Mycoplasma-latex agglutination reaction

Morton, Harry E. (University of Pennsylvania, Philadelphia). Mycoplasma-latex agglutination reaction. J. Bacteriol. 92:1196-1205. 1966.-The building up of Mycoplasma cell mass through adsorption to carrier particles as a method for enhancing the agglutination reaction to identify Mycoplasma is described. Mycoplasma cells of human, avian, swine, goat, sewage, and tissue-culture origin were adsorbed to latex particles (0.81 mu) and then were agglutinated by immune sera. The adsorption was demonstrated by electron microscopy. Either the cells or their antibodies, depending on which came into contact with the latex particles first, were adsorbed. The test, completed in less than 2 hr, consisted of serially diluting immune sera with buffered saline, adding the antigen, incubating in a water bath, centrifuging, and reading the reaction under 50 x microscope magnification. The antigen in each reaction tube, representing the growth from about 1.6 ml of culture, was estimated to contain 23 mug of protein (approximately one-tenth the amount of Mycoplasma cells needed for a direct agglutination reaction). In the sera from rabbits undergoing immunization with Mycoplasma antigens, the presence of anti-Mycoplasma antibodies was detected much sooner in the Mycoplasma-latex agglutination reaction test than in the agar-gel diffusion reaction and the growth inhibition tests. Four different lots of latex particles showed excellent uniformity of behavior and stability during storage and testing.     

Detection of a lactate threshold during incremental exercise?

An important question in the study of the exercise response is the real or imaginary nature of the anaerobic threshold, and mathematical modeling techniques have been invoked to assist in resolving this issue. Two opposing views with competing data models recently published in this journal are criticized. One view suggests a segmented model with a discontinuous first derivative at the threshold. The other suggests a continuous model over the whole work load range, implying the anaerobic threshold to be imaginary. However, neither group of authors has devoted proper rigorous attention to the models they use. Had this been done, some of the divergence of opinion may have been avoided. Ideal data from an alternate segmented model that has a continuous first derivative at the threshold are considered for comparative purposes. This suggests that the log-log transformation method may well lead to improved detection of a threshold when one exists, although the estimates of the threshold value obtained are unreliable. Modeling methodology is a useful approach to the resolution of scientific issues, but there exist fundamental implications and alternatives that must be fully recognized.     

CO2 sensitivity changes during the menstrual cycle

A study of the changes in CO2 sensitivity at rest was undertaken in 20 regularly menstruating females in an attempt to determine the influence of the menstrual cycle on this variable. A biphasic oral temperature graph was used to signify fertility and demarcate three phases of the cycle. A CO2-rebreathing test was conducted 3 times/wk for 6 wk to obtain CO2 sensitivity and CO2 threshold measures. An analysis of variance was used to compare the results collected in each phase of the cycle for each of the variables. A significant increase was found in the sensitivity to CO2 between the follicular and luteal phases, a significant decrease between the luteal and menstrual phases, and no significant difference between the follicular and menstrual phases. The change between follicular and luteal phases was attributed to the effect of progesterone, which is elevated during the luteal phase. No significant change was found in the CO2 threshold level.     

A common theme in the amino acid sequences of actin and many actin-binding proteins?

The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?     

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.     

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine