Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Core and surface mutations affect folding kinetics, stability and cooperativity in IL-1 beta: does alteration in buried water play a role?

Interleukin-1 beta (IL-1 beta) is a cytokine and a member of the beta-trefoil superfamily of protein structures. An interesting feature in the folding of IL-1 beta, shared with some other members of the same topological family, is the existence of a slow step in folding to the native conformation from a discrete intermediate. Wanting to probe the nature of this slow step in the folding of WT IL-1 beta (tau(1)=45 seconds), we made ten sequence variants of IL-1 beta (L10A, T9Q, T9G, C8S, C8A, N7G, N7D, L6A, R4P, and R4Q), where all mutations are located along strand 1. This strand is not protected from hydrogen exchange until late in folding. Most of the mutations showed little effect on the kinetics of folding for IL-1 beta. However, C8 is clearly involved in both the late and the early steps in folding, while sequence variants at L10 and L6 affect only late events in folding. The value of the slowest relaxation time, tau(1), which is associated with the rate of native protein formation, increased for the refolding of C8S, while C8A, L6A, and L10A showed smaller but systematic increases in the value of tau(1.)For both C8S and C8A, the value of the step associated with formation of the intermediate, tau(2), was independent of denaturant concentration. In addition, mutations in the hydrophobic core (L10A, C8A, C8S, and L6A) and, surprisingly, along the surface (T9G, T9Q, and N7G) alter the stability. The most destabilizing mutations show changes in equilibrium unfolding cooperativity, which is atypical for destabilizing mutations in IL-1 beta. Crystallographic studies indicate that mutations along strand 1 may alter the number of ordered water molecules within the core. Thus, side-chain replacement in this region can disrupt essential main-chain interactions mediated by ordered water contacts in a highly cooperative network of hydrogen bonding.     

A novel high molecular weight fibrinogenase from the venom of Bitis arietans

A fibrinogenase (Ba100) with an apparent molecular mass of 100 kDa under non-reducing conditions and a pI of 5.4 was purified from the venom of the African puff adder (Bitis arietans) by fibrinogen affinity chromatography. Under reducing conditions the protease dissociates into subunits of 21 kDa and 16 kDa. N-Terminal amino acid sequencing showed these two chains to have 66.7% homology and homology to C-type lectins. The fibrinogenase activity of Ba100 cleaves the Aalpha and Bbeta chain of fibrinogen rendering the molecule unable to polymerise into fibrin clots. Ba100 inhibited platelet aggregation in platelet rich plasma, and clot formation in whole blood, in a concentration dependent manner.     

The molecular basis for protein kinase A anchoring revealed by solution NMR

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.     

Attenuation of neointima formation through the inhibition of DNA repair enzyme PARP-1 in balloon-injured rat carotid artery

Increased oxidative stress is a major characteristic of restenosis after angioplasty. The oxidative stress is mainly created by oxidants such as reactive oxygen species (ROS), which are assumed to play an important role in neointima formation after angioplasty. DNA is a sensitive target for oxidants; however, oxidative DNA damage remains a poorly examined field in the pathogenesis of restenosis. In the present study, we demonstrated that the expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was quickly increased in rat carotid arteries after balloon injury. It reached its peak at 14 days after injury and still kept high expression at 28 days after injury. The immunostaining of 8-oxo-dG was present predominantly in the neointima. In response to oxidative DNA damage, the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was significantly increased after balloon injury. The time course change and location of PARP-1 is similar to that of 8-oxo-dG. Daily injections of the PARP-1 inhibitor PJ34 (5 mg.kg(-1).day(-1) ip) attenuated neointima formation by approximately 40% at 7, 14, and 28 days after balloon injury. Treatment with PJ34 inhibited leukocyte infiltration and improved both anatomic (reendothelialization) and functional (endothelial function) recovery of endothelial cells after balloon injury. In conclusion, levels of oxidative DNA damage and the DNA repair enzyme PARP-1 are increased in vessels after balloon injury. Inhibition of PARP-1 attenuates neointima formation through inhibition of leukocyte infiltration and improvement of endothelial cell recovery after balloon injury. Targeting of the DNA repair enzyme might be a therapeutic strategy for restenosis.     

Formation of ochratoxin A and aflatoxins following the use of propionic acid as a grain preservative for storing damp barley

The growth of storage moulds was studied in barley at 22% and approximately 28% moisture content treated with the recommended and reduced commercial doses of propionic acid over a 6 month storage period at 20°C. Experimental sample size was 5 kg barley per lot. Barley was fully protected against the growth ofA. flavus and aflatoxin formation when the recommended dose was applied. However, the treatment was less effective in controlling growth ofP. verrucosum and preventing ochratoxin A formation such that by 4 to 6 months of storage, the fungus had started to develop and toxin had formed even in some of the samples treated with propionic acid. The risk of the development of ochratoxin A during storage increased as the optimum dose was reduced, particularly for barley at 22% moisture content.     

Radial arm maze performance of rats following repeated low level microwave radiation exposure

We examined the possibility of changes in "working" memory of rats following whole body exposure to microwave (MW) radiation. During each of 10 days, we exposed rats within circularly polarized waveguides for 45 min to 2450 MHz fields at whole body SARs of 0.6 W/kg (2 micros pulses, 500 pps), followed by testing in a 12 arm, radial arm maze (RAM). Rats received a preexposure injection of one of three psychoactive compounds or saline, to determine whether a compound would interact with MW exposure to affect performance in the maze. Error rate, i.e., reentry into arms already visited, and time to criterion data for 10 consecutive days of testing were analyzed by a three way analysis of variance (ANOVA) using main effects of "exposure" and "drug" and a repeated factor of "test day." Our alpha limit for significance was P <.05. Analyzes of error rates revealed no significant exposure effect, no significant drug effect and no significant interaction between the two main factors. There was a significant difference in test days, as expected, with repeated test-trial days, which indicates that learning was accomplished. There was no significant interaction of test day and the other two factors. The results of our analyzes of time to criterion data included no significant exposure effect, a significant drug effect, a significant test day effect, and a significant interaction between drug and test day factors. Post hoc analyzes of the drug factor revealed that rats treated with either physostigmine or nalrexone hydrochloride, took significantly longer to complete the maze task than rats pretreated with saline or with naloxone methodide. We conclude that there is no evidence from the current study that exposure to of MW radiation under parameters examined caused decrements in the ability of rats to learn the spatial memory task.     

Effects of a Peracetic Acid Disinfection Protocol on the Biocompatibility and Biomechanical Properties of Human Patellar Tendon Allografts

Patellar tendon allografts, retrieved from cadaveric human donors, are widely used for replacement of damaged cruciate ligaments. In common with other tissue allografts originating from cadaveric donors, there are concerns regarding the potential for disease transmission from the donor to the recipient. Additionally, retrieval and subsequent processing protocols expose the graft to the risk of environmental contamination. For these reasons, disinfection or sterilisation protocols are necessary for these grafts before they are used clinically. A high-level disinfection protocol, utilising peracetic acid (PAA), has been developed and investigated for its effects on the biocompatibility and biomechanics of the patellar tendon allografts. PAA disinfection did not render the grafts either cytotoxic or liable to provoke an inflammatory response as assessed in vitro . However, the protocol was shown to increase the size of gaps between the tendon fibres in the matrix and render the grafts more susceptible to digestion with collagenase. Biomechanical studies of the tendons showed that PAA treatment had no effect on the ultimate tensile stress or Young's modulus of the tendons, and that ultimate strain was significantly higher in PAA treated tendons.     

Real-time NMR kinetic studies provide global and residue-specific information on the non-cooperative unfolding of the beta-trefoil protein,interleukin-1beta

The interleukin-1beta (IL-1beta) structural motif is a beta-trefoil super fold created by six two-stranded beta-hairpins. Turns are thus particularly important in creating the topology and the arrangement of beta-strands in this structural motif. In contrast to the signals observed in optical studies, real-time NMR kinetic investigations of the denaturant-induced unfolding of interleukin-1beta provide direct, global, and residue-specific information on the structural nature of the unfolding reaction. Heterogeneity in the individual amino acid residue kinetics reveals a rugged unfolding landscape. The relative kinetic stability of native-like turns supports low temperature molecular dynamics predictions of turn-controlled unfolding.