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Biomedical subjects

M Yeager

Publications and source records attributed to M Yeager.

At least 19 recordsLinked to original sources
PubMed2001

Localization of membrane permeabilization and receptor binding sites on the VP4 hemagglutinin of rotavirus: implications for cell entry.

The surface of rotavirus is decorated with 60 spike-like projections, each composed of a dimer of VP4, the viral hemagglutinin. Trypsin cleavage of VP4 generates two fragments, VP8*, which binds sialic acid (SA), and VP5*, containing an integrin binding motif and a hydrophobic region that permeabilizes membranes and is homologous to fusion domains. Although the mechanism for cell entry by this non-enveloped virus is unclear, it is known that trypsin cleavage enhances viral infectivity and facilitates viral entry. We used electron cryo-microscopy and difference map analysis to localize the binding sites for two neutralizing monoclonal antibodies, 7A12 and 2G4, which are directed against the SA-binding site within VP8* and the membrane permeabilization domain within VP5*, respectively. Fab 7A12 binds at the tips of the dimeric heads of VP4, and 2G4 binds in the cleft between the two heads of the spike. When these binding results are combined with secondary structure analysis, we predict that the VP4 heads are composed primarily of beta-sheets in VP8* and that VP5* forms the body and base primarily in beta-structure and alpha-helical conformations, respectively. Based on these results and those of others, a model is proposed for cell entry in which VP8* and VP5* mediate receptor binding and membrane permeabilization, and uncoating occurs during transfer across the lipid bilayer, thereby generating the transcriptionally active particle.

Animals↗
PubMed2001

Transmembrane alpha-helices in the gap junction membrane channel: systematic search of packing models based on the pair potential function.

Recent progress in the field of electron cryo-microscopy and image analysis has shown that there is an overwhelming need to interpret medium resolution (5 to 10 A) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling programs must be supplemented with further analysis. We have used a potential of mean force (PMF) method, derived from Boltzmann statistics in protein structure, to generate models for the packing of alpha-helices, using pairwise potentials between amino acid residues. The approach was tested using the three-dimensional map of a recombinant cardiac gap junction membrane channel provided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) which had a resolution of 7.5 A in the membrane plane and 21 A in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility SymServ (Macke et al., 1998) to build hexameric connexons with 24 transmembrane alpha-helices. Canonical alpha-helices were aligned to the axes of the rods of density and translated along the density so that the center of masses coincided. The PMF function was used to evaluate 162,000 conformations for each of the 24 possible alpha-helical packing models. Since the different packing models yielded different energy distributions, the pair potential function appears to be a promising tool for evaluating the packing of alpha-helices in membrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed by hydrophobic residues in contact with the lipids.

Connexins↗
PubMed2001

Nosocomial bloodstream infections in patients with implantable left ventricular assist devices.

BACKGROUND: Implantable left ventricular assist devices (LVAD) are used as a bridge to transplantation but are associated with a high risk of infection including nosocomial bloodstream infections (BSI). METHODS: We retrospectively reviewed the medical records of all patients with implantable LVAD at the Cleveland Clinic with 72 hours or longer of LVAD support from January 1992 through June 2000, to determine the attack rate, incidence, and impact of nosocomial BSI in patients with LVAD. A nosocomial BSI was defined using Centers for Disease Control and Prevention definition. An LVAD-related BSI was defined as one where the same pathogen is cultured from the device and the blood with no other obvious source. Two hundred fourteen patients were included in the study (17,831 LVAD-days). RESULTS: One hundred forty BSI were identified in 104 patients for an attack rate of 49% and incidence of 7.9 BSI per 1000 LVAD-days. Thirty-eight percent of the BSI were LVAD associated. The most common pathogens causing BSI were coagulase-negative staphylococci (n = 33), Staphylococcus aureus, and Candida spp. (19 each), and Pseudomonas aeruginosa (16 each). A Cox proportional hazard model found BSI in patients with LVAD to be significantly associated with death (hazard ratio = 4.02, p < 0.001). Fungemia had the highest hazard ratio (10.9), followed by gram-negative bacteremia (5.1), and gram-positive bacteremia (2.2). CONCLUSIONS: Patients with implantable LVAD have a high incidence of BSI, which are associated with a significantly increased mortality. Strategies for prevention of infection in LVAD recipients should focus on the drive line exit site until technical advances can achieve a totally implantable device.

Bacteremia↗
PubMed2001

The pathobiology of pulmonary hypertension. Endothelium.

Dysfunctional endothelial cells have a central and critical role in the initiation and progression of severe pulmonary hypertension. The elucidation of the mechanisms involved in the control of endothelial cell proliferation and cell death in the pulmonary vasculature, therefore, is fundamentally important in the pathogenesis of severe pulmonary hypertension and of great interest for a better understanding of endothelial cell biology. Because the intravascular growth of endothelial cells resulting in tumorlets is unique to severe pulmonary hypertension, this phenomenon can teach researchers about the factors involved in the formation and maintenance of the normal endothelial cell monolayer. Clearly, in severe pulmonary hypertension, the "law of the endothelial cell monolayer" has been broken. The ultimate level of such a control is at the altered gene expression pattern that is conducive to endothelial cell growth and disruption of pulmonary blood flow. Secondary pulmonary hypertension certainly also is associated with proliferated pulmonary endothelial cells and plexiform lesions that are histologically indistinguishable from those in PPH. What is then the difference in the mechanisms of endothelial cell proliferation between primary and secondary pulmonary hypertension? The authors believe that PPH is a disease caused by somatic mutations in key angiogenesis- or apoptosis-related genes such as the TGF-beta receptor-2 and Bax. The loss of these important cell growth control mechanisms allows for the clonal expansion of endothelial cells from a single cell that has acquired a selective growth advantage. On the other hand, the proliferated endothelial cells in secondary pulmonary hypertension are polyclonal. It follows from this finding that local (vascular) factor(s) (such as increased shear stress), rather than mutations, play a major role in triggering endothelial cell proliferation. In PPH and secondary pulmonary hypertension, the researcher can postulate that the pulmonary vascular bed contains progenitor-like cells with the capacity of dysregulated growth. The main difference in the pathogenesis of primary and secondary pulmonary endothelial cell proliferation therefore may be the initial mechanism involved in the recruitment of an endothelial progenitor-like cell. In PPH, anorexigen-associated, and familial PPH, the proliferation of endothelial cells occurs from a mutated single cell, whereas in secondary pulmonary hypertension, several progenitor-like cells would be activated to grow. The abnormal endothelial cells in both forms of severe pulmonary hypertension expand because of the expression of angiogenesis-related molecules such as VEGF, VEGFR-2, HIF-1 alpha, and HIF-beta. Also important for the expansion of these cells is the down-regulation of expression of apoptosis-related mediators such as TGF-beta receptor-2 or Bax. The success of any therapy for severe pulmonary hypertension requires that the underlying process of endothelial cell proliferation could be controlled or reversed.

Animals↗
PubMed2001

The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA.

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.

Amino Acid Sequence↗
PubMed2000

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Amino Acid Sequence↗
PubMed2000

3D domain swapping modulates the stability of members of an icosahedral virus group.

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Amino Acid Sequence↗
PubMed2000

Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein.

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.

Amino Acid Sequence↗
PubMed2000

Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV).

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.

Animals↗
PubMed2000

Class I and class II MHC bind self peptide sets that are strikingly different in their evolutionary characteristics.

Comparison of peptides eluted from human class I and class II major histocompatibility complex (MHC) molecules and the proteins from which they are derived (source proteins) revealed that class I MHC bind peptides derived from proteins that are highly conserved, hydrophilic, and universally expressed, while the peptides themselves are hydrophobic and even more conserved than their source proteins. In contrast, source proteins for class II-bound peptides were not significantly more conserved than a random sample of proteins. Class II-bound peptides were generally more conserved than their source proteins but were significantly less conserved than class I-bound peptides. The characteristics of class I-bound peptides can probably be explained by the selectivity of processing and transport of peptides for binding by class I, while the relative lack of selectivity of peptide binding for class II may explain the high incidence of autoimmune diseases associated with alleles of these molecules.

Alleles↗
PubMed1999

Projection structure of a plant vacuole membrane aquaporin by electron cryo-crystallography.

The water channel protein alpha-TIP is a member of the major intrinsic protein (MIP) membrane channel family. This aquaporin is found abundantly in vacuolar membranes of cotyledons (seed storage organs) and is synthesized during seed maturation. The water channel activity of alpha-TIP can be regulated by phosphorylation, and the protein may function in seed desiccation, cytoplasmic osmoregulation, and/or seed rehydration. Alpha-TIP was purified from seed meal of the common bean (Phaseolus vulgaris) by membrane fractionation, solubilization in diheptanoylphosphocholine and anion-exchange chromatography. Upon detergent removal and reconstitution into lipid bilayers, alpha-TIP crystallized as helical tubes. Electron cryo-crystallography of flattened tubes demonstrated that the crystals exhibit plane group p2 symmetry and c222 pseudosymmetry. Since the 2D crystals with p2 symmetry are derived from helical tubes, we infer that the unit of crystallization on the helical lattice is a dimer of tetramers. A projection density map at a resolution of 7.7 A revealed that alpha-TIP assembles as a 60 A x 60 A square tetramer. Each subunit is formed by a heart-shaped ring comprised of density peaks which we interpret as alpha-helices. The similarity of this structure to mammalian plasma membrane MIP-family proteins suggests that the molecular design of functionally analogous and genetically homologous aquaporins is maintained between the plant and animal kingdoms.

Amino Acid Sequence↗
PubMed1999

Expression, two-dimensional crystallization, and electron cryo-crystallography of recombinant gap junction membrane channels.

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Animals↗
PubMed1999

Three-dimensional structure of a recombinant gap junction membrane channel.

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Animals↗
PubMed1999

Electron cryo-crystallography of a recombinant cardiac gap junction channel.

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

Amino Acid Sequence↗
PubMed1999

Coevolution of the mammalian chemokines and their receptors.

A phylogeny of mammalian chemokines revealed two major clusters, corresponding to the CC and CXC chemokines; the C chemokines appeared to be more closely related to the former. In a phylogeny of chemokine receptors, there were also two major clusters: one containing CC chemokine receptors plus other receptors of unknown function and another containing CXC receptors and other receptors of unknown function. However, within the CC receptors, there was not a close correspondence between the phylogenies of chemokines and their receptors. The CC chemokines contained two major subfamilies: (1) the MIP subfamily (including MIP-1alpha, MIP-1beta, and RANTES); and (2) the MCP subfamily (including MCP-1,-2,-3, and -4 and eotaxin). Receptors having preferred ligands in the MCP subfamily did not constitute a monophyletic group but rather evolved twice independently. Reconstruction of ancestral amino acid sequences suggested that these two groups of MCP receptors did not convergently evolve any amino acid residues; rather, they convergently lost sequence features found in the third and fourth extracellular domains of known receptors for MIP-subfamily chemokines.

Amino Acid Sequence↗