Analysis of metabolism by mathematical programming techniques

A method is presented for analyzing metabolic interactions by procedures based on mathematical programming techniques. In the procedures described it is assumed that the organism has (through natural selection) maximized (within the constraints imposed on it by its genetic constitution) its fitness to the environment. A practical experimental procedure is described through which the constraints imposed on reaction rates can be observed and from which the metabolic objective function which, it is presumed, metabolism has optimized can be calculated. A method for testing the validity of the objective function is given. Discussion is carried out in terms of a two-dimensional example but the procedures are valid for any number of dimensions. The results of the procedures are expressed by statements of the sort: the metabolic interactions of the cell are such that Q is maximized where Q = a₁x₁ + a₂x₂ + ... + a_nx_n, where a₁, ..., a_n are constants and x₁, ..., x_n are reaction rates. Some possible uses of metabolic objective functions are given.     

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H₂O₂ generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.     

Determination of the specific radloactivity of proteins separated by two-dimensional gel electrophoresis

Tritlum-labeled proteins, separated by two-dimensional gel electrophoresis, can be quantitatively extracted using sodium dodecyl sulfate (SDS)-urea buffer and subsequently acid-precipitated in the presence of serum albumin as carrier at low temperature (0–4°C). Their radioactivity can be counted efficiently under these conditions. Besides a better efficiency of counting, this method has some other advantages over the classical procedures using H₂O₂. The amounts of the eluted proteins can be easily measured in the SDS solution, using the Lowry method, and therefore specific radioactivity can be calculated. Also SDS can be removed easily, and the proteins can be used for further experiments.     

Writing and Low-Temperature Characterization of Oxide Nanostructures

Oxide nanoelectronics is a rapidly growing field which seeks to develop novel materials with multifunctional behavior at nanoscale dimensions. Oxide interfaces exhibit a wide range of properties that can be controlled include conduction, piezoelectric behavior, ferromagnetism, superconductivity and nonlinear optical properties. Recently, methods for controlling these properties at extreme nanoscale dimensions have been discovered and developed. Here are described explicit step-by-step procedures for creating LaAlO₃/SrTiO₃ nanostructures using a reversible conductive atomic force microscopy technique. The processing steps for creating electrical contacts to the LaAlO₃/SrTiO₃ interface are first described. Conductive nanostructures are created by applying voltages to a conductive atomic force microscope tip and locally switching the LaAlO₃/SrTiO₃ interface to a conductive state. A versatile nanolithography toolkit has been developed expressly for the purpose of controlling the atomic force microscope (AFM) tip path and voltage. Then, these nanostructures are placed in a cryostat and transport measurements are performed. The procedures described here should be useful to others wishing to conduct research in oxide nanoelectronics.     

A Site-Specific Approach for the Evaluation of Natural Attenuation at Metals-Impacted Sites

Consideration of monitored natural attenuation (MNA) as a remedy component for metals-contaminated sites can be achieved using a site-specific screening approach, followed by application of one or a series of sequential extraction measurements. Hazardous waste sites contaminated with metals can be screened for the implementation of monitored natural attenuation on the basis of contaminant-specific soil chemical characteristics (i.e., K_d's, solubilities, and nonexchangeable sorbed fraction). Field cases are used to demonstrate the screening approach and to outline the primary considerations involved in accurately applying sequential extraction procedures to support the of MNA for site remediation. The results of these case studies provide strong evidence that site-specific screening and the use of sequential extraction procedures are effective methods for evaluating natural attenuation for metals impacted sites.     

Institutional Diagnostic Reference Levels and Peak Skin Doses in selected diagnostic and therapeutic interventional radiology procedures

PurposeInstitutional (local) Diagnostic Reference Levels for Cerebral Angiography (CA), Percutaneous Transhepatic Cholangiography (PTC), Transarterial Chemoembolization (TACE) and Percutaneous Transhepatic Biliary Drainage (PTBD) are reported in this study.Materials and methodsData for air kerma-area product (P_KA), air kerma at the patient entrance reference point (K_a,r), fluoroscopy time (FT) and number of images (NI) as well as estimates of Peak Skin Dose (PSD) were collected for 142 patients. Therapeutic procedure complexity was also evaluated, in an attempt to incorporate it into the DRL analysis.ResultsLocal P_KA DRL values were 70, 34, 189 and 54 Gy.cm² for CA, PTC, TACE and PTBD respectively. The corresponding DRL values for K_a,r were 494, 194, 1186 and 400 mGy, for FT they were 9.2, 14.2, 27.5 and 22.9 min, for the NI they were 844, 32, 602 and 13 and for PSD they were 254, 256, 1598 and 540 mGy respectively. P_KA for medium complexity PTBD procedures was 2.5 times higher than for simple procedures. For TACE, the corresponding ratio was 1.6. PSD was estimated to be roughly 50% of recorded K_a,r for procedures in the head/neck region and 10% higher than recorded K_a,r for procedures in the body region. In only 5 cases the 2 Gy dose alarm threshold for skin deterministic effects was exceeded.ConclusionProcedure complexity can differentiate DRLs in Interventional Radiology procedures. PSD could be deduced with reasonable accuracy from values of K_a,r that are reported in every angiography system.     

Beta2-microglobulin removal by extracorporeal renal replacement therapies

There is increasing evidence that end-stage renal disease patients with lower beta₂-microglobulin plasma levels and patients on convective renal replacement therapy are at lower mortality risk. Therefore, an enhanced beta₂-microglobulin removal by renal replacement procedures has to be regarded as a contribution to a more adequate dialysis therapy. In contrast to high-flux dialysis, low-flux hemodialysis is not qualified to eliminate substantial amounts of beta₂-microglobulin. In hemodialysis using modern high-flux dialysis membranes, a beta₂-microglobulin removal similar to that obtained in hemofiltration or hemodiafiltration can be achieved. Several of these high-flux membranes are protein-leaking, making them suitable only for hemodialysis due to a high albumin loss when used in more convective therapy procedures. On-line hemodiafiltration infusing large substitution fluid volumes represents the most efficient and innovative renal replacement therapy form. To maximize beta₂-microglobulin removal, modifications of this procedure have been proposed. These modifications ensure safer operating conditions, such as mixed hemodiafiltration, or control albumin loss at maximum purification from beta₂-microglobulin, such as mid-dilution hemodiafiltration, push/pull hemodiafiltration or programmed filtration. Whether these innovative hemodiafiltration options will become accepted in clinical routine use needs to be proven in future.     

VO(acac)(2)/H(2)O(2)/NaI: a mild and efficient combination for the cleavage of dithioacetal derivatives of sugars

A wide variety of dithioacetal derivatives of sugars can be cleaved easily into the corresponding open-chain aldehydo sugars using an efficient combination of VO(acac)₂/H₂O₂/NaI at 0–5 °C. Some of the salient features of this protocol are mild reaction conditions, good yields, short reaction times, easy work-up procedures, and non-involvement of toxic chemicals.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G_2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')₂ fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3–4 h of incubation at 37°C. The F(ab')₂ fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')₂ fragments for the diagnosis of cancer in patients.     

Reciprocal Giemsa staining of late DNA replicating regions produced by low and high pH sodium phosphate

A procedure is described whereby late replicating, BUdR-substituted chromosome regions stain intensely with Giemsa, thus producing the reciprocal staining patterns compared to those obtained by all other BUdR-Giemsa procedures where BUdR-substituted regions appear pale staining. This method may be more convenient than pre-existing techniques for demonstrating late replicating chromosome regions, and may provide a higher degree of resolution of the late replicating regions. The finding that BUdR-substituted regions can be made to stain either intensely or palely with Giemsa, depending on the pH of the pretreatment NaH₂PO₄ solution, may have important implications concerning the mechanism of BUdR-induced chromosome differentiation.     

Early termination of pregnancy: A comparative study of intrauterine prostaglandin F2α and vacuum aspiration

The relative safety and effectiveness of vacuum aspiration and the intrauterine administration of 5 mg PGF_2α for terminating pregnancies within two weeks of a missed menstrual period were evaluated in a study where subjects were randomly assigned to procedures; 100 patients were aborted with vacumm aspiration and 100 patients were aborted with PGF_2α. All PGF_2α-treated patients were premedicated with meperidine, diazepam and atropine. Complications were infrequent with either of the procedures. Vomiting occurred more frequently during the PGF_2α procedure (30.0%) than during the vacuum aspiration procedure (9.0%). The intrauterine instillation of PGF_2α successfully terminated all pregnancies. One patient continued to be pregnant after the vacuum aspiration procedure. Based on the results of this study both study procedures appeared to be safe and effective for terminating early first trimester pregnancies.     

Purification of Proteins by the Use of Hydrophobic Zeolite Y

Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH₄)₂SO₄ or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be adsorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH₄)₂SO₄ fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5.     

High performance liquid chromatography of prostaglandins: Biological applications

Two procedures are described for separation and purification of prostaglandins by high performance liquid chromatography. Both systems show excellent resolution of PGA₂, PGE₂ and PGF_2α. Peak definition on the micro-particle silicic acid system is particularly good with the PGs appearing in 2–3 ml of organic effluent. Studies on reproducibility showed that PGE₂ and PGF_2α could be recovered with a retention volume of 54.2±0.76 ml and 64±0.6 ml, respectively (n=7, mean ±SD) with good recovery. The column can be run in about one hour and can be regenerated indefinitely (>200 times). The degree of purification is compatible with analysis by gas chromatography-mass spectrometry. Examples showing the application of this chromatographic method to human seminal fluid, human renal tissue, platelet rich plasma and human urine samples indicate that it makes possible analysis of these samples even at low levels.     

Additive main effects and multiplicative interaction (AMMI) analysis of genotype-location interaction in variety trials repeated over years

Genotype-location (GL) interaction effects are of special interest for breeding programmes to identify adaptation targets, adaptive traits and test sites. These effects, generally having relatively low repeatability between years, should be studied on a multiyear basis in annual crops. Their assessment by additive main effects and multiplicative interaction (AMMI) analysis is currently defined for this situation. Two procedures based on cross validations are proposed for testing the GL-interaction principal component axes, exploiting the utilities of the computer programme MATMODEL. The use of Gollob’s F test, F _GH2 test, F _R test and the heuristic criterion based on the signal-to-noise ratio is also envisaged. The consistency of results provided by the testing procedures was verified on four data sets of different cereal crops. Gollob’s test tended to be the most liberal, while the F _GH2 test appeared somewhat more liberal than the F _R test. The signal-to-noise ratio gave results consistent with the F _R test considered at a P?0.01 level of significance. These criteria disagreed in two data sets with the conclusions provided by the two cross-validation procedures which, in turn, also disagreed in one data set. Preference could be given to different testing procedures depending on the number of test years, locations and genotypes.     

Preparation of deuterated arachidonic acid

Arachidonic acid is transformed to a wide variety of oxygenated metabolites. The limiting factor in the quantitation of these metabolites by GC/MS using selected ion monitoring (SIM) is the availability of suitable stable-isotope labelled internal standards. We report a convenient procedure for the preparation of ²H₈-arachidonic acid, from which the desired deuterated metabolite standards can be produced by a combination of chemical and biological procedures.     

Two simple volumetric methods for respiration measurements with oxygen supply to the sample

Summary Two easy and inexpensive procedures suitable especially for the measurements of aerobic respiration activity of numerous soil samples are described. Size of samples can be changed within broad limits. The first of the two methods is based on measurements of the sum of oxygen consumed during a prolonged period of time. Actual rate of O₂ consumption over short-time intervals is determined by the second method.     

An enzymatic method for the histochemical localization of free and esterified cholesterol separately

Synopsis An enzymatic method for the histochemical localization of cholesterol is presented. It makes possible the localization of free cholesterol, cholesterol esters, or both and is compatible with routine histological staining procedures. The method is based on the production of H₂O₂ from free cholesterol by cholesterol oxidase. Sites of peroxide production are visualized by a brown reaction product formed in a peroxidase-catalysed reaction between diaminobenzidine and H₂O₂. cholesterol esters can be demonstrated as cholesterol after hydrolysis by cholesterol ester hydrolase. Some examples of the application of the method are given.     

The Use of Permeabilized Cells to Assay Protein Phosphorylation and Catecholamine Release

A number of approaches can be used to determine the protein kinases and protein phosphatases acting on particular phosphoproteins in vivo. Cell permeabilization represents one such approach. In this overview we discuss the different permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on the extent of digitonin-permeabilization, protein phosphorylation and catecholamine release are also discussed. The factors include the permeabilization medium, the ions such as calcium, and the second messengers, such as cAMP, IP₃, cADPR and calmodulin. The effect of specific peptide inhibitors of protein kinases on tyrosine hydroxylase phosphorylation is illustrated. Advantages and disadvantages of cell permeabilization procedures are discussed throughout the text.