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J D Harper

Publications and source records attributed to J D Harper.

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Microtubule-organizing centers and nucleating sites in land plants.

Microtubule-organizing centers (MTOCs) are morphologically diverse cellular sites involved in the nucleation and organization of microtubules (MTs). These structures are synonymous with the centrosome in mammalian cells. In most land plant cells, however, no such structures are observed and some have argued that plant cells may not have MTOCs. This review summarizes a number of experimental approaches toward the elucidation of those subcellular sites involved in microtubule nucleation and organization. In lower land plants, structurally well-defined MTOCs are present, such as the blepharoplast, multilayered structure, and polar organizer. In higher plants, much of the nucleation and organization of MTs occurs on the nuclear envelope or other endomembranes, such as the plasmalemma and smooth (tubular) endoplasmic reticulum. In some instances, one endomembrane may serve as a site of nucleation whereas others serve as the site of organization. Structural and motor microtubule-associated proteins also appear to be involved in MT nucleation and organization. Immunochemical evidence indicates that at least several of the proteins found in mammalian centrosomes, gamma-tubulin, centrin, pericentrin, and polypeptides recognized by the monoclonal antibodies MPM-2, 6C6, and C9 also recognize putative lower land plant MTOCs, indicating shared mechanisms of nucleation/organization in plants and animals. The most recent data from tubulin incorporation in vivo, mutants with altered MT organization, and molecular studies indicate the potential of these research tools in investigation of MTOCs in plants.

Centromere

Observation of metastable Abeta amyloid protofibrils by atomic force microscopy.

BACKGROUND: Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the beta-amyloid protein (Abeta). As Abeta amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however. RESULTS: Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Abeta variants Abeta1-40 and Abeta1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Abeta1-42 aggregates much more rapidly than Abeta1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Abeta amyloid protofibrils. CONCLUSIONS: Abeta protofibrils are likely to be intermediates in the in vitro assembly of Abeta amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Abeta aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Abeta protofibrils may be a useful therapeutic strategy.

Alzheimer Disease

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein.

BACKGROUND: Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy. RESULTS: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed. CONCLUSIONS: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.

Alzheimer Disease

Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins.

Ordered protein aggregation in the brain is a hallmark of Alzheimer's disease and scrapie. The disease-specific amyloid fibrils comprise primarily a single protein, amyloid beta, in Alzheimer's disease, and the prion protein in scrapie. These proteins can be induced to form aggregates in vitro that are indistinguishable from brain-derived fibrils. Consequently, much effort has been invested in the development of in vitro model systems to study the details of the aggregation processes and the effects of endogenous molecules that have been implicated in disease. Selected studies of this type are reviewed herein. A simple mechanistic model has emerged for both processes that involves a nucleation-dependent polymerization. This mechanism dictates that aggregation is dependent on protein concentration and time. Furthermore, amyloid formation can be seeded by a preformed fibril. The physiological consequences of this mechanism are discussed.

Alzheimer Disease

A simple and rapid technique for the immunofluorescence confocal microscopy of intact Arabidopsis root tips.

Visualisation of immunofluorescence labelling of Arabidopsis roots has previously been limited to single cell layers. A simple, rapid method has been devised in which the whole root can be processed to allow antibody penetration into several cell layers. When optically sectioned using confocal microscopy, cellular arrangements of microtubules, callose, calmodulin and a phosphoprotein epitope have been visualised using this technique. As the root is not physically sectioned, information regarding the three-dimensional position of individual cells in relation to each other and the tissue as a whole is retained. Using this technique, we have assessed the effect of brefeldin A on the frequency of mitotic arrays in root tip cortical and epidermal cells, and found that the occurrence of phragmoplasts increases significantly with brefeldin A treatment. This study demonstrates the possible future use of the whole root technique to assess rapidly the developmental, mutational and inhibitor-induced changes in the organisation of cellular components in Arabidopsis.

Arabidopsis

A gamma-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas.

gamma-Tubulin is a putative component of microtubule initiating material. To further explore its subcellular distribution in plant and animal cells, we have raised a polyclonal antibody, Rb27, directed towards a conserved region (EEFATEGTDRKDVFFY) of the gamma-tubulin molecule. Immunoblotting of cell protein extracts with Rb27 reveals a polypeptide band of M(r) 49 kD in HeLa and a 58 kD band in Chlamydomonas. Although these polypeptides are comparable in size to forms of gamma-tubulin detected previously in mammalian and plant protein extracts by other antibodies to gamma-tubulin, by immunofluorescence microscopy Rb27 gives localization patterns not previously seen. It localizes specifically with the centrioles in HeLa cells and with the basal body complex in Chlamydomonas. Other gamma-tubulin antibodies label pericentriolar material. Because of the similarities in the size of the polypeptides recognized by our and other gamma-tubulin antibodies, and a restricted co-localization with known microtubule-organizing centres in evolutionarily distant organisms, we propose that Rb27 recognizes a novel conserved gamma-tubulin isotype.

Amino Acid Sequence

Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii.

The antiphosphoprotein monoclonal antibody MPM-2 was used to investigate protein phosphorylation during flagellar regeneration in Chlamydomonas reinhardtii. MPM-2 recognizes a phosphorylated epitope and detects several Chlamydomonas proteins by Western immunoblot analysis. Two MPM-2 reactive proteins (34 and 90 kD) increase in Western immunoblot intensity after flagellar excision and decrease in intensity during flagellar regeneration. Immunofluorescence and immunogold labeling revealed MPM-2 staining within the nucleus, especially towards the nuclear periphery, the flagellar basal apparatus, and the nucleus-basal body connector after flagellar excision. Comparison of MPM-2 reactivity in wild-type cells and in the mutant bald-2, which lacks functional basal bodies, demonstrates that the 34-kD protein is localized in the nucleus and the 90-kD protein is localized in the flagellar basal region. MPM-2 reactivity is observed in cells competent for flagellar regeneration. However, when cells were treated with the kinase inhibitor, staurosporine, MPM-2 reactivity did not increase after flagellar excision and flagellar regeneration was impaired. These observations suggest that phosphorylation of the 34- and 90-kD proteins may be important for flagellar regrowth. Possible roles for phosphorylation in flagellar regeneration include transcriptional activation and transport of flagellar precursors to the base of the growing flagella.

Animals

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.

Actins

The mitosis-specific monoclonal antibody MPM-2 recognizes phosphoproteins associated with the nuclear envelope in Chlamydomonas reinhardtii cells.

The monoclonal antibody MPM-2 recognizes a family of phosphorylated proteins present in mitotic cells. In a number of organisms it stains nuclei and also cytoskeletal structures which contain or organize tubulin. In mitotic Chlamydomonas reinhardtii cells MPM-2 reacts with phosphoproteins associated with the nuclear envelope (NE). Staining of the NE region appears in preprophase, reaches a maximum intensity in metaphase/anaphase and disappears rapidly in telophase. Localized hyperphosphorylation of the anterior NE region is apparent in many cells throughout mitosis. The distribution and timing of MPM-2 labeling suggests that in Chlamydomonas MPM-2 may be interacting with lamin-like phosphoproteins.

Antibodies, Monoclonal

Tartrazine.

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Azo Compounds

Evaluation of a multiple-variable thin-layer and reversed-phase thin-layer chromatographic scheme for identification of basic and neutral drugs in an emergency toxicology setting.

An analytical scheme composed of one normal-phase thin-layer chromatographic (TLC) method, one reversed-phase thin-layer chromatographic (RPTLC) method, and sequential analyte detection through four stages of color reactions is described. Eighty-one basic or neutral drugs were analyzed with this scheme and seventy-four were uniquely characterized with 95% confidence. Six of the remaining seven formed three unresolved pairs. The scheme was evaluated by mean list length analysis and shown to offer analyte resolution similar to that of a scheme of TLC and gas chromatography (GC) with nonspecific detection. Nine out of ten unknowns from the field of eighty-one drugs were uniquely identified with 95% confidence by the TLC/RPTLC scheme. The tenth unknown was not completely resolved from its isomer, but was statistically the more probable candidate.

Chromatography, Thin Layer