Effect of a Single Dose of β,β''-Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro

The biochemical properties of neurofilaments isolated from control and iminodipropionitrile-treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross-linking between neurofilament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile-treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament-associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilament-associated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament-associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.     

On the mechanism of hydrogen peroxide formation during precipitation of hemoglobin with perchloric acid

During acid precipitation of hemoglobin, hydrogen peroxide is formed. The oxidant is generated from hemoglobin-bound as well as from physically dissolved oxygen, provided divalent hemoglobin-iron is present. The extent of hydrogen peroxide formation seems to be dependent on the precipitating reagent. From experiments on GSH oxidation during precipitation of red cells and from a literature survey formation of at least one second reactive oxygen species has to be assumed. Different reaction pathways for generating these oxidants during acid precipitation are discussed: Hydroperoxide which is probably liberated by proton catalysis may contribute to the observed oxidation reactions. At pH approximately 1 it rapidly disproportionates yielding oxygen and hydrogen peroxide, which might produce reactive oxidants with hemoglobin intermediates formed during precipitation. In addition a new hypothesis is presented explaining the ability of N-ethylmaleimide to inhibit GSH oxidation during precipitation of red cells. Accordingly, the SH-reagent rapidly penetrates the cell membrane and reacts with GSH before access of protons prevents alkylation.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Mother-infant bonding

A study of the research on postpartum mother-infant bonding shows that results from poorly constructed research programs were published in major journals and became a part of hospital policy because the bonding concept was politically useful in the struggle between advocates of natural childbirth and managers of the medical model of birth. The concept was also uncritically accepted because it was consistent with a longstanding ideology of motherhood that sees women as the prime architects of their children’s personalities. Diane Eyer earned her Ph.D. in developmental psychology from the University of Pennsylvania. She is currently writing a book on the ways in which the concepts of bonding and attachment have affected our understanding of appropriate early childcare.     

Reactions of the Wurster's red radical cation with hemoglobin and glutathione during the cooxidation of N,N-dimethyl-p-phenylenediamine [correction of phenlenediamine] and oxyhemoglobin in human red cells.

N,N-Dimethyl-p-phenylenediamine (DMPD) reacted directly with oxyhemoglobin under formation of ferrihemoglobin and, presumably, the N,N-dimethyl-p-phenylenediamine radical cation (DMPP.+). The apparent second-order rate constant of this reaction was 1 M-1 s-1 (pH 7.4, 37 degrees C). The reaction rate was diminished by catalase (by 1/3) and by superoxide dismutase (by 1/5). The apparent second-order rate constant of ferrihemoglobin formation by DMPD.+ was 5 x 10(3) M-1 s-1. Since DMPD.+ is disproportionated by 50% at pH 7.4, the quinonediimine could not be excluded as the ultimate ferrihemoglobin forming oxidant. To prove this hypothesis, the disproportionation equilibrium was shifted to the radical side by addition of excess DMPD. Ferrihemoglobin formation was thereby increased, indication that the radical was the responsible oxidant. In contrast to ferrihemoglobin formation, reactions with glutathione occurred predominantly with the quinonediimine. The second-order rate constant of this reaction was 4 x 10(5) M-1 s-1 which approaches the value obtained with p-benzoquinone. In contrast to the corresponding reactions of the N,N,N',N'-tetramethyl-p-phenylenediamine radical cation, the disporportionation reaction of DMPD.+ was very fast, k = 2 x 10(6) M-1 s-1. Formation of glutathione disulfide was negligible and the main reaction products were two isomeric glutathione adducts, 2- and 3-(glutathione-S-yl)-N,N-dimethyl-p-phenylenediamine. In human erythrocytes, DMPD produced many equivalents of ferrihemoglobin, diminished glutathione and produced both thioethers. In contrast to ferrihemoglobin formation, DMPD and glutathione disappearance as well as thioether appearance occured only after a marked lag phase. The calculated steady state concentration of DMPD.+ was only 4 x 10(-6) the DMPD concentration, as long as ferrihemoglobin was low. At increasing ferrihemoglobin higher steady state concentrations of the radical are attained. In fact, preformed ferrihemoglobin in red cells significantly accelerated DMPD and glutathione disappearance. This effect was completely prevented in the presence of ferrihemoglobin-complexing cyanide. The presented experiments once more appoint blood as a metabolically competent organ for the biotransformation of aromatic amines.     

Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite

Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. This relationship becomes more complicated in social species, such as social insects, since the chance of disease transmission increases with the frequency of interactions among individuals. However, social insects may benefit from social immunity, whereby individual physiological defenses may be bolstered by collective‐level immune responses, such as grooming or sharing of antimicrobial substance through trophallaxis. We set out to determine whether differences in genetic diversity between colonies of the subterranean termite, Reticulitermes flavipes, accounts for colony survival against pathogens. We sampled colonies throughout the United States (Texas, North Carolina, Maryland, and Massachusetts) and determined the level of inbreeding of each colony. To assess whether genetically diverse colonies were better able to survive exposure to diverse pathogens, we challenged groups of termite workers with two strains of a pathogenic fungus, one local strain present in the soil surrounding sampled colonies and another naïve strain, collected outside the range of this species. We found natural variation in the level of inbreeding between colonies, but this variation did not explain differences in susceptibility to either pathogen. Although the naïve strain was found to be more hazardous than the local strain, colony resistance was correlated between two strains, meaning that colonies had either relatively high or low susceptibility to both strains regardless of their inbreeding coefficient. Overall, our findings may reflect differential virulence between the strains, immune priming of the colonies via prior exposure to the local strain, or a coevolved resistance toward this strain. They also suggest that colony survival may rely more upon additional factors, such as different behavioral response thresholds or the influence of a specific genetic background, rather than the overall genetic diversity of the colony.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.     

Characterization of NFH-LacZ transgenic mice with the SHIRPA primary screening battery and tests of motor coordination,exploratory activity,and spatial learning

NFH-LacZ transgenic mice express a fusion protein between a truncated form of the endogenous neurofilament of heavy molecular weight and the complete E. coli beta-galactosidase. NFH-LacZ transgenic mice could be distinguished from controls in the SHIRPA neurological battery by the appearance of action tremor and hindlimb clasping and a lower body weight. Despite normal exploratory activity and spatial learning, NFH-LacZ transgenic mice were deficient in stationary beam, coat-hanger, and rotorod tests of motor coordination. These results are concordant with neuropathological findings in spinal motoneurons and the cerebellum and indicate that despite the absence of paralysis, these transgenic mice may serve as an experimental model of the early stage of amyotrophic lateral sclerosis.