Magnesium chelatase subunit D from pea: characterization of the cDNA,heterologous expression of an enzymatically active protein and immunoassay of the native protein

Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes – BchI, D and H – encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl₂. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.     

Aminoimidazoles as bioisosteres of acylguanidines: novel, potent, selective and orally bioavailable inhibitors of the sodium hydrogen exchanger isoform-1

Inhibition of the sodium hydrogen exchanger isoform-1 (NHE-1) has been shown to limit damage to the myocardium under ischemic conditions in animals. While most known NHE-1 inhibitors are acylguanidines, this report describes the design and synthesis of a series of heterocyclic inhibitors of NHE-1 including aminoimidazoles with undiminished in vitro activity and oral bioavailability.     

In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells

The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.     

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.     

VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells

Glomerular epithelial cells (GEC) are aknown site of vascular endothelial growth factor (VEGF) production. Weestablished immortalized rat GEC, which retained the ability to produceVEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells culturedon GEC-conditioned matrix, an indicator of the permeability ofmonolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased byVEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showedthat VEGF results in a rapid appearance of transcellular elongatedstructures decorated with caveolin. Transmission electron microscopy ofendothelial cells showed that caveolae undergo rapid internalizationand fusion 30 min after application of VEGF-165. Later (36 h),endothelial cells pretreated with VEGF developed fenestrae and showed adecrease in electrical resistance. Immunoelectron microscopy ofglomeruli confirmed VEGF localization to podocytes and in the basementmembrane. In summary, immortalized GEC retain the ability to synthesizeVEGF. Matrix-deposited and soluble VEGF leads to the enhancement ofcaveolae expression, their fission and fusion, formation of elongatedcaveolin-decorated structures, and eventual formation of fenestrae,both responsible for the increase in endothelial permeability.

     

In vivo imaging of embryonic vascular development using transgenic zebrafish

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.     

Identification of a MAD2-binding protein,CMT2, and its role in mitosis

MAD2 is a key component of the spindle checkpoint that delays the onset of anaphase until all the kinetochores are attached to the spindle. It binds to human p55CDC and prevents it from promoting destruction of an anaphase inhibitor, securin. Here we report the characterization of a novel MAD2-binding protein, CMT2. Upon the completion of spindle attachment, formation of the CMT2-MAD2 complex coincides with dissociation of the p55CDC-MAD2 complex. Overexpression of CMT2 in cells arrested by the spindle checkpoint causes premature destruction of securin and allows exit from mitosis without chromosome segregation. Depletion of CMT2 induces cell death following a transient delay in the onset of anaphase. These results indicate that CMT2 interacts with the spindle checkpoint and coordinates cell cycle events in late mitosis.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.