Alcohol
 Consumption 
and 
Risk 
Attitude”):
 October 
2008.Many risky actions are carried out under the influence of alcohol. However, the effect of alcoholic intoxication over the willingness to take risks is complex and still remains unclear. We conduct an economic field experiment in a natural, drinking and risk-taking environment to analyze how both actual and self-estimated blood alcohol concentration (BAC) levels influence subjects’ choices over monetary lotteries. Our results reveal a negative impact of both actual and self-estimated BAC levels on risk-taking. However, for male and young subjects, we find a positive relationship between BAC underestimation (a pattern of estimation error which mainly occurs at high BAC levels) and the willingness to choose riskier lotteries. Our findings suggest that a risk compensation mechanism is activated only when individuals’ own intoxication level is consciously self-perceived to be high. We conclude therefore that human propensity to engage in risky activities under the influence of alcohol is not due to an enhanced preference for risky choices. In addition to the suggestion in the existing literature that such propensity is due to a weakened ability to perceive risks, our results indicate that an impaired self-perception of own intoxication level may also be an important factor.Financial
 support 
from 
the 
Spanish 
Ministry
 of 
Science
 and Innovation (SEJ2010‐17049/ECON),
 the
 Regional
 Programs SEJ‐02547,
 SEJ‐340
 and
 SEJ‐023
 of
 the
 Regional
 Government 
of 
Andalusia


Disautonomía felina

En el presente trabajo, se efectúa una revisión completa del síndrome disautonomía felina, así como la descripción de un caso clínico correspondiente a un gato macho de 11 meses de edad, junto con los medios de diagnóstico utilizado.At the present research a review ol the dysautonomia feline syndrome is included with the description of a clinical case in a male cat of 11 months of age

Effect on Health-related Quality of Life of changes in mental health in children and adolescents

<p>Abstract</p> <p>Background</p> <p>The objective of the study was to assess the effect of changes in mental health status on health-related quality of life (HRQOL) in children and adolescents aged 8 - 18 years.</p> <p>Methods</p> <p>A representative sample of Spanish children and adolescents aged 8-18 years completed the self-administered KIDSCREEN-52 questionnaire at baseline and after 3 years. Mental health status was measured using the Strengths and Difficulties Questionnaire (SDQ). Changes on SDQ scores over time were used to classify respondents in one of 3 categories (improved, stable, worsened). Data was also collected on gender, undesirable life events, and family socio-economic status. Changes in HRQOL were evaluated using effect sizes (ES). A multivariate analysis was performed to identify predictors of poor HRQOL at follow-up.</p> <p>Results</p> <p>Response rate at follow-up was 54% (n = 454). HRQOL deteriorated in all groups on most KIDSCREEN dimensions. Respondents who worsened on the SDQ showed the greatest deterioration, particularly on Psychological well-being (ES = -0.81). Factors most strongly associated with a decrease in HRQOL scores were undesirable life events and worsening SDQ score.</p> <p>Conclusions</p> <p>Changes in mental health status affect children and adolescents' HRQOL. Improvements in mental health status protect against poorer HRQOL while a worsening in mental health status is a risk factor for poorer HRQOL.</p