The effects of different HIV type 1 strains on human thymic function

Studies of HIV-1-infected humans indicate that the thymus can be infected by HIV-1. In some of these patients, there is a significant CD4(+) T cell decline and a faster disease progression. This phenomenon is more evident in pediatric patients who depend heavily on their thymus for generation of new T cells. We hypothesize that HIV-1 causes T cell regenerative failure within the thymus, which has a profound impact on disease progression. Building on our established human thymopoiesis model, we include dynamic interactions between different HIV-1 strains (R5 and X4) and thymocytes. Our results predict that thymic infection with different HIV-1 strains induces thymic dysfunction to varying degrees, contributing to differences in disease progression as observed in both HIV-1-infected children and adults. Thymic infection in children is more severe than in adults, particularly during X4 infection. This outcome is likely due to both a higher viral load and a more active thymus in pediatric patients. Our results also indicate that a viral strain switch from R5 to X4 induces further deterioration in thymopoiesis. We predict that both viral and host factors play key roles in controlling thymic infection, including strain virulence and health status of the thymus.     

Human Sco1 functional studies and pathological implications of the P174L mutant

The pathogenic mutant (P174L) of human Sco1 produces respiratory chain deficiency associated with cytochrome c oxidase (CcO) assembly defects. The solution structure of the mutant in its Cu(I) form shows that Leu-174 prevents the formation of a well packed hydrophobic region around the metal-binding site and causes a reduction of the affinity of copper(I) for the protein. K(D) values for Cu(I)WT-HSco1 and Cu(I)P174L-HSco1 are approximately 10(-17) and approximately 10(-13), respectively. The reduction potentials of the two apo proteins are similar, but slower reduction/oxidation rates are found for the mutant with respect to the WT. The mitochondrial metallochaperone in the partially oxidized Cu(1)(I)Cox17(2S-S) form, at variance with the fully reduced Cu(4)(I)Cox17, interacts transiently with both WT-HSco1 and the mutant, forming the Cox17/Cu(I)/HSco1 complex, but copper is efficiently transferred only in the case of WT protein. Cu(1)(I)Cox17(2S-S) indeed has an affinity for copper(I) (K(D) approximately 10(-15)) higher than that of the P174L-HSco1 mutant but lower than that of WT-HSco1. We propose that HSco1 mutation, altering the structure around the metal-binding site, affects both copper(I) binding and redox properties of the protein, thus impairing the efficiency of copper transfer to CcO. The pathogenic mutation therefore could (i) lessen the Sco1 affinity for copper(I) and hence copper supply for CcO or (ii) decrease the efficiency of reduction of CcO thiols involved in copper binding, or both effects could be produced by the mutation.     

Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer

The human protein Cox17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S-S bonds and two reduced cysteines (HCox17(2S-S)). HCox17(2S-S) is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCox17(2S-S), i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCox17(3S-S), which can be then reduced by glutathione to apoHCox17(2S-S). The HSco1/HCox17(2S-S) redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper-electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein-protein complex, which is instead observed in the Cu(I)HCox17(2S-S)/HSco1 interaction.     

Isolation and characterization of the major centipede allergen Sco m 5 from Scolopendra subspinipes mutilans

BackgroundAllergic reactions have been observed following both direct centipede bites and the clinical use of centipede-containing medicines, such as traditional Chinese medicines utilizing Scolopendra subspinipes mutilans; however, no natural centipede allergen has yet been characterized.MethodsAn allergen was purified from S. s. mutilans venom using Superdex 75 gel filtration and RESOURCE S ion chromatography, and its primary structure was determined via a combination of LC-MS-MS, MALDI-TOF/TOF and protein sequencing techniques. Its potential allergenicity was evaluated by immunoblotting, ELISAs, skin prick tests (SPTs) and mast cell activation assays.ResultsA novel allergen Sco m 5 (210 amino acids long) was successfully purified from crude S. s. mutilans venom. Sco m 5 could promote the degranulation of a human mast cell line, HMC-1. Among centipede-allergic patients, Sco m 5 showed an 83.3% IgE-binding frequency and a 66.7% positive reaction frequency, as detected by immunoblotting and SPTs, respectively. Sco m 5 IgE-binding frequencies of common Chinese population was found to be 9%–16%. Sera positive for Sco m 5 IgE-binding was cross-reactive against venom from the wasp Vespa mandaeinia.ConclusionsThe present study isolated and characterized a novel allergen termed as Sco m 5 from the centipede S. s. mutilans. The use of Sco m 5 to identify centipede-allergic individuals could be important, given the high potential allergenicity of Sco m 5 among the general Chinese population, along with the likely possibility of cross-reactivity against wasp venom among centipede-allergic patients.     

A simple model for calculating the kinetics of protein folding from three-dimensional structures

An elementary statistical mechanical model was used to calculate the folding rates for 22 proteins from their known three-dimensional structures. In this model, residues come into contact only after all of the intervening chain is in the native conformation. An additional simplifying assumption is that native structure grows from localized regions that then fuse to form the complete native molecule. The free energy function for this model contains just two contributions-conformational entropy of the backbone and the energy of the inter-residue contacts. The matrix of inter-residue interactions is obtained from the atomic coordinates of the three-dimensional structure. For the 18 proteins that exhibit two-state equilibrium and kinetic behavior, profiles of the free energy versus the number of native peptide bonds show two deep minima, corresponding to the native and denatured states. For four proteins known to exhibit intermediates in folding, the free energy profiles show additional deep minima. The calculated rates of folding the two-state proteins, obtained by solving a diffusion equation for motion on the free energy profiles, reproduce the experimentally determined values surprisingly well. The success of these calculations suggests that folding speed is largely determined by the distribution and strength of contacts in the native structure. We also calculated the effect of mutations on the folding kinetics of chymotrypsin inhibitor 2, the most intensively studied two-state protein, with some success.     

NMR structures of two designed proteins with high sequence identity but different fold and function

How protein sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal sequence identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, G_A88 and G_B88, with 88% sequence identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963–11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the sequence identity level from 16 to 88%, G_A88 and G_B88 maintain their distinct wild-type 3-α and α/β folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high sequence identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-α and α/β conformations. Further mutation of a subset of these nonidentities, guided by the G_A88 and G_B88 structures, leads to proteins with even higher levels of sequence identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.     

The processing and biological function of the human amyloid precursor protein (APP): lessons from different cellular models

One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since gamma-secretase is as yet not identified; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilinsellipsis). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.     

A simplified method for the assessment of C1 esterase inhibitor function

From the result that the activated form of C1-s(C1-s) prolonged the kinetics of hemolysis via complement, this assay was applied to assess C1 esterase inhibitor (C1INH) function. In the kinetic assay, the complement hemolytic activity was evaluated by the time which required to cause 50% reduction of the initial turbidity of sensitized sheep erythrocytes, and was expressed as T1/2. (1) T1/2 of pooled normal human sera (p-NHS) showed dose-dependent prolongation by the addition of various amounts of C1-s. (2) Preincubation of various amounts of functionally pure C1INH with the constant amounts of C1-s inhibited dose-dependently the prolongation of T1/2 by C1-s. (3) The C1INH activity of NHS was 840 +/- 80 units/ml (n = 6) and that of the C1INH deficient serum was 80 units/ml, which were calculated from the standard curve established by the addition of various amounts of purified C1INH. This test requiring only C1-s and sensitized sheep erythrocytes is simple technically and high in sensitivity, and seems to be useful for the routine assay for C1INH function of human sera.     

A novel anti-inflammatory function of human galectin-1: inhibition of hematopoietic progenitor cell mobilization

OBJECTIVE: The immunosuppressive and anti-inflammatory activity of mammalian galectin-1 (Gal-1) has been well established in experimental in vivo animal models and in vitro studies. Since the proliferation and migration of leukocytes represent a necessary and important step in response to the inflammatory insult, we have investigated whether Gal-1 affects the mobilization of hematopoietic progenitor cells (HPC) induced by cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). METHODS: Bone marrow HPCs were mobilized with CY/G-CSF or CY/G-CSF plus human recombinant Gal-1 in BDF1 mice. Bone marrow (BM) and blood cells were taken at different time points and analyzed for their in vivo repopulating ability in lethally irradiated syngeneic animals. The number of myeloid progenitor cells in BM and blood samples was determined by colony-forming cell assay. Expression of surface markers (Sca-1, CD3epsilon, CD45R/B220, Ter-119, GR-1, and CD11b) on nucleated marrow cells was measured by flow cytometry. The lymphocytes, granulocytes, and monocytes in blood samples were counted after Giemsa staining. RESULTS: Gal-1 dramatically inhibited CY/G-CSF-induced HPC migration to the periphery as well as decreased peripheral neutrophilia and monocytosis in a dose- and time-dependent manner. In contrast, Gal-1 itself stimulated HPC expansion and accumulation within the BM. The presence of the lectin for inhibition of HPC mobilization was essential during the second half of the treatment. Moreover, Gal-1 inhbited transendothelial migration of BM-derived HPCs in response to SDF-1 in vitro. CONCLUSION: Gal-1 blocked BM progenitor cell migration induced by CY/G-CSF treatment, indicating a novel anti-inflammatory function of the lectin. We suggest that the inhibition of HPC mobilization occurs mainly via obstructing the transendothelial migration of BM-derived cells including primitive hematopoietic and committed myeloid progenitor cells and mature granulocytes and monocytes.     

Cleavage mechanism of human Mus81-Eme1 acting on Holliday-junction structures

Recombination-mediated repair plays a central role in maintaining genomic integrity during DNA replication. The human Mus81-Eme1 endonuclease is involved in recombination repair, but the exact structures it acts on in vivo are not known. Using kinetic and enzymatic analysis of highly purified recombinant enzyme, we find that Mus81-Eme1 catalyzes coordinate bilateral cleavage of model Holliday-junction structures. Using a self-limiting, cruciform-containing substrate, we demonstrate that bilateral cleavage occurs sequentially within the lifetime of the enzyme-substrate complex. Coordinate bilateral cleavage is promoted by the highly cooperative nature of the enzyme and results in symmetrical cleavage of a cruciform structure, thus, Mus81-Eme1 can ensure coordinate, bilateral cleavage of Holliday junction-like structures.     

Anticoagulant function of a 24-Kd fragment isolated from human fibrinogen A alpha chains

A fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent M(r) approximately 24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro- gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen A alpha chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen A alpha chains, the N-terminal of which starts at alpha Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin's amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.     

Anti-apoptogenic function of TGFbeta1 for human synovial cells: TGFbeta1 protects cultured synovial cells from mitochondrial perturbation induced by several apoptogenic stimuli

OBJECTIVE: To investigate anti-apoptogenic mechanism of transforming growth factor beta1 (TGFbeta1) towards synovial cells. METHODS: Isolated synovial cells, treated or not with TGFbeta1, were cultured in the presence or absence of anti-Fas IgM, proteasome inhibitor Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of synovial cells was examined by the presence of hypodiploid DNA(+) cells, the presence of terminal deoxy (d)-UTP nick end labelling(+) cells (TUNEL(+) cells), activation of caspases, and disruption of mitochondrial transmembrane potential (DeltaPsim). RESULTS: Activation of caspase-9 and DeltaPsim was found in anti-Fas IgM treated synovial cells. The increment of both hypodiploid DNA(+) cells and TUNEL(+) cells accompanied by the activation of caspase-8 and caspase-3 was also determined in anti-Fas IgM treated synovial cells. These hallmarks for apoptosis induced by anti-Fas IgM were significantly suppressed in TGFbeta1 treated synovial cells. LLL-CHO, etoposide, and C2-ceramide also caused DeltaPsim, the increment of both hypodiploid DNA(+) cells and TUNEL(+) cells, and the activation of both Leu-Glu-His-Asp ase (LEHDase; caspase-9 like activity) and Asp-Glu-Val-Asp ase (DEVDase; caspase-3 like activity) in synovial cells. As determined in anti-Fas IgM treatment, TGFbeta1 significantly reduced apoptotic cell death of synovial cells induced by the above chemicals. CONCLUSIONS: The protective effect of TGFbeta1 for mitochondrial homoeostasis may be important in the anti-apoptogenic function of TGFbeta1 for synovial cells.     

Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts

Methods of rapidly extracting chromosomal DNA from human pathogenic yeasts were used in mitochondrial DNA (mtDNA) studies. This paper is concerned with rapid and reliable methods of extracting mtDNA for sequence analysis for species or strain identification, and epidemiological study of medically important fungi and fungal infections. To determine the optimal method of mtDNA extraction without isolating mitochondria, we examined three commonly used methods: 1). boiling, 2). glass bead disruption, and 3). a commercially available kit. We assessed the amount and quality of DNAs using a spectrophotometer and specific polymerase chain reaction (PCR). The DNA yield depended on the extraction method used and the yeast species. An adequate amount of mtDNA was obtained with both glass beads and a commercially available kit to amplify the mitochondrial gene using PCR without isolating the mitochondria. These techniques are convenient for extracting DNA from a variety of small-scale samples.