Serum proteomic signature of human chagasic patients for the identification of novel potential protein biomarkers of disease

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.     

Process simulation for recombinant protein production: cost estimation and sensitivity analysis for heparinase I expressed in Escherichia coli

Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.     

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.     

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H⁺ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H⁺ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.     

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.     

Patterns of variation in desiccation resistance in a set of recombinant inbred lines in Drosophila melanogaster

Desiccation, resulting from extremely dry environmental conditions, is a serious obstacle to the survival of organisms. Water is vital for the maintenance of intracellular structure and prevents the irreversible formation of aggregates, an occurrence leading to loss of cellular function. To characterize genetic variation in desiccation stress resistance (DSR) in Drosophila melanogaster Meigen, an intercontinental set of recombinant inbred lines (RIL) is used. Flies are exposed to a low humidity environment (<10% relative humidity) at a constant temperature of 25 °C. Desiccation stress resistance is higher in RIL derived from a backcross to the parental stock sensitive to heat stress (from Denmark) than in RIL derived from the reciprocal backcross to the heat‐stress resistant stock (from Australia). Composite interval mapping reveals significant quantitative trail loci (QTL) for DSR in the set of RIL. Both major and minor effects QTL are detected, suggesting a complex genetic architecture. When compared with a previous investigation performed on the same set of RIL, the present study indicates that not all traits of resistance to environmental stressors are affected in the same direction by segregating co‐localized QTL.     

Body proportions,microhabitat selection,and adaptive radiation of Liolaemus lizards in central Chile

Summary A biometric analysis of body proportions with presumably functional meaning for microhabitat selection was made on 12 species of Liolaemus lizards in central Chile. Characters studied were forelimb length, hindlimb length, tail length (all standardized by the corresponding snout-vent length), and the ratio forelimb/hindlimb length. It is shown that irrespective of terrestrial, saxicolous, or arboreal habits, Liolaemus species are remarkably similar in body proportions. The only exceptions are: L. lemniscatus, an open ground-dweller which exhibits significantly shorter limbs; and L. chiliensis and L. schroederi, both shrub-climbers which exhibit significantly longer tail. It is concluded that the adaptive radiation of Liolaemus lizards in central Chile has been accomplished mainly by diversification of activity time, food size, and microhabitat type. Morphological divergence in body proportions seems to have played an unimportant role.     

Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIV-induced cell fusion.

HIV use the CD4 molecule as their primary cellular receptor. Residues in the N-terminal domain (D1) of CD4 are crucial to HIV attachment through the gp120 envelope component. However, other regions of CD4 appear to be required subsequently for virus- and cell-cell fusion. Little is understood of the post-binding steps which may differ between HIV variants. We report a novel anti-CD4 mAb that does not block CD4/gp120 binding, but that does efficiently block both viral infection and cell-cell syncytia formation, and define its contact site as residues in CD4 D2 using both mouse/human CD4 chimeras and CD4 substitution mutants. We also investigated the basis for its antiviral effect. Using the CD4 D2 specific mAb, we identify another conserved step in HIV infection, as evidenced by its ability to neutralize a broad range of primary isolates and T cell-line passaged strains. Monovalent forms of the mAb were used to determine if its activity was due to masking of the D2 epitope, to steric inhibition, or bivalency. Our data indicate that both binding site and bivalency of the mAb underlie its potency. The need for bivalency is not simply explained by affinity, because monovalent forms can displace the intact mAb and reverse its protective effect. These results provide evidence that binding of the D2-specific mAb prevents structural alterations necessary for membrane fusion.