Quantitative chimeric analysis of six specificity determinants that differentiate Escherichia coli aspartate from tyrosine aminotransferase

The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower K(M)s for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered k(cat)s for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k(cat)/K(M)) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK(a)s, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a K(D) for dimer dissociation of 350 nM compared with the wild type K(D) of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on k(cat)/K(M)(Asp). Effects on k(cat)/K(M)(Phe) are more nearly additive and context independent.     

Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction

Dengue virus (DV) is an important re-emerging arthropod-borne virus of global significance. The defining characteristic of DV infection-associated pathology is haemorrhagic fever, which often leads to a fatal shock-like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose-dependent manner, that DV-infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)-9-and to a lesser extent MMP-2-which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti-MMP-9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM-1) and vascular endothelium (VE)-cadherin cell adhesion molecules and redistribution of F-actin fibres. These in vitro observations were confirmed in an in vivo vascular-leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever-inducing viruses, and identify a new therapeutic approach for the treatment of viral-induced vascular leakage by specifically targeting gelatinolytic metalloproteases.     

Do hydration dynamics follow the structural perturbation during thermal denaturation of a protein: a terahertz absorption study

We investigate the thermal denaturation of human serum albumin and the associated solvation using terahertz (THz) spectroscopy in aqueous buffer solution. Far- and near-ultraviolet circular dichroism spectroscopy reveal that the protein undergoes a native (N) to extended (E) state transition at temperature ≤55°C with a marginal change in the secondary and tertiary structure. At 70°C, the protein transforms into an unfolded (U) state with significant irreversible disruption of its structures. We measure the concentration- and temperature-dependent THz absorption coefficient (α) of the protein solution using a p-Ge THz difference spectrometer (2.1–2.8 THz frequency range), thereby probing the collective protein-water network dynamics. When the solvated protein is heated up to 55°C and cooled down again, a reversible change in THz absorption is observed. When increasing the temperature up to 70°C, we find a dramatic irreversible change of THz absorption. The increase in THz absorption compared to bulk water is attributed to a blue shift in the spectrum of the solvated protein compared to bulk water. This is supported by measurements of THz absorption coefficients using THz time-domain spectroscopy (0.1–1.2 THz frequency range). We also use picosecond-resolved fluorescence spectroscopy of the tryptophan 214 moiety of human serum albumin. All experimental observations can be explained by a change in the hydration dynamics of the solvated protein due to the additional exposure of hydrophobic residues upon unfolding.     

Physiological and biochemical adrenergic regulation of the stunned myocardium

A dual approach was employed to study -adrenergic receptor signal transduction in post ischemic (stunned) myocardium, examining physiological interventions in awake, chronically instrumented pigs and biochemical, cellular mechanisms in sarcolemmal preparations from the stunned hearts using the contralateral non-ischemic zone as a control. Ten min of coronary artery occlusion (CAO) and 30 min coronary artery reperfusion (CAR) resulted in depressed posterior wall-thickening (myocardial stunning). Isoproterenol increased transmural wall thickening more in stunned myocardium than in non-ischemic myocardium. In contrast, the responses of wall thickening to forskolin, actually decreased during stunning compared with control. NKH 477, a water soluble forskolin derivative, that does not activate cardiac nerves, increased wall thickening in non-ischemic tissue similarly to the effects on stunned myocardium. Increasing cardiac neural tone reflexly with inferior venal caval occlusion (IVCO) elicited similar results to forskolin, i.e., stunned myocardium responded with less of an increase in wall thickening as compared with non-ischemic myocardium. -adrenergic receptor density, as determined with ¹²⁵I-cyanopindolol binding, was significantly increased in stunned subendocardium and subepicardium compared with respective values in non-ischemic myocardium. There were no differences in the response of adenylyl cyclase to isoproterenol in stunned and non-ischemic myocardium. The enhanced responsiveness of the -adrenergic receptor to isoproterenol stimulation in stunned myocardium corresponded to the increase in -adrenergic receptor density. The combination of enhanced responses to isoproterenol, and decreased responses to forskolin and to IVCO and preserved responsiveness to NKH 477, suggest that stunned myocardium is characterized by transient sympathetic neural stunning. The enhanced sensitivity to -adrenergic receptor stimulation has important clinical implications, both in terms of therapy of stunned myocardium and detection of stunned and /or hibernating myocardium, i.e., low dose dobutamine echocardiography.     

Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Km^r) transconjugants, a majority of which were gentamicin sensitive (Gm^s). A small proportion (about 0.8%) of the Km^r Gm^s clones were either auxotrophic or failed to catabolize galacturonate (Gtu⁻). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Km^r Gm^r transconjugant, but not in Km^r Gm^sE. chrysanthemi strains carrying Tn5-induced mutations. In Hfr crosses, Km^r (Tn5) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Km^s, except for Leu⁺ and Arg⁺ recombinants which were 30 to 50% Km^s. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met⁺, Leu⁺, or His⁺ clones, were Km^s. We conclude from both genetic and physical data that Tn5 transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.     

Evaluation of an in vitro assay system as a potential alternative to current histamine sensitization test for acellular pertussis vaccines

The histamine sensitization test (HIST) is a lethal test for batch release of acellular pertussis or its combination vaccines (ACV). Large numbers of animals have been used and it is difficult to standardize. Therefore there is an urgent need to develop an in vitro alternative to HIST.An in vitro test system has been developed as a potential alternative to HIST, to examine both the functional domains of PT based on a combination of enzyme coupled-HPLC (E-HPLC) and carbohydrate binding assays. We describe here an international collaborative study, which involved sixteen laboratories from 9 countries to assess the methodology transferability of the in vitro test system and its suitability for the testing of three different types of ACV products that are currently used worldwide. This study also evaluated further the relationship between the in vivo activity by HIST and the in vitro assay system.The results showed that the methodology of the E-HPLC and carbohydrate binding assays are transferable between laboratories worldwide and is suitable for the three types of ACV products included in the study. Although direct correlation between the in vitro assay system and the in vivo HIST (temperature reduction assay) for each individual vaccine lot cannot be established due to the large variation in the HIST results, the observation that the mean estimates of the in vitro and in vivo activities gave the same rank order of the three vaccine types included in the study is encouraging. The in vitro systems provide reproducible product specific profiles which supports their use as a potential alternative to the HIST.     

The number, size, and type of axons in rat subcortical white matter on left and right sides: A stereological, ultrastructural study

Abundant evidence indicates important functional differences between the two cerebral hemispheres of humans, although the cellular basis of these differences is unknown. A recent hypothesis proposes that these functional differences depend on differences between sides in the “repertoire” of axonal conduction delays for cortico-cortical axons. In morphological terms this corresponds to differences in caliber, or proportion, of myelinated versus unmyelinated axons. Several behavioural studies have indicated that cerebral asymmetry occurs in rodents, in which rigorous morphological analysis is possible. The hypothesis was therefore tested for the first time in adult male Wistar rats, using transmission electron microscopy and stereological methods. Subcortical white matter was compared between left and right sides in three regions (frontal, parietal, and occipital). The average caliber and numerical density of unmyelinated and myelinated axons was compared between sides and between regions. All data were corrected for shrinkage. No significant differences between sides were found in the average caliber of either type of axon in any region. The numerical density of either type of axon also yielded no significant differences between sides in any region. Significant differences were evident between regions in both caliber and numerical density of the two axonal types, and these quantitative data are reported. The proportion of unmyelinated axons in the lateral white matter was also higher than in previous studies of hemispheric white matter that studied the corpus callosum. The present study provides no evidence supporting the hypothesis that functional hemispheric specialization is due to differences in axonal number, caliber or type.     

Liposome Damage and Modeling of Fragments of Human Islet Amyloid Polypeptide (IAPP) Support a Two-Step Model of Membrane Destruction

A key factor in the development of Type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide (hIAPP) is believed to play a crucial role in this process by forming small aggregates that disrupt the cellular membrane. During Type II diabetes mellitus, human IAPP (hIAPP) fibrillizes to form amyloid deposits. However, the role of various regions of the 37 amino acid peptide in the process of membrane disruption has yet to be fully elucidated. Therefore, several fragments (10–19, 20–29, 10–29, 1–19) of hIAPP were synthesized and compared to full length hIAPP for their effects on model PC/PS bilayers. These fragments were also modeled using density functional methods and analyzed by circular dichroism, to determine possible correlations between activity and available conformations and charge distribution. Results from dye leakage and Thioflavin T fluorescence assays confirmed that the hIAPP fragments disrupt the lipid bilayer to varying extents, despite their inability to form amyloid fibrils. The longer and more positively charged fragments were most active in the assay (1–19 > 10–29 > 10–19 > 20–29), though none rivaled the activity of the native full length peptide. This may reflect their relative abilities to interact with the negatively charged membrane. Data support a two-step model for membrane disruption: insertion by the N-terminus followed by fibrillization mediated by the middle to C-terminal region.