Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.     

A numeric study of the noise-induced tremor in a mathematical model of the stretch reflex

A mathematical model of the stretch reflex for the cat soleus muscle is presented. The time-delay differential equations of the model are solved using the fourth-order Runge-Kutta algorithm, introducing a Gaussian-noise term to simulate the environmental noise. The muscle response dynamics are then studied under various levels of average muscle activation. Finally, the feasibility of explaining the so-called physiological tremor from the properties of the stretch reflex mechanisms is discussed by comparing our results with reported experimental evidence.     

Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain

Prenatal stress greatly influences the ability of an individual to manage stressful events in adulthood. Such vulnerability may result from abnormalities in the development and integration of forebrain dopaminergic and glutamatergic projections during the prenatal period. In this study, we assessed the effects of prenatal stress on the expression of selective dopamine and glutamate receptor subtypes in the adult offsprings of rats subjected to repeated restraint stress during the last week of pregnancy. Dopamine D₂-like receptors increased in dorsal frontal cortex (DFC), medial prefrontal cortex (MPC), hippocampal CA₁ region and core region of nucleus accumbens (NAc) of prenatally stressed rats compared to control subjects. Glutamate NMDA receptors increased in MPC, DFC, hippocampal CA₁, medial caudate-putamen, as well as in shell and core regions of NAc. Group III metabotropic glutamate receptors increased in MPC and DFC of prenatally stressed rats, but remained unchanged in all other regions examined. These results indicate that stress suffered during the gestational period has long lasting effects that extend into the adulthood of prenatally stressed offsprings. Changes in dopamine and glutamate receptor subtype levels in different forebrain regions of adult rats suggest that the development and formation of the corticostriatal and corticolimbic pathways may be permanently altered as a result of stress suffered prenatally. Maldevelopment of these pathways may provide a neurobiological substrate for the development of schizophrenia and other idiopathic psychotic disorders.     

Efficient minimization of angle-dependent potentials for polypeptides in internal coordinates

Angular potentials play an important role in the refinement of protein structures through angle-dependent restraints (e.g., those determined by cross-correlated relaxations, residual dipolar couplings, and hydrogen bonds). Analytic derivatives of such angular potentials with respect to the dihedral angles of proteins would be useful for optimizing such restraints and other types of angular potentials (i.e., such as we are now introducing into protein structure prediction) but have not been described. In this article, analytic derivatives are calculated for four types of angular potentials and integrated with the efficient recursive derivative calculation methods of Gō and coworkers. The formulas are implemented in publicly available software and illustrated by refining a low-resolution protein structure with idealized vector-angle, dipolar-coupling, and hydrogen-bond restraints. The method is now being used routinely to optimize hydrogen-bonding potentials in ROSETTA.     

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

动态心电图在小儿心律失常与心率变异性诊断中的意义

目的：研究动态心电图（DCG）诊断小儿心律失常及心率变异性（HVR）的临床价值。方法：560 例患者均进行常规心电图和动 态心电图的检查,由两组专业人员分别对心电图（ECG）和动态心电图（DCG）的检查结果进行（评价）,比较两种检查手段的不同 类型的心律失常的诊出率;评价不同类型心律失常的HVR。结果：ECG对各型心律失常的总诊出率为25.00%,对不同类型心率失 常的诊出率分别为5.71%、1.43%、9.29%、6.43%和2.14%;DCG 总诊出率为62.85%,对不同类型心率失常的诊出率分别为 16.43%、6.43%、17.86%、14.29%和7.86%,二者具有统计学差异（P＜0.05）。不同类型的心律失常的HVR不同,其中室性心动过速 和室性期前收缩的各项指标均明显低于其他类型（P＜0.05）。结论：DCG对不同心律失常的诊出率明显高于ECG,并能有效反应 不同类型心率失常的HVR,在小儿心律失常的诊断中具有一定的临床价值。     

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000 ng/ml, and with cTnI from 25 to 1000 ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07–0.11) and a y-intercept of −7.79 (−17.87–2.29) ng/L (n = 65, r = 0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.     

Modelling the optimal timing in metabolic pathway activation—Use of Pontryagin's Maximum Principle and role of the Golden section

The time course of enzyme concentrations in metabolic pathways can be predicted on the basis of the optimality criterion of minimizing the time period in which an essential product is generated. This criterion is in line with the widely accepted view that high fitness requires high pathway flux. Here, based on Pontryagin's Maximum Principle, a method is developed to solve the corresponding constrained optimal control problem in an almost exclusively analytical way and, thus, to calculate optimal enzyme profiles, when linear, irreversible rate laws are assumed. Three different problem formulations are considered and the corresponding optimization results are derived. Besides the minimization of transition time, we consider an operation time in which 90% of the substrate has been converted into product. In that case, only the enzyme at the lower end of the pathway rather than all enzymes are active in the last phase. In all cases, biphasic or multiphasic time courses are obtained. The biological meaning of the results in terms of a consecutive just-in-time expression of metabolic genes is discussed. For the special case of two-enzyme systems, the role of the Golden section in the solution is outlined.     

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 μg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.