NMR analysis shows that a b-type variant of Hydrogenobacter thermophilus cytochrome c552 retains its native structure

Conversion of Hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E. J., and Ferguson, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5156-5160). To understand the effects of this change on the structure and dynamics of the protein, hetero-nuclear (15)N-edited NMR techniques have been used to characterize this b-type variant. The backbone (15)N, (1)H(N), and (1)H(alpha), and (1)H(beta) resonances of the protein have been assigned. Analysis of (3)J(HN)alpha coupling constants, nuclear Overhauser enhancement intensities, and chemical shift index data demonstrates that the four alpha-helices present in the wild-type protein are retained in the b-type variant. Comparison of the chemical shifts for the b-type and wild-type proteins indicates that the tertiary structures of the two proteins are closely similar. Some subtle differences are, however, observed for residues in the N-terminal region and in the vicinity of the heme-binding pocket. Hydrogen exchange studies show that there are 25 backbone amide protons that exchange very slowly in the b-type variant and confirm that the fluctuations within the b-type protein are of a similar extent to those in the wild-type protein. These data demonstrate the notable retention of the native secondary structure and tertiary fold despite the absence of covalent linkages between the heme group and the protein.     

Structural consequences of cysteine substitutions C1977Y and C1977R in calcium-binding epidermal growth factor-like domain 30 of human fibrillin-1

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.     

Atrial BNP endocrine function during chronic unloading of the normal canine heart

The goal of the study was to define the effect of chronic unloading of the normal heart on atrial endocrine function with a focus on brain natriuretic peptide (BNP), specifically addressing the role of load and neurohumoral stimulation. Although produced primarily by atrial myocardium in the normal heart, controversy persists with regard to load-dependent vs. neurohumoral mechanisms controlling atrial BNP synthesis and storage. We used a unique canine model of chronic unloading of the heart produced by thoracic inferior vena caval constriction (TIVCC), which also resulted in activation of plasma endothelin (ET-1), ANG II, and norepinephrine (NE), known activators of BNP synthesis, compared with sham. TIVCC was produced by banding of the inferior vena cava for 10 days (n = 6), whereas in control (n = 5) the band was not constricted (sham). In a third group (n = 7), the band was released on day 11, thus acutely reloading the heart. Chronic TIVCC decreased cardiac output and right atrial pressure with a decrease in atrial mass index consistent with atrial atrophy. Atrial BNP mRNA decreased compared with sham. Immunoelectron microscopy revealed an increase in BNP in atrial granules consistent with increased storage. Acute reloading increased cardiac filling pressures and resulted in an increase in plasma BNP. We conclude that chronic unloading of the normal heart results in atrial atrophic remodeling and in suppression of atrial BNP mRNA despite intense stimulation by ET, ANG II, and NE, underscoring the primacy of load in the control of atrial endocrine function and structure.     

Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload

Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.     

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.     

A model for the selective loss of major histocompatibility complex self-restricted T cell immune responses during the development of acquired immune deficiency syndrome (AIDS)

Functional analyses of peripheral blood leukocytes (PBL) from high risk HTLV-III antibody-negative and antibody-positive donors, as well as from patients with acquired immune deficiency syndrome (AIDS), lymphadenopathy syndrome (LAS), and AIDS-related complex (ARC) were performed by the in vitro generation of cytotoxic T lymphocyte (CTL) and proliferative responses to the HLA self-restricted antigens of influenza virus (S + X) and to nonself restricted HLA alloantigens (ALLO). All 40 antibody-negative donors tested responded to both S + X and ALLO in the CTL response, whereas six of 14 antibody-positive, two of three LAS, four of five ARC, and seven of 17 AIDS patients exhibited a selective absence of CTL to S + X, but generated normal or elevated CTL responses to ALLO. Of the remaining 10 AIDS patients, nine did not respond to either S + X or ALLO, and one responded to both S + X and ALLO. A similar selective loss of the proliferative response to S + X was found. We also observed antibody-positive donors who initially generated CTL responses to S + X and ALLO, but lost the S + X response as a function of time. We were able to restore the selective loss of S + X CTL activity in vitro by the addition of IL 2 and, to some extent, by co-stimulation with S + X plus ALLO. Depletion of CD4+ T helper cells and removal of autologous antigen-presenting cells from the PBL of healthy antibody-negative donors indicated that distinct T helper cell subsets exist in human PBL, and that S + X responses must use a CD4+ T helper population, whereas ALLO responses can utilize an alternate CD4- T helper pathway. A model is presented indicating the selective depletion of CD4+ T helper function in the developmental stages of AIDS. The functional test for T helper activity to self restricted antigens may be the earliest indicator of immune functional loss in the development of AIDS, and may precede a reduction in the absolute number of CD4+ cells.     

Evolution of Bacterial Transformation: Is Sex with Dead Cells Ever Better than No Sex at All?

Computer simulations of bacterial transformation are used to show that, under a wide range of biologically reasonable assumptions, transforming populations undergoing deleterious mutation and selection have a higher mean fitness at equilibrium than asexual populations. The source of transforming DNA, the amount of DNA taken up by each transforming cell, and the relationship between number of mutations and cell viability (the fitness function) are important factors. When the DNA source is living cells, transformation resembles meiotic sex. When the DNA source is cells killed by selection against mutations, transformation increases the average number of mutations per genome but can nevertheless increase the mean fitness of the population at equilibrium. In a model of regulated transformation, in which the most fit cells of a transforming population do not transform, transforming populations are always fitter at equilibrium than asexual populations. These results show that transformation can reduce mutation load.     

NMR study of nitrogen-15-labeled Escherichia coli valine transfer RNA.

1,3-15N-Labeled uracil was synthesized chemically and used to prepare labeled Escherichia coli tRNA(Val) biosynthetically. 500-MHz measurements of 15N and proton chemical shift were obtained, for all uridine and uridine-related bases, by heteronuclear multiple-quantum coherence spectroscopy. All the uracil NH group resonances were assigned and were in agreement with previous proton-only assignments. The temperature dependence of intensities of resonances was used to infer the relative stability of parts of the molecule. The acceptor stem was the least thermally stable structural feature, while the anticodon and T loop were relatively more stable.