George C. Ruben

Cell-free DNA in human blood plasma : Length measurements in patients with pancreatic cancer and healthy controls

The amount of non-cell-associated DNA free in blood plasma from pancreatic cancer patients usually exceeds that from healthy donors. We have evaluated the plasma DNA by gel electrophoresis and measured the variation in length of soluble DNA fragments by electron microscopy in plasma from three patients with pancreatic cancer and from three healthy controls. Whereas electrophoresis of nick-translated DNA isolated from plasma obtained from healthy controls showed autoradiographic bands at sizes equivalent to whole-number multiples (1-5x) of nucleosomal DNA (185-200 bp), in the samples obtained from pancreatic cancer patients, stronger ladder patterns appeared. Likewise, strand length distributions of DNA (DNA-SL) in the two groups differ. The DNA-SL distribution data include 2,752 measurements made from cancer patient plasma and 3,291 for control plasma. The shortest DNA-SL measured approximately 30 nm (approximately 88 bp calculated at 0.34 nm/bp) and the largest approximately 28,000 nm (>80,000 bp), with 50% of all lengths measuring between 100 and 900 nm long. The average plasma DNA-SL in controls (311 nm; median, 273 nm) exceeded that in cancer patients (231 nm; median, 185 nm). Small excesses of DNA at approximately 63, approximately 126, approximately 189, approximately 252, and approximately 315 nm, corresponding to small multiples of lengths associated with nucleosomes, were more prominent in the cancer patient plasma than in the healthy control plasma. This study provides evidence indicating differences in non-cell-associated DNA in plasma between cancer patients and healthy controls and indicates that a significant amount of this DNA is probably derived from apoptosis in neoplastic and/or normal cells.

Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane

A large heparan sulfate proteoglycan of low buoyant density (p = 1.32 to 1.40 g/cm3 in 6 M-guanidine.HCl) was extracted from a tumor basement membrane with denaturing solvents and purified by chromatography and CsCl gradient centrifugation. Chemical, immunological, physical and electron microscopical analyses have demonstrated a high degree of purity and have allowed us to propose a structural model for this proteoglycan. It is composed of an 80 nm long protein core formed from a single polypeptide chain (Mr about 500,000) with intrachain disulfide bonds. This core is folded into a row of six globular domains of variable size as shown by electron microscopy after rotary shadowing and negative staining. A multidomain structure was confirmed by protease digestion experiments that allowed the isolation of a single heparan sulfate-containing peptide segment representing less than 5% of the total mass of the protein core. Electron microscopy has visualized generally three heparan sulfate chains in each molecule close to each other at one pole of the protein core. The molecular mass and length (100 to 170 nm) of the heparan sulfate chains were found to vary consistently between different preparations. The mass per length ratio (350 nm-1) indicated an extended conformation for the heparan sulfate side-chains. These structural features are distinctly different from those of the high density proteoglycan, suggesting that both forms of basement membrane heparan sulfate proteoglycan are genetically distinct and not derived from a common precursor.

Imaging aerogels at the molecular level

Aerogels are a special class of open-cell foams that have an ultrafine cell/pore size (<50 nm), high surface area (400–1000 m2 g−1), and an ultrastructure composed of interconnected colloidal-like particles or polymeric chains with characteristic dimensions of 10 nm. The purpose of this paper is to directly image a series of resorcinol-formaldehyde (RF) and silica aerogels by high resolution transmission electron microscopy (HRTEM). A new vertical replication technique allows us to examine aerogels at the molecular level in situ so that differences in polymeric and colloidal aerogels can be visualized. Such information is crucial for nano-engineering the structure and properties of these novel materials.

Conformation of Lac repressor tetramer in solution, bound and unbound to operator DNA

We tested whether the Steitz et al. [(1974) Proc. Natl. Acad. Sci. U.S.A., 71:593–597] model of lactose repressor (LacR) (14 × 6.0 × 4.5 nm) represented the shape of free or operator‐bound LacR in solution. The model predicts a 14 nm length for bound LacR. Direct measurement, using Pt‐C shadow width standards, was 9.6 ± 0.2 nm long. Using the Steitz model, we generated a distribution of measurements and converted them into a distribution of shadow widths using gold ball standards. Direct measurement of LacR produced a narrower shadow width distribution with a larger mean size than the Steitz model predicted. Measurement along two orthogonal axes of negatively stained LacR images generated a size distribution, also converted into a shadow width distribution using the gold ball standards. Since the experimental shadow width distribution exactly matched the shadow width distribution derived theoretically from negatively stained LacR, our negative‐stained images are representative of LacRs conformation in solution. Approximately 56% of negatively stained LacR had a V‐shaped fold around an axis orthogonal to its length, bringing the DNA binding domains of each dimer adjacent. This open end of the V binds single operator DNA. The other 44% of the LacR tetramer is in the extended form with its DNA binding sites at opposite ends. Although the V‐shaped conformation has a closed hinge with the dimers associated along a side, the extended open‐hinged state remains important since LacR must bind two distant operator sites for full repression. Our measurements predict the normal presence of both conformations in nearly equal amounts, suggesting that both are equally active in repressing the lac operon. Microsc. Res. Tech. 36:400–416, 1997.

High-resolution transmission electron microscopy of the nanostructure of melamine-formaldehyde aerogels

Abstract The transparency of melamine-formaldehyde (MF) aerogels strongly depends on the solution pH during polymerization. Two MF aerogels were studied. MF #1 was transparent and was formed at pH ⋍ 2 with a bulk density of 160 kg/m3 and a Brunauer, Emmett and Teller (BET) surface area of 970 m2/gm. MF #2 was an opaque aerogel formed at pH ⋍ 0.5 with a bulk density of 130 kg/m3 and a surface area of 850 m2/gm. Both MF aerogels have a polymeric nanostructure. Melamine and formaldehyde formed single polymeric chains of ⋍ 0.95 nm in diameter. The MF #1 aerogel connectors were frequently composed of two or three polymeric chains with the connector diameter averaging ⋍ 2.0 and ⋍ 2.8 nm which usually showed branching every 12.6 ± 4.5 nm thereby defining the connector length. The side chains along the connectors in MF #1 formed a reticular MF matrix. The MF #1 matrix pores averaged 10.4 ± 5.5 nm and pores between sections of the MF matrix averaged 28.8 ± 19 nm. In MF #2, the connectors also formed side chains but these side chains did not form a reticular matrix. As a consequence, the connector diameter, 8.8 ± 5.8 nm, includes the side chains projecting from a core chain of 1.9 ± 0.7 nm. In addition to the average pore size of 47.7 ± 30 nm, MF #2 contained cavities or voids within the aerogel with length dimensions averaging 328 ± 130 nm, responsible for its opacity. The high BET surface areas of both MF aerogels correlate with their polymeric structure.

Studies of DNA Organization in Hydrated Spermidine-Condensed DNA Toruses and Spermidine-DNA Fibres

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Mannings counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.

A Study of øX-174 DNA Torus and Lambda DNA Torus Tertiary Structure and the Implications For DNA Self-Assembly

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.

The organization of the microtubule associated protein tau in Alzheimer paired helical filaments

The structural relationship of the microtubule associated protein tau to paired helical filaments (PHF) was examined by high resolution transmission electron microscopy (TEM) without treatment with any chemical fixatives. Neurofibrillary tangles (NFT) were isolated in the absence of detergent from Alzheimer diseased brains, were freeze-dried, and were vertically platinum-carbon replicated for TEM. The PHF we observed made one helical turn (L) in 74 +/- 8.5 nm and had a wide region (W) of 14.8 +/- 0.6 nm similar to PHF previously modeled with a periodic morphology. The PHF thin region (T) measured approximately 2.4 nm, approximately 4.9 nm, approximately 7.4 nm and approximately 9.7 nm and the most often observed width was approximately 2.4 nm. No surface features regularly divide the PHF into two filaments. Morphologically the PHF are thin helical ribbons with an often observed thickness of approximately 2.4 nm. At high magnification, approximately 1.0 nm and some approximately 0.4 nm strands identical to normal and denatured tau monomer covered PHF surfaces and were aggregated in non-periodic fashion. Bovine tau polymer approximately 2.1 nm diameter filaments, trapped on a filter, were partially heat denatured and showed some of the morphological features of PHF.

Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze-etch electron microscopy study.

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported.

Alzheimer neurofibrillary tangles contain 2.1 nm filaments structurally identical to the microtubule-associated protein τ: a high-resolution transmission electron microscope study of tangles and senile plaque core amyloid

Alzheimer neurofibrillary tangles (NFT) and senile plaque core amyloid (SPCA) isolated from the brain of patients with Alzheimers disease were freeze-dried and replicated with a new platinum-carbon (Pt-C) vertical deposition method for high-resolution transmission electron microscopy (TEM). The resolution of this vertical Pt-C replication method is superior to either of the more conventional 20 degrees rotary replication or 45 degrees unidirectional replication methods and is dependent on the Pt-C film thickness coating the specimen. The paired helical filaments (PHF) observed within the tangles were right-handed helices with a fairly regular twist period averaging 79.3 +/- 5.9 nm and a fairly regular maximum width averaging 14.9 +/- 1.0 nm. The PHF regions of minimum width were not regular and fell into three size categories: 2.4 +/- 0.3 nm, 4.9 +/- 0.6 nm and 9.6 +/- 1.4 nm. In addition to the PHF found in the tangles, a new filament was found within all the tangles. These 2.1 +/- 0.2 nm diameter filaments were triple-stranded left helices with 1.0 +/- 0.2 nm diameter strands with a structure identical to bovine tau. Like bovine tau polymer a number of filaments (130 nm to 238 nm) were longer than a fully stretched tau monomer of 96 nm. Images of neuritic senile plaque core amyloid (SPCA) showed that amyloid had a more solid appearance than the NFT and its branched filament structures were unlike the approximately 2.1 nm diameter filaments or the PHF found in NFT.