Structure of C protein purified from cardiac muscle

C protein is a component of the thick filament of striated muscles. Although the function of C protein remains unknown, a variety of evidence suggests that C protein may regulate actin-myosin interaction or be involved in structural support or elasticity of the sarcomere. We have previously proposed (Hartzell, H. C., 1984, J. Gen. Physiol., 83:563-588) that C protein is involved in regulating twitch relaxation in cardiac muscle. To gain further insight into the function of C protein, we have studied the structure of C protein purified from chicken heart. C protein was purified from extracts of detergent-washed myofibrils by sequential hydroxylapatite and DEAE-Sephacel chromatography. C protein was judged greater than 95% pure by SDS PAGE. The polypeptide subunit had a molecular weight of 155,000 and the native molecule sedimented on linear sucrose or glycerol gradients at 4-5S. For electron microscopy, purified C protein was dialyzed and diluted into a volatile buffer in 50% glycerol, aspirated onto mica, dried under vacuum, and rotary platinum-shadowed. Replicas revealed particles of relatively homogeneous overall dimensions. Over half of the particles were V-shaped. The "arm" lengths of the V-shaped particles were 22 +/- 4.5 nm (SD). Gel filtration on Sephacryl S-300 demonstrated that purified C protein had a Stokes' radius of 5.07 nm. Measurements of viscosity gave an intrinsic viscosity of 16.5 cm3/g. These data are consistent with the electron microscopic data and suggest that C protein in heart muscle is asymmetric. The C protein molecule is large enough to extend from the surface of a thick filament to adjacent thin or thick filaments.     

The magnetic susceptibility of cytochrome oxidase in the 4.2-1.5 K range.

Sixteen low temperature measurements on eight independent cytochrome oxidase samples from two separate laboratories have yielded magnetic susceptibility data compatible with a model of spin-coupled iron and copper ions, as presented in the preceding paper (Tweedle, M.F., Wilson, L.J., García-I?iguez, L., Babcock, G. T., and Palmer, G. (1978) J. Biol. Chem. 253, 8065-8071). The data in the 1.5-77 K range match those attained at higher temperatures and the predictions of the spin-coupled model. Measurements on reduced samples confirm the high spin nature of one iron atom. No obvious uncoupling of the antiferromagnetic Fe-Cu interaction is detected in partly reduced samples.     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate VMP001/AS01B in Malaria-Naive Adults: Safety,Immunogenicity, and Efficacy

Background

A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001), a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP) and a truncated repeat region that contains repeat sequences from both the VK210 (type 1) and the VK247 (type 2) parasites, was developed as a vaccine candidate for global use.

Methods

We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI) of VMP001 formulated in the GSK Adjuvant System AS01_B. A total of 30 volunteers divided into 3 groups (10 per group) were given 3 intramuscular injections of 15μg, 30μg, or 60μg respectively of VMP001, all formulated in 500μL of AS01_B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls.

Results

The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period.

Significance

This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.     

Cutaneous and presumed visceral leishmaniasis in a soldier deployed to Afghanistan

Leishmaniasis has been frequently diagnosed in US military personnel returning from duty in Southwest Asia. The majority of cases have demonstrated cutaneous disease, although a few cases of visceral disease have been documented. We present the case of an immunocompetent, HIV-negative, US Army soldier who suffered both visceral and cutaneous manifestations of leishmaniasis after returning from deployment in Afghanistan. Overlap of cutaneous and visceral involvement is rare and has not been reported in our cohort. Latent Plasmodium vivax infection may have been an exacerbating cofactor. We discuss this case and present a review of the literature.     

Expression of beta‐galactosidase and pig leptin gene in vitro by recombinant adenovirus

Abstract

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing β‐galactosidase reporter gene (AdCMVLac‐Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac‐Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac‐Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac‐Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac‐Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long‐term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.