Biomedical subjects

Richard D Leapman

Publications and source records attributed to Richard D Leapman.

11 recordsLinked to original sources

PubMed2006

Evaluation of bioreactor-cultivated bone by magnetic resonance microscopy and FTIR microspectroscopy.

We present a three-dimensional mineralizing model based on a hollow fiber bioreactor (HFBR) inoculated with primary osteoblasts isolated from embryonic chick calvaria. Using non-invasive magnetic resonance microscopy (MRM), the growth and development of the mineralized tissue around the individual fibers were monitored over a period of 9 weeks. Spatial maps of the water proton MRM properties of the intact tissue, with 78 microm resolution, were used to determine changes in tissue composition with development. Unique changes in the mineral and collagen content of the tissue were detected with high specificity by proton density (PD) and magnetization transfer ratio (MTR) maps, respectively. At the end of the growth period, the presence of a bone-like tissue was verified by histology and the formation of poorly crystalline apatite was verified by selected area electron diffraction and electron probe X-ray microanalysis. FTIR microspectroscopy confirmed the heterogeneous nature of the bone-like tissue formed. FTIR-derived phosphate maps confirmed that those locations with the lowest PD values contained the most mineral, and FTIR-derived collagen maps confirmed that bright pixels on MTR maps corresponded to regions of high collagen content. In conclusion, the spatial mapping of tissue constituents by FTIR microspectroscopy corroborated the findings of non-invasive MRM measurements and supported the role of MRM in monitoring the bone formation process in vitro.

Animals↗

PubMed2006

Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas.

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an approximately 8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells.

Antineoplastic Agents↗

PubMed2005

Mass of the postsynaptic density and enumeration of three key molecules.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils.

Amyloid fibrils commonly exhibit multiple distinct morphologies in electron microscope and atomic force microscope images, often within a single image field. By using electron microscopy and solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue beta-amyloid peptide of Alzheimer's disease (Abeta(1-40)), we show that different fibril morphologies have different underlying molecular structures, that the predominant structure can be controlled by subtle variations in fibril growth conditions, and that both morphology and molecular structure are self-propagating when fibrils grow from preformed seeds. Different Abeta(1-40) fibril morphologies also have significantly different toxicities in neuronal cell cultures. These results have implications for the mechanism of amyloid formation, the phenomenon of strains in prion diseases, the role of amyloid fibrils in amyloid diseases, and the development of amyloid-based nano-materials.

Amino Acid Motifs↗

PubMed2004

Organization of designed nanofibrils assembled from alpha-helical peptides as determined by electron microscopy.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.

Microscopy, Electron, Scanning↗

PubMed2004

Novel techniques in electron microscopy.

Emerging techniques in electron microscopy promise to yield a wide range of new information about the nervous system. Aided by the development of detectors, electron optics, energy filters, computer automation and preparative methods, electron tomography now provides three-dimensional structures over a scale ranging from single receptor molecules to synapses and neurons. To relate structure to function, a variety of correlative methods are being developed, including protein tags observable both by light microscopy of living cells and, subsequently, by electron microscopy. It is also becoming possible to measure functionally important ions like Ca(2+) in cellular compartments at a scale of about 10 nm by exploiting new advances in electron energy loss and X-ray spectroscopic imaging.

Animals↗

PubMed2004

Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras.

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.

Chromatography, Gel↗

PubMed2003

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2.

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, approximately 5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.

Bacterial Proteins↗

PubMed2002

Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance.

We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1)(-)(42)) and by residues 10-35 of the full-length peptide (Abeta(10)(-)(35)). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of beta-sheets present in the characteristic amyloid cross-beta structural motif and the assembly of these beta-sheets into a fibril. EM images of negatively stained Abeta(10)(-)(35) fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Abeta(10)(-)(35) fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-beta layer. Abeta(10)(-)(35) fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Abeta(1)(-)(42) fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-beta layer are also formed by Abeta(1)(-)(42) and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Abeta(10)(-)(35) fibrils, using multiple-quantum (13)C NMR, (13)C-(13)C dipolar recoupling, and (15)N-(13)C dipolar recoupling techniques, support the in-register parallel beta-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this beta-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Abeta(1)(-)(42) fibrils, which represent the first NMR data on Abeta(1)(-)(42) fibrils, also indicate an in-register parallel beta-sheet organization. These results, along with previously reported data on Abeta(1)(-)(40) fibrils, suggest that the supramolecular structures of Abeta(10)(-)(35), Abeta(1)(-)(40), and Abeta(1)(-)(42) fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.

Alzheimer Disease↗

PubMed2002

A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR.

We present a structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-beta structural motif established by x-ray fiber diffraction and satisfies constraints on Abeta(1-40) fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Abeta(1-40) are structurally disordered in the fibrils. Residues 12-24 and 30-40 adopt beta-strand conformations and form parallel beta-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two beta-sheets in contact through sidechain-sidechain interactions. A single cross-beta unit is then a double-layered beta-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-beta units with their hydrophobic faces juxtaposed.

Amino Acid Sequence↗

PubMed2002

Oligomeric structure of the human immunodeficiency virus type 1 envelope protein on the virion surface.

The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.

Biopolymers↗