Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Detection of membrane-associated antigens on lymphoid cells by antibody coupled to staphylococcal protein A.

A simple technique is described for the detection of membrane-associated antigens on lymphoid cells. It is based on the observation that the protein A component of staphylococci binds to the Fc pieces of IgG molecules. Lymphocytes from various sources (mouse, rat, and human tissues) were incubated with hyperimmune antisera directed against surface determinants. Subsequent treatment with a suspension of staphylococci containing protein A permitted visualization of both the presence and distribution of determinants on the cell membrane. The method had comparable sensitivity to the fluorescent sandwich technique and could be used to detect a variety of membrane antigens on both T cells and B cells.     

Evaluating and predicting risk to a large reptile (Varanus varius) from feral cat baiting protocols

Control of introduced predators to mitigate biodiversity impacts is a pressing conservation challenge. Across Australia feral cats (Felis catus) are a major threat to terrestrial biodiversity. Currently feral cat control is hindered by the limited utility of existing predator baiting methods. Further proposed control methods include use of the novel poison para-aminopropiophenone (PAPP) which may present a hazard to some native animal populations. Here we used experimental and predictive approaches to evaluate feral cat bait take by a large native Australian predatory reptile the Lace monitor (Varanus varius). These lizards would be expected to readily detect, ingest and consume a lethal dose (depending on toxin) from surface-laid baits intended for feral cat control if a precautionary approach was not adopted when baiting. We modelled V. varius bait take using experimental and predictive biophysical modelling approaches to evaluate temporal effects of climate variables on V. varius activity and hence potential for bait removal. Finally we conducted a pre-PAPP baiting site occupancy assessment of V. varius within Wilson Promontory National Park (WPNP) to provide a basis for monitoring any longer term population effects of cat baiting. V. varius removed 7 % of deployed baits from 73 % of bait stations across another study area in Far Eastern Victoria. Daily bait removal was positively correlated with maximum temperature and solar radiation. Biophysical modelling for Far Eastern Victoria predicted that maximum temperatures <19.5 °C prevented V. varius activity and hence opportunity for bait removal. V. varius in WPNP was undetectable suggesting aerial baiting posed limited hazard to this species at this location. Depending how climate influences annual activity patterns and the specific poison, surface-laid baits could pose a significant mortality risk to V. varius. However, use of biophysical models to predict periods of V. varius inactivity may provide a novel means to reduce non-target bait take by this predator.     

Determinants of inter-specific variation in basal metabolic rate

Basal metabolic rate (BMR) is the rate of metabolism of a resting, postabsorptive, non-reproductive, adult bird or mammal, measured during the inactive circadian phase at a thermoneutral temperature. BMR is one of the most widely measured physiological traits, and data are available for over 1,200 species. With data available for such a wide range of species, BMR is a benchmark measurement in ecological and evolutionary physiology, and is often used as a reference against which other levels of metabolism are compared. Implicit in such comparisons is the assumption that BMR is invariant for a given species and that it therefore represents a stable point of comparison. However, BMR shows substantial variation between individuals, populations and species. Investigation of the ultimate (evolutionary) explanations for these differences remains an active area of inquiry, and explanation of size-related trends remains a contentious area. Whereas explanations for the scaling of BMR are generally mechanistic and claim ties to the first principles of chemistry and physics, investigations of mass-independent variation typically take an evolutionary perspective and have demonstrated that BMR is ultimately linked with a range of extrinsic variables including diet, habitat temperature, and net primary productivity. Here we review explanations for size-related and mass-independent variation in the BMR of animals, and suggest ways that the various explanations can be evaluated and integrated.     

The contribution of conformational adjustments and long-range electrostatic forces to the CD2/CD58 interaction

CD2 is a T cell surface molecule that enhances T and natural killer cell function by binding its ligands CD58 (humans) and CD48 (rodents) on antigen-presenting or target cells. Here we show that the CD2/CD58 interaction is enthalpically driven and accompanied by unfavorable entropic changes. Taken together with structural studies, this indicates that binding is accompanied by energetically significant conformational adjustments. Despite having a highly charged binding interface, neither the affinity nor the rate constants of the CD2/CD58 interaction were affected by changes in ionic strength, indicating that long-range electrostatic forces make no net contribution to binding.     

Buffers for the reconstitution of aspartate aminotransferase

The cofactor activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase was studied in various buffers. Cationic buffers are shown to allow maximal reconstitution in the pH range of 5.0 to 9.0. Anionic buffers made up of mono- and dicarboxylates are found to affect reconstitution in a pH-dependent manner. At low pH, the carboxylates strongly inhibit reconstitution, but at high pH, they show less effect. In contrast, the more potent inhibitor Pi shows the opposite pH profile. Dicarboxylates are considerably more inhibitory than monocarboxylates. Substantial protection against inhibition by a number of carboxylates may be achieved by the addition of sodium chloride.     

The efficacy and sterilisation of human decellularised dermal allografts with combinations of cupric ions and hydrogen peroxide

Decellularised tissue allografts have been used in reconstructive surgical applications and transplantation for many years. Some of the current methods of sterilisation have a detrimental effect on the tissue graft structure and function. The anti-microbial activity of cupric ions and hydrogen peroxide (H₂O₂) are well known however their combined application is not currently utilised as a decontamination agent in the tissue banking world sector. The aim of this study was to determine the combined concentrations of copper chloride (CuCl₂) and H₂O₂ that have the optimal bactericidal and sporicidal activity on decellularised (dCELL) human dermis. The first part of this study established the decimal reduction time (D-value) of CuCl₂ (0.1 mg/L and 1 mg/L) together with H₂O₂ (0.01, 0.1, 0.5 and 1%) for Staphylococcus epidermidis, Escherichia coli and Bacillus subtilis spores. The second part of this study identified the most effective CuCl₂ and H₂O₂ concentration that decontaminated dCELL human dermis inoculated with these pathogens. Of all the concentrations tested, 0.1 mg/L CuCl₂ in combination with 1% H₂O₂ had the shortest D-value; S. epidermidis D = 3.15 min, E. coli D = 2.62 min and B. subtilis spores D = 18.05 min. However when adsorbed onto dCELL dermis, S. epidermidis and E. coli were more susceptible to 1 mg/L CuCl₂ together with 0.5% H₂O₂. These studies show promise of CuCl₂–H₂O₂ formulations as potential sterilants for decellularised dermal allografts.