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J F Hainfeld

Publications and source records attributed to J F Hainfeld.

At least 19 recordsLinked to original sources
PubMed1998

The location of the carboxy-terminal region of gamma chains in fibrinogen and fibrin D domains.

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.

Fibrin↗
PubMed1998

Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy.

Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. By using Fab' fragments, the entire probe is smaller than a whole IgG molecule. A simple fluorescence assay was used to investigate the fluorescence properties of the new probes. They were used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy, and also for labeling leukocyte microtubules; labeling was imaged using fluorescence microscopy and, after silver enhancement, by a variety of optical methods and by electron microscopy. Combined Nanogold and Texas Red, Cy3, Lissamine Rhodamine B, and AMCA probes were also prepared, and in preliminary experiments show similar properties to the combined fluorescein and gold cluster probes. The fluorescent and gold cluster probes enable a new degree of correlation between fluorescence and electron microscopy, and may also be used to check labeling of specimens before processing for electron microscopy.

Cell Nucleus↗
PubMed1997

Paired helical filaments in corticobasal degeneration: the fine fibrillary structure with NanoVan.

Paired helical filaments (PHF) composed of hyperphosphorylated tau proteins are characteristic findings in neurodegenerative disorders, including Alzheimer's disease (AD) and corticobasal degeneration (CBD). The filaments in CBD differ from those in AD by a reduced number of tau isoforms and less stable ultrastructure. To further compare the ultrastructure of both filaments, we employed a novel staining reagent, NanoVan, as well as aurothioglucose and uranyl acetate. With commonly used uranyl acetate, both kinds of filaments appeared as twisted ribbons 15-20-nm and 21-23-nm wide, respectively, without significant internal substructure. With application of aurothioglucose, only few structural details were apparent. With NanoVan, AD filaments showed similar structure to that with uranyl acetate but CBD filaments displayed a highly heterogeneous appearance consistent with the dissociation of the 20-25-nm-wide filaments along two longitudinal axes. This was evident by the presence of thinner, 12-13-nm-wide filaments and filaments that splayed into two 20-25-nm-wide components at one or both ends. Moreover, detection of a prominent, 7-8-nm-wide axial region distinguished up to four protofilaments per one filament. Each protofilament appeared to contain two 3-5-nm-wide fibrils separated by an approximately 1-nm-wide axial region. The results suggest that 3-5-nm fibrils are the smallest structural subunits of filaments in CBD and that NanoVan may be an unique reagent in detecting eight-fibril organization in these less stable filaments.

Aged↗
PubMed1997

Binding of amyloid beta protein to the 20 S proteasome.

Neurodegenerative disorders of aging are characterized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conjugates are degraded by cellular particles known as proteasomes. We have previously shown that amyloid beta protein (Abeta) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we found that Abeta binds the 20 S proteasome and forms a proteasome-Abeta complex. The complex was detected by Western blot with anti-Abeta antibodies. Using a 1.4 nm Nanogold-labeled Abeta, we visualized proteasome-Abeta complexes by scanning transmission electron microscopy (STEM). Analysis of the side-on oriented proteasome-Abeta complexes revealed a single gold particle, corresponding to one gold-labeled Abeta, in the middle portion of the proteasome. On end-on views of proteasome-Abeta complexes, gold was detected within the area delimited by the proteasome circular projection. Both STEM views are consistent with Abeta localization inside the proteasome along the peptide channel. Direct interaction of Abeta with the inner catalytic compartment of the proteasome may explain the generation of ubiquitin-containing lesions in Alzheimer's disease and other neurodegenerative disorders. In addition, detection of Nanogold-labeled peptide inside the 20 S eukaryotic proteasome suggests that conformational constraints for protein degradation in eukaryotic proteasomes are different from those in archaebacteria proteasomes.

Amyloid beta-Peptides↗
PubMed1997

A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy.

Immunoprobes that combine a fluorescent label with a 1.4-nm gold cluster compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporation of one or more fluorescein moieties into the coordinated ligands of the 1.4-nm Nanogold gold cluster label yields a stable, dual-function immunolabel in which fluorescence quenching is negligible. In a second synthetic strategy, Nanogold and fluorescein were separately covalently conjugated to Fab' fragments to yield a probe with very similar properties. With the use of Fab' fragments, the entire probe is smaller than a whole IgG molecule, and it exhibited excellent penetration properties. It was used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy.

Fluorescein↗
PubMed1997

Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-Nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus.

The usefulness of standard in situ hybridization for viral nucleic acid detection is occasionally limited by its sensitivity limit of 10 to 50 copies per cell. A modified version of the recently described signal amplification method, catalyzed reporter deposition (CARD), and its application to formalin-fixed cells and tissue sections is presented. Deposition of the reporter is facilitated by using horseradish peroxidase catalyzing the deposition of biotinylated tyramide on the location of the probe target. The biotin accumulation created is usually detected with streptavidin-labeled enzymes or fluorochromes. In the present investigation, this step was replaced by streptavidin-Nanogold and combined with silver acetate autometallography. This resulted in deep-black precipitation at positive in situ hybridized reaction sites. The sensitivity of this new approach was tested with a biotinylated, genomic probe specific for human papillomavirus (HPV)-16/18. SiHa cells, a cervical carcinoma-derived cell line with one to two HPV16 copies per cell, and 10 histologically confirmed cervical carcinomas were used for the study. All samples were previously HPV16 positive with solution polymerase chain reaction, but only two of the cervical carcinomas were positive with standard in situ hybridization with barely visible signals. When employing CARD-Nanogold, SiHa cells and 9 of 10 biopsies proved positive with marked signals. It is concluded that this nonisotopic method can detect single viral copies in situ in routinely fixed material and may have the potential to replace in situ polymerase chain reaction in many applications.

Acetates↗
PubMed1996

Labeling with nanogold and undecagold: techniques and results.

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope.

Animals↗
PubMed1995

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

Binding Sites↗
PubMed1995

Gold, electron microscopy, and cancer therapy.

Radioactive gold has properties suitable for radiotherapy and can provide lethal irradiation to cells. If the gold is conjugated to a targeting molecule, such as an antibody, it may be possible to specifically deliver the dose to tumor cells. Various gold particles are possible candidates and include gold colloids with adsorption of antibodies or gold clusters with covalent attachment. Different sizes of gold particles are available and some may be preferred for certain situations. Problems with intravenous injection and in vivo delivery are numerous, and a more tractable application is the direct instillation into the urinary bladder of radiogold immunoconjugates to treat superficial bladder carcinoma. Preliminary studies indicate the feasibility of this approach.

Animals↗
PubMed1995

Orientation of the carboxy-terminal regions of fibrin gamma chain dimers determined from the crosslinked products formed in mixtures of fibrin, fragment D, and factor XIIIa.

There are two schools of thought regarding the orientation of the intermolecular epsilon-amino-(gamma-glutamyl) lysine isopeptide bonds formed between gamma chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains ('DD-long'), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands ('DD-transverse'). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent 'D:E' interaction between each fibrin E domain and two D fragments ('D:fibrin:D'). If carboxy-terminal gamma chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers ('D-D') will result and the fibrin 'template' will not become crosslinked to the associated D fragments. If instead, gamma chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result. SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that gamma chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that gamma chain crosslinks are also positioned transversely in an assembled fibrin polymer.

Fibrin↗
PubMed1994

Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster labels and scanning transmission electron microscopy.

This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E2E3 subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively. The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3. Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex. Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.

Escherichia coli↗
PubMed1994

Structure of phosphorylase kinase. A three-dimensional model derived from stained and unstained electron micrographs.

Phosphorylase kinase, the first protein kinase discovered, is a key regulatory enzyme in glycogen metabolism. Although its biochemical properties are well characterized, details of its three-dimensional structure and subunit topology are yet to be elucidated. This study describes four characteristic views of the hexadecameric holoenzyme (alpha 4 beta 4 gamma 4 delta 4) as observed in both negatively stained and unstained electron micrographs. The predominant views are the widely reported "butterfly" with two wing-like lobes connected by thin bridges, and the previously described "chalice", composed of "cup" and "stem" segments. Two additional views, a "cube", similar to the previously reported "tetrad", and a "cross" or "X" are less common, but illustrate the overall geometry of the particle. Based on these images, the first three-dimensional model of the enzyme has been constructed. It is composed of four identical protomers that associate with D2 symmetry to form the two major structural elements (the two lobes). Two protomers in a head to head arrangement make up each symmetrical lobe; to complete the holoenzyme, one lobe is inverted and placed perpendicular to the other. Thus, the overall structure has three 2-fold axes of symmetry, and the arrangement of the four protomers approximates a tetrahedron. Each lobe of the model corresponds to a wing of the butterfly projection. Two projections form the chalice: in the intra-lobe orientation, one lobe forms the cup and the other forms the stem, and in the inter-lobe view, one-half of each lobe contributes to each segment of the image. The cube and cross projections result from 90 degrees rotations from the butterfly orientation. In the cube, the distal portions of each lobe are projected separately. In the cross, one lobe is crossed over and is above the other. This model both accounts for and predicts all of the observed microscopic images.

Animals↗
PubMed1994

An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.

An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

Amino Acid Sequence↗
PubMed1993

Evidence for a second type of fibril branch point in fibrin polymer networks, the trimolecular junction.

Fibrin molecules polymerize to double-stranded fibrils by intermolecular end-to-middle domain pairing of complementary polymerization sites, accompanied by fibril branching to form a clot network. Mass/length measurements on scanning transmission electron microscopic images of fibrils comprising branch points showed two types of junctions. Tetramolecular junctions occur when two fibrils converge, creating a third branch with twice the mass/length of its constituents. Newly recognized trimolecular junctions have three fibril branches of equal mass/length, and occur when an extraneous fibrin molecule initiates branching in a propagating fibril by bridging across two unpaired complementary polymerization sites. When trimolecular junctions predominate, clots exhibit nearly perfect elasticity.

Blood Coagulation↗
PubMed1993

A polypeptide bound by the chaperonin groEL is localized within a central cavity.

Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state. Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter. We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters. In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings. In some cases, two gold densities were observed in the same groEL complex. We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings. In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.

Bacterial Proteins↗
PubMed1992

Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy.

A method is described to deliver 235U to tumors; the isotope would then be fissioned by incident neutrons, producing localized lethal radiation sufficient for therapy. Apoferritin was loaded with an average of approximately 800 238U atoms per molecule. Stability of the loaded apoferritin in solution was improved, so that only 8% loss of uranium occurred after 8 days at pH 7. Fab' antibody fragments were covalently attached to the uranium-loaded apoferritin, and the immunoreactivity of the conjugate was 92% of that for antibody alone. Such bio-uranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy.

Apoferritins↗
PubMed1992

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.

Animals↗