Use of an in vitro flat-bed biofilm model to measure biologically active anti-odour compounds

The objective of this study was to demonstrate the utility of a modified flat-bed perfusion biofilm matrix system for testing toothpaste formulations directly, without dilution, as a layer in direct contact with the biofilm matrix surface. Final biofilm yields and volatile sulphur compounds (VSC) biogenesis were measured to show the relative efficacy of toothpaste formulations. Diffusion characteristics of the flat-bed system to exposure with Meridol® tooth and tongue gel (TTG; 1,400 ppm F^? from amine fluoride/stannous fluoride, 0.5 % zinc lactate, oral malodour counteractives) was assessed using a bioluminescent target species Escherichia coli Nissle 1917/pGLITE coupled with a low-light photon camera to visualise the kill kinetics. Tongue-flora derived, mixed culture biofilms (n?=?4) received 5, 15 and 30 min treatment with TTG, respectively, to determine the optimum time of exposure. VSC biogenesis was measured from headspace samples by gas chromatography prior to and following treatment of two daily applications for 4 days of treatment (TTG), positive control (CHX gel) and negative controls (placebo and sham treatment). Viable counts were performed at the end of experiments by destructive sampling of the biofilms and plating onto selective and non-selective agar. Following a single treatment with TTG, the E. coli biofilm with lux target gave >50 % reduction of luminescence within 2 to 3 h before recovering to a steady state over 10 h, suggesting biofilm cidal activity rather biostasis. For mixed culture biofilms, 15- and 30-min treatment exposure with TTG gave almost identical reductions in final biofilm yields. For comparing efficacy of treatments, biofilms treated with TTG gave greatest reductions in both pre–post levels of H₂S (P?<?0.01) and CH₃SH (P?<?0.05) and population yields at the end of the experiments (P?<?0.001) compared to placebo and positive control. The in vitro flat-bed perfusion model may be used to replicate many of the activities and reactions believed to be occurring by the tongue biofilm microflora within a real mouth, including VSC biogenesis and its inhibition by exposure to active agents as components of toothpastes and gels applied in direct contact with the biofilm.     

Sensitivity analysis of a cemented hip stem to implant position and cement mantle thickness

Patient-specific finite element models of the implanted proximal femur can be built from pre-operative computed tomography scans and post-operative X-rays. However, estimating three-dimensional positioning from two-dimensional radiographs introduces uncertainty in the implant position. Further, accurately measuring the thin cement mantle and the degree of cement–bone interdigitation from imaging data is challenging. To quantify the effect of these uncertainties in stem position and cement thickness, a sensitivity study was performed. A design-of-experiment study was implemented, simulating both gait and stair ascent. Cement mantle stresses and bone–implant interface strains were monitored. The results show that small variations in alignment affect the implant biomechanics, especially around the most proximal and most distal ends of the stem. The results suggest that implant position is more influential than cement thickness. Rotation around the medial–lateral axis is the dominant factor in the proximal zones and stem translations are the dominant factors around the distal tip.     

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.     

Use of the harmonic mean to the determination of dissociation constants of stereoisomeric mixtures of biologically active compounds

Abstract

Herein we introduce the derivation of a mathematical expression to evaluate the dissociation constant of a mixture of stereoisomers in equal amounts (K_dMIX), when the corresponding dissociation constants (K_d) or medium response (MR₅₀) of the pure stereoisomers are known; the final equation takes the form of the harmonic mean. In order to validate the equation, we carried out a bibliographic search of experimental data of enantiomeric molecules with biological activity, considering the K_d’s or MR₅₀’s of the isolated enantiomers as well as that of the racemate. The comparisons between the experimental dissociation constants of the mixtures (K_dEXP or MR_50EXP) and the calculated values (K_dMIX or MR_50MIX) were consistent; the similarity between these values is supported through statistical analyses of group comparison and simple linear correlation. The equation we obtained, which corresponds to the harmonic mean, was used to predict the values of K_dMIX (or MR_50MIX) or K_d (or MR₅₀) in systems when only two of the experimental values are known: either the dissociation constants of both enantiomers or the K_d (or MR₅₀) of one of the enantiomers and dissociation constant of the racemate.     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

Current-voltage relations of the basolateral membrane in tight amphibian epithelia: Use of nystatin to depolarize the apical membrane

Summary Exposure of the mucosal side of toad(Bufo bufo) urinary bladder and frog(Rana ridibunda) skin to the polyene ionophore nystatin, resulted in stable preparations in which the apical resistance was negligible compared to the basolateral resistance. The preparations support passive K currents in both directions and an amiloride-insensitive Na current in the apicalserosal direction which is blocked by ouabain. The nystatintreated toad bladder was used to study the electrical properties of the basolateral membrane by means of current-voltage curves recorded transepithelially. The K current showed strong rectification at cellular potentials negative with respect to the interstitial space. The ouabain-sensitive current increased with membrane voltage at negative voltages but saturated above+20 mV.     

Self-consistent model of an inductive RF plasma source in an external magnetic field

A theory is developed that makes it possible to calculate RF power absorption in an inductive plasma source. Conditions are determined under which most of the power is deposited in the plasma. It is shown that these conditions correspond to the excitation of spatial waves (an oblique Langmuir wave and a helicon wave). A simple self-consistent model of a plasma source is proposed that describes all of the experimentally observed distinctive properties of plasma sources well.     

Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes

Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics.     

Use of chiral auxiliaries in the asymmetric synthesis of biologically active compounds: A review

This review article describes the use of some of the most popular chiral auxiliaries in the asymmetric synthesis of biologically active compounds. Chiral auxiliaries derived from naturally occurring compounds, such as amino acids, carbohydrates, and terpenes, are considered essential tools for the construction of highly complex molecules. We highlight the auxiliaries of Evans, Corey, Yamada, Enders, Oppolzer, and Kunz, which led to remarkable progress in asymmetric synthesis in the last decades and continue to bring advances until the present day.     

Use of the individual data of the a-bomb survivors for biologically based cancer models

All recent analyses of the data on solid cancer incidence of the atomic bomb survivors are corrected for migration and random dose errors. In the usual treatment with grouped data and regression calibration, the calibration of doses depends on the used dose response. For solid cancers, it usually is linear. Here, an individual likelihood is presented which works without further adjustment for all dose responses. When the same assumptions are made as in the usual Poisson regression, equivalent results are obtained. But, the individual likelihood has the potential to use more detailed models for dose errors and to estimate non-linear dose responses without recalibration. As an example for the potential of the individual data set for the analysis of risk at low doses, signals for a saturating bystander effect are investigated.     

Use of magnetic resonance to measure molecular diffusion within the brain extracellular space

Ion-selective microelectrode measurements of molecular diffusion have provided unique information about the structural characteristics of the extracellular compartment of brain tissue. Magnetic resonance (MR) techniques can also be used to perform diffusion measurements in living tissue in situ. In MR applications, the challenge to study a particular physiological compartment lies in achieving the appropriate specificity in the experimentally-observed MR signal, and many strategies have been used to provide measurements that reflect molecular diffusion within the extracellular space. This review describes how magnetic resonance and microelectrode diffusion measurements are performed, and applications using the MR technique are summarized. Comparisons of experimental results obtained from the two techniques indicate that their use in combination may further augment what is known about extracellular space structure.     

Induction of tubules in rat metanephrogenic mesenchyme in the absence of an inductive tissue

Differentiation of metanephrogenic mesenchyme to renal tubular epithelium requires induction by the ureteric bud in vivo or any of several embryonic tissues in vitro. In an effort to eliminate the tissue requirement in embryonic induction, extracellular matrices and soluble factors were analyzed individually or in combination for their ability to stimulate tubulogenesis in uninduced metanephrogenic mesenchyme from 13-gestation-day rat embryos. These evaluations have established that pituitary extract and epidermal growth factor (EGF) in concert with a matrix can promote morphogenesis of mesenchymal rudiments in culture. While type I collagen, laminin, or fibronectin matrices all promoted tubulogenesis in the presence of pituitary extract and EGF, type IV collagen proved the most effective. Under these conditions, tubules were induced in 23/24 mesenchymal rudiments by 9 days in culture. Mesenchyme was not induced prior to explanation since it formed no tubules when cultured in a medium that allowed tubulogenesis in intact embryonic kidneys. Preliminary characterization of the undefined factor in pituitary extract was consistent with a protein of molecular weight greater than 100,000 but less than 300,000. When uninduced metanephrogenic mesenchyme from mouse was used instead of rat tissue, a similar pattern of morphogenesis was not observed, suggesting that the described medium is inappropriate for promoting differentiation in mouse or, less likely, that different mechanisms mediate differentiation in rat and mouse. These studies show that embryonic induction can occur in explanted rat renal mesenchyme in an appropriate environment and does not require the presence of an inductive tissue.