A simple method for the determination of the kinetic constants of membrane enzymes utilizing hydrophobic substrates: ubiquinol cytochrome c reductase

We have devised a method to determine the true Km of membrane enzymes for hydrophobic substrates dissolved in lipid bilayers, and the lipid/water partition coefficients, by simple steady-state kinetic measurements at varying membrane phospholipid fractional volumes in the assay medium. The method has been applied to mitochondrial ubiquinol cytochrome c reductase, using short-chain ubiquinols as reductants at saturating cytochrome c. The partition coefficients of the quinols, as obtained by this method, are in good agreement with those determined directly by other procedures; Km values obtained by this method, when expressed as concentrations in the lipid bilayer, are in the millimolar range. The kinetics of the ubiquinol analog duroquinol are independent of phospholipid concentration, as expected from its partition coefficient close to unity.     

A new method for decay-associated fluorescence spectroscopy. Application to the tryptophan zwitterion

We describe a new method for decay associated fluorescence spectroscopy using synchrotron radiation as the excitation pulse and a photon counting technique. This method is based on the determination of the difference between the barycenters of the exciting pulse and of the fluorescence response at several wavelengths. It is applicable to the case where individual decay times are independent of emission wavelength. Coupled to the analysis of the decay curve at only one emission wavelength, this method reduces the time devoted to the numerical analysis and avoids the spectral distortion due to the lamp profile. The results obtained by this method on indole, the tryptophan zwitterion, and N-acetyl-tryptophan are presented. Results are compared to those obtained by two other methods: Determination of the fluorescence decay parameters by deconvolution analysis at several emission wavelengths. Photon counting of the fluorescence spectrum emitted during a selected time window after the excitation pulse.     

Genetic changes across language boundaries in Europe

By means of three different methods we investigated whether 59 allele frequencies and ten cranial variables show increased change at 29 language-family boundaries in Europe. The quadrat-variance method compares variances of map quadrats crossed by language-family boundaries to variances of quadrats that are not crossed. The rate-of-change method examines the directional derivative of surfaces of the variables perpendicular to a language-family boundary and compares these derivatives to the same quantities obtained by randomly placing the language boundaries on the map of Europe. The difference method tests whether these variables differ more across language-family boundaries than across randomly placed boundaries. These special data-analytic techniques had to be developed to avoid the problem of spatial autocorrelation of both language and biological data. All three methods indicate increased genetic change at language-family boundaries. Clearer and more pronounced results are obtained by the first two methods than by the difference method. Thirteen language-family boundaries show significant gene frequency change by at least one of the methods. Changes are more marked in gene frequencies than in cranial variables. Different allele frequencies mark the increased change at different language boundaries. A model, based on the known history of each language-family boundary, was constructed to predict whether given boundaries should exhibit increased genetic change. The model is in good agreement with the observed results.     

The labelled inhibitor method in cytoenzymology

Synopsis The principles of a histoenzymological method based on the use of labelled inhibitors are reviewed. The detection of inhibitor-labelled enzyme complexes by histoautoradiography allows enzymes to be localized quantitatively in cells and tissue structures. The present state and future possibilities of the method are discussed.This review was presented in a shorter form at the International Symposium on Autoradiography organized by the Royal Microscopical Society at Nottingham in July 1971.     

Determining blood pressure in infants: use of the flush technique

The flush technique that recently has come into use is probably the most satisfactory method available for measuring blood pressures in infants and is of great aid in diagnosis of diseases associated with hypertension or hypotension. The method is practical and highly accurate. Readings obtained by this technique are not influenced by the width of the cuff, are usually higher at the wrist than at the ankle, and approximate the mean rather than the systolic blood pressure.     

DETERMINING BLOOD PRESSURE IN INFANTS—Use of the Flush Technique

The flush technique that recently has come into use is probably the most satisfactory method available for measuring blood pressures in infants and is of great aid in diagnosis of diseases associated with hypertension or hypotension. The method is practical and highly accurate. Readings obtained by this technique are not influenced by the width of the cuff, are usually higher at the wrist than at the ankle, and approximate the mean rather than the systolic blood pressure.     

A simplified method for the rapid preparation of peroxidase-anti-peroxidase (PAP) complexes

Summary A modified method is described for the rapid production of peroxidase-antiperoxidase complexes to be used in immunocytochemistry. In this method anti-peroxidase antibodies are precipitated from crude serum with peroxidase at equivalence and subsequently resolubilized at low pH with excess peroxidase. The complexes are isolated from unbound immunoglobulin and peroxidase by gelfiltration. The method combines the advantages of both previously described preparation procedures. The resulting PAP-complex, when tested in indirect immunocytochemistry, is comparable to that obtained in established preparation procedures.Supported by a grant from the Queen Wilhelmina Cancer Foundation (grant no. PA 77/52)     

THE BIOLOGY OF THE RICE WEEVIL, CALANDRA ORYZAE (L.)

The length of the developmental instars of Calandra oryzae has been estimated at relative humidities of 50, 60, 70 and 80% at 21° C., at 70% at 18, 25 and 30° C., and at 80% at 18° C., by the dissection daily of samples of infested wheat-grains. The results are expressed as a median obtained by the method of probit analysis. Comparison of this method, with estimates of median and mean obtained by orthodox arithmetical methods from similar work on Rhizopertha , show that the probit method gives good estimates.
About 90% of the eggs laid are fertile. Normally only one adult will develop in a grain, all other individuals being destroyed by cannibalism. The sex ratio is unity. It was not possible to cross C. oryzae and C. granaria.
The daily oviposition rate of C. oryzae at 17, 21 and 25° C. increases with relative humidity. There is a critical point at about 60% r.h . below which egg laying declines rapidly, and mortality is high. At 100% r.h . the oviposition rates per female per day are approximately 1.3, 2.5 and 3.4 at 17, 21 and 25° C. respectively.
In experimental conditions most eggs per grain are obtained by giving isolated females one grain each, but more eggs are laid by females given more than one grain. Daily egg output is reduced by grouping females or including males. In culture, depths of grain up to 7 cm. do not discourage egg laying.     

Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery

We report on the development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery. The method consists of depletion of albumin and IgG and multi-lectin affinity chromatography (M-LAC), followed by nanoLC-MS/MS analysis of digested proteins and label-free comparative quantitation of proteins. The performance of the method is monitored by multiple quality control points to ensure reproducibility of the analysis. The method identifies proteins that are reported to be present in normal plasma at concentrations of 10-100 ng/mL and that may be of particular interest when studying a variety of disease conditions. Numerous tissue leakage proteins of potentially even lower concentrations are also identified. When the method was used in a study to identify potential biomarkers of psoriasis, the differential abundance of proteins present at low mug/mL level was quantitated and later verified by ELISA measurements.     

超临界CO2萃取红景天中红景天苷、苷元酪醇的研究

采用超临界CO₂ 萃取法和乙醇常温浸提法相比较, 研究从红景天中提取红景天苷、苷元酪醇的工艺条件, 结论是:采用超临界CO₂ 萃取法能萃取出红景天生药中红景天苷的1.2%, 提取率不高, 但该方法能萃取出80%的苷元酪醇, 萃取液中苷元酪醇的相对含量可达45.68%;乙醇常温浸提法能将红景天苷、苷元酪醇同时有效萃取, 且得率较高, 但是萃取液中两物质相对含量较低, 进一步分离纯化将有难度。本研究结果表明, 将超临界CO₂ 萃取法和乙醇常温浸提法有效结合, 可实现两物质的有效分离, 推进红景天有效成分的产业化进程。     

A comparative analysis of strategies for isolation of matrix vesicles

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.     

A new histogenetic method for minor histocompatability antigen typing.

A new method for typing minor H antigens is described. The method is based on the observation that effector T cells, sensitized in vivo to minor H mitigens and then rechallenged in vitro, will lyse labeled target cells in the CML assay, provided that the target, the sensitizing, and the responding cells share the same allele at H-2K or H-2D loci. In this method, two congenic lines differing at a minor H locus are cross-immunized (by skin grafting, injection of lymphoid cells, or, preferably, by a combination of both treatments), rechallenged in vitro, and tested against a panel of strains carrying the same H-2 haplotype as the two lines. Target cells of the panel carrying the same minor H antigens as the strain against which the effector cells were sensitized are lysed; target cells not carrying these antigens are unaffected. The feasibility of the method was demonstrated by testing for H-3 antigens of a panel fo H-2b-bearing strains. The new method is faster and more economical than the classicial F1 method which has been used until recently.     

Metabolic activity of bacterial cells enumerated by direct viable count

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Metabolic activity of bacterial cells enumerated by direct viable count.

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Quick Counting Method for Estimating the Number of Viable Microbes on Food and Food Processing Equipment

A rapid method for estimating the extent of microbial contamination on food and on food processing equipment is described. Microbial cells are rinsed from food or swab samples with sterile diluent and concentrated on the surface of membrane filters. The filters are incubated on a suitable bacteriological medium for 4 hr at 30 C, heated at 105 C for 5 min, and stained. The membranes are then dried at 60 C for 15 min, rendered transparent with immersion oil, and examined microscopically. Data obtained by the rapid method were compared with counts of the same samples determined by the standard plate count method. Over 60 comparisons resulted in a correlation coefficient of 0.906. Because the rapid technique can provide reliable microbiological count information in extremely short times, it can be a most useful tool in the routine evaluation of microbial contamination of food processing facilities and for some foods.     

Analytical study of the effects of some soil and plant parameters on root growth due to absorption of one mobile ion: A free-boundary model

The object of our study is: a model for root growth through a free-boundary problem and the effects resulting from differences in nutrient availability and transport of only one mobile nutrient between the root surface and the rhizosphere produced by an absorption Michaelis-Menten for low and high concentrations. The model equations are solved by two methods: the quasi-stationary method and the balance integral method. The numerical solutions are used to compute radial root growth. Curves of nutrient concentration at the root-soil interface, curve as a function of root radius as well as curves representing root radius as a function of time are plotted. The parameters which are varied are the root absorption power, flux velocity at the root surface, efflux, rhizosphere radius, diffusion coefficient, buffer power, and maximum influx. The two methods show the theoretical results for radial root growth in the range of low and high concentrations. The balance integral method provides more detailed information.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 1000 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients on variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at −25°C prior to analysis.     

Online resistance monitoring during autometallographic enhancement of colloidal Au labels for DNA analysis

DNA diagnostics at the point-of-care requires biosensors that rely on highly sensitive transducers and are producible at low cost. A promising candidate technology is based on direct electrical detection of autometallographically enhanced Au labeled analytes. We present a substantial improvement to the previously used method by introducing online DC resistance monitoring during the autometallographic enhancement process. Since multi-step enhancement, washing, drying, and measurement cycles are eliminated, our method takes the direct electrical detection method a step further to applicability in a point-of-care environment. The feasibility of the novel method is demonstrated by its application in a simple DNA hybridization assay and the analysis of a single nucleotide polymorphism (SNP) using allele-specific hybridization. Unequivocal discrimination of all possible base pairing combinations in the SNP assay has been achieved. The SNP assay in particular indicates the potential of the method for analyte quantification.     

Retinal Vessel Width Measurement at Branchings Using an Improved Electric Field Theory-Based Graph Approach

The retinal vessel width relationship at vessel branch points in fundus images is an important biomarker of retinal and systemic disease. We propose a fully automatic method to measure the vessel widths at branch points in fundus images. The method is a graph-based method, in which a graph construction method based on electric field theory is applied which specifically deals with complex branching patterns. The vessel centerline image is used as the initial segmentation of the graph. Branching points are detected on the vessel centerline image using a set of detection kernels. Crossing points are distinguished from branch points and excluded. The electric field based graph method is applied to construct the graph. This method is inspired by the non-intersecting force lines in an electric field. At last, the method is further improved to give a consistent vessel width measurement for the whole vessel tree. The algorithm was validated on 100 artery branchings and 100 vein branchings selected from 50 fundus images by comparing with vessel width measurements from two human experts.     

Evaluation of methods for storage of marine macroorganisms with optimal recovery of bacteria

Marine macroorganisms are a potential source for new bioactive substances. In many cases marine microorganisms--especially bacteria--associated with these macroorganisms are actually producing the bioactive substances. One often is not able to immediately isolate microorganisms from collected macroorganismic materials; we therefore evaluated different methods for storage of such material, e.g., on board research vessels. These methods were the following: storage of macerates in sintered glass beads and 5% trehalose at -20 degrees C (SGT method); storage of sections in 5% dimethyl sulfoxide at -70 degrees C (SD method); storage of macerates at -20 degrees C using the commercial ROTI-STORE system (RS method); storage of macerates at -20 degrees C in 50% glycerol (GC method); and storage of macerates covered by mineral oil at 4 degrees C (MO method). The SGT and SD methods resulted in numbers of and especially diversity of recoverable bacteria that were higher than for the other methods. Data for the RS method indicated its potential usefulness, too. The MO method resulted in growth during storage, thereby enriching a few selected microorganisms; the GC method resulted in a survival and diversity of recovered bacteria that was too low.