Disc Plate Method of Microbiological Antibiotic Assay: I. Factors Influencing Variability and Error 1

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.     

Investigation of the Peculiarities of Two-joint Muscles Using a 3 DOF Model of the Human Upper Limb in the Sagittal Plane: an Optimization Approach

Abstract

The purpose of this paper is an investigation of the peculiarities of biarticular muscles by means of modelling and analytical solution of the indeterminate problem. The basic model includes 10 muscle elements performing flexio/extensio in the shoulder, elbow and wrist. Four of them are biarticular muscles. Two modifications of the model with only monoarticular muscles are developed. The indeterminate problem is solved analytically using the objective criterion σc_iF_i ² where F( is the module of the i-th muscle force and Cj is a weight coefficient. The predicted muscle forces, joint reactions and moments are compared in-between the basic model and its two modifications for different joint angles, external loading and weight coefficients. The main conclusions are: it is impossible to formulate strict advantages of the biarticular muscles under quasistatical conditions, their peculiarities depend on limb position, external loading and neural control; in general, monoarticular muscles are more powerful than biarticular ones; the biarticular muscles fine tune muscle coordination, their control is more precise and graceful; the presence of biarticular muscles leads to an increase of the joint reactions and moments, thus stabilizing the limb.     

Studies on the Reduction of 2,6-Dichlorophenolindophenol by 6-Substituted 3-Hydroxy-4-Pyrones,Using a Stopped-flow Method

The reduction of 2,6-dichlorophenolindophenol (DCIP) by 6-substituted 3-hydroxy-4-pyrones was studied in the pH range of 3.0 to 7.8, using a stopped-flow apparatus.

1) Kinetic characteristics of 6-substituted 3-hydroxy-4-pyrones are shown as the apparent first-order rate constant, K_app, or the second-order rate constant, k. The plot of log k against pH was different from those obtained for the reactions of L-ascorbic acid and triose reductone with DCIP.

2) The log (k/k_H) of 6-substituted 3-hydroxy-4-pyrone solutions at acidic pHs have been correlated with Δδ_C?3 values for 3-hydroxy-4-pyrone.     

Prediction of Individual Muscle Forces Using Lagrange Multipliers Method - A Model of the Upper Human Limb in the Sagittal Plane: II. Numerical Experiments and Sensitivity Analysis

Using the method of Lagrange multipliers an analytical solution of the optimization problem formulated for a two-dimensional, 3DOF model of the human upper limb has been described in Part I of this investigation. The objective criterion used is the following: [formula: see text], where F(i) -s are the muscle forces modelled and c(i) -s are unknown weight factors. This study is devoted to the numerical experiments performed in order to investigate which sets of the weight factors may predict physiologically reasonable muscle forces and joint reactions. A sensitivity analysis is also presented. The influence of: the gravity forces, different external loads applied to the hand, changes of the weight factors and of joint angle on the optimal solution is studied. A general conclusion may be drawn: using the above mentioned objective criterion, practically all motor tasks performed by the human upper limb may be described if the c(i) -s are properly chosen. These weight factors generally depend on the joint moments and must be different (their magnitudes as well as their signs) for agonistic muscles and for their antagonists.     

Distinct Specificities of pRb Phosphorylation by CDK4 Activated by Cyclin D1 or Cyclin D3: Differential Involvement in the Distinct Mitogenic Modes of Thyroid Epithelial Cells

Two distinct mitogenic modes coexist in the physiologically relevant model ofprimary cultures of dog thyroid epithelial cells. The differentiation-associated mitogenicstimulation by TSH and cAMP specifically requires the assembly and activation of cyclin D3-cyclin-dependent kinase (CDK)4 associated to p27kip1, while the dedifferentiatingproliferation induced by growth factors is associated with induction of cyclin D1. Here, wesuggest that the related CDK “inhibitors” p21cip1 and p27 are differentially utilized as positiveCDK4 regulators in these mitogenic stimulations. p21 was induced by EGF+serum, butrepressed by TSH, which, as previously shown, up-regulates p27. In response to EGF+serum,p21 supported the nuclear localization, phosphorylation and pRb-kinase activity of CDK4.Unexpectedly, partly different site-specificities of pRb-kinase activity, leading to similardifferences in the phosphorylation pattern of pRb in intact cells, were associated with cyclinD3-CDK4 bound to p27 in TSH-stimulated cells, or with CDK4 bound to p21 in growthfactor-stimulated cells. These differences were ascribed to the predominant association of thelatter complex to cyclin D1. Indeed, in different cell types and species, cyclin D1 varied fromcyclin D3 by more efficiently driving the phosphorylation of pRb at sites (Ser807/811 andThr826) required for its electrophoretic mobility shift. Therefore, different D-type cyclinscould differently impact some pRb functions, which should be considered not only in theunderstanding of the relationships between cell cycle and differentiation expression in thedistinct mitogenic modes of thyroid cells, but also in various development or differentiationmodels associated with dramatic switches in the expression of individual D-type cyclins.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Enhanced External Counterpulsation Treatment May Intervene The Advanced Atherosclerotic Plaque Progression by Inducing The Variations of Mechanical Factors: A 3D FSI Study Based on in vivo Animal Experiment

Growing evidences suggest that long-term enhanced external counterpulsation (EECP) treatment can inhibit the initiation of atherosclerotic lesion by improving the hemodynamic environment in aortas. However, whether this kind procedure will intervene the progression of advanced atherosclerotic plaque remains elusive and causes great concern in its clinical application presently. In the current paper, a pilot study combining animal experiment and numerical simulation was conducted to investigate the acute mechanical stress variations during EECP intervention, and then to assess the possible chronic effects. An experimentally induced hypercholesterolemic porcine model was developed and the basic hemodynamic measurement was performed in vivo before and during EECP treatment. Meanwhile, A 3D fluid-structure interaction (FSI) model of blood vessel with symmetric local stenosis was developed for the numerical calculation of some important mechanical factors. The results show that EECP augmented 12.21% of the plaque wall stress (PWS), 57.72% of the time average wall shear stress (AWSS) and 43.67% of the non-dimensional wall shear stress gradient (WSSGnd) at throat site of the stenosis. We suggest that long-term EECP treatment may intervene the advanced plaque progression by inducing the significant variations of some important mechanical factors, but its proper effects will need a further research combined follow-up observation in clinic.     

The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis

The cAMP-dependent mitogenic stimulation elicited by thyroid-stimulating hormone (TSH) in primary cultures of canine thyroid epithelial cells is unique as it upregulates the cyclin-dependent kinase (CDK) inhibitor p27kip1 but not D-type cyclins. TSH and cAMP promote the assembly of required cyclin D3-CDK4 complexes and their nuclear import. Here, the nuclear translocation of these complexes strictly correlated in individual cells with the enhanced presence of nuclear p27. p27, like cyclin D3, supported the TSH-stimulated pRb-kinase activity of the CDK4 complex and, as demonstrated using the high-resolution power of the two-dimensional (2D) gel electrophoresis, the phosphorylation of CDK4, presumably by the nuclear CDK-activating kinase. In the presence of TSH, transforming growth factor beta (TGFbeta) did not affect the assembly of cyclin D3-CDK4, but it strongly inhibited the pRb-kinase activity associated with both cyclin D3 and p27, not only by preventing the nuclear import of cyclin D3-CDK4 and its binding to p27, but also by inhibiting CDK4 phosphorylation within residual p27-bound cyclin D3-CDK4 complexes. No alterations of the relative abundance of multiple (un)phosphorylated forms of cyclin D3 and p27 demonstrated by 2D-gel electrophoresis were associated with these processes. This study suggests a crucial positive role of p27 in the TSH-stimulated nuclear import, phosphorylation, and catalytic activity of cyclin D3-bound CDK4. Moreover, it demonstrates a technique to directly assess the in vivo phosphorylation of endogenous CDK4, which might appear as a last regulated step targeted by the antagonistic cell cycle effects of TSH and TGFbeta.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

Method for simultaneous measurement of norepinephrine, 3-methoxy-4-hydroxyphenylglycol and 3,4-dihydroxyphenylglycol by liquid chromatography with electrochemical detection: application in rat cerebral cortex and plasma after lithium chloride treatment

An assay was developed to quantify norepinephrine (NE) and its metabolites (MHPG and DHPG) by high-performance liquid chromatography with electrochemical detection method (HPLC-ECD) in brain tissue and plasma of rats treated by LiCl. Separation on C(18) column was obtained by a mobile phase consisting of 4.5% methanol in buffer (0.1 M sodium acetate, 0.2 M citric acid) containing 0.2 mM ethylenediaminetetraacetic acid disodium salt (EDTA Na(2)) and 0.4 mM sodium octylsulfate, operated at a flow rate of 0.8 ml/min. A potential of +0.78 V was applied across the working and reference electrodes of the detector. The precision was in the range 2.88-4.35% for NE, 5.94-11.0% for MHPG and 1.97-4.40% for DHPG. Accuracy was 98.8-99.3% for NE, 97.4-100% for MHPG and 96.1-101% for DHPG. The limit of detection was 0.6 ng/ml for NE, 0.5 ng/ml for MHPG and 0.2 ng/ml for DHPG. The linearity is over the range 20-60 ng/ml for NE, 7-23 ng/ml for MHPG and 6-20 ng/ml for DHPG. The assay has been applied successfully to measure simultaneously cortex and plasmas concentrations of these three catecholamines in rats.     

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.5 degrees C. which corresponded to denaturation of C- and N-domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N-domain or active N-domain with inactive C-domain within parent somatic ACE. Selective denaturation of C-domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29-133 in amino acid sequence of the N-part of the molecule as responsible for thermostability of the N-domain.