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Biomedical subjects

M A Gee

Publications and source records attributed to M A Gee.

11 recordsLinked to original sources
PubMed2000

Distinct but overlapping sites within the cytoplasmic dynein heavy chain for dimerization and for intermediate chain and light intermediate chain binding.

Cytoplasmic dynein is a molecular motor complex consisting of four major classes of polypeptide: the catalytic heavy chains (HC), intermediate chains (IC), light intermediate chains (LIC), and light chains (LC). Previous studies have reported that the ICs bind near the N terminus of the HCs, which is thought to correspond to the base of the dynein complex. In this study, we co-overexpressed cytoplasmic dynein subunits in COS-7 cells to map HC binding sites for the ICs and LICs, as well as HC dimerization. We have found that the LICs bind directly to the N terminus of the HC, adjacent to and overlapping with the IC binding site, consistent with a role for the LICs in cargo binding. Mutation of the LIC P-loop had no detectable effect on HC binding. We detected no direct interaction between the ICs and LICs. Using triple overexpression of HC, IC and LIC, we found that both IC and LIC are present in the same complexes, a result verified by anti-IC immunoprecipitation of endogenous complexes and immunoblotting. Our results indicate that the LICs and ICs must be located on independent surfaces of cytoplasmic dynein to allow each to interact with other proteins without steric interference.

Animals↗
PubMed1998

Make room for dynein.

Three classes of cytoskeletal motor protein have been identified--myosins, kinesins and dyneins. Together, these proteins are now thought to be responsible for the remarkable variety of movements that occur in eukaryotic cells and that are essential for reproduction and survival. Crystallographic analysis of the myosin and kinesin motor domains at atomic resolution has provided insight into their mechanism of force production. However, because of its relative intractability to molecular manipulation, definition of the dynein motor domain, let alone progress in understanding how it works, has been slower. Evidence now indicates that the microtubule-binding domain of dynein is spatially isolated from the ATPase domain at the tip of a projecting coiled coil. As proposed here, this curious arrangement might serve to accommodate multiple copies of the outsized and functionally complex motor heads on the microtubule surface.

Animals↗
PubMed1997

An extended microtubule-binding structure within the dynein motor domain.

Flagellar dynein was discovered over 30 years ago as the first motor protein capable of generating force along microtubules. A cytoplasmic form of dynein has also been identified which is involved in mitosis and a wide range of other intracellular movements. Rapid progress has been made on understanding the mechanism of force production by kinesins and myosins. In contrast, progress in understanding the dyneins has been limited by their great size (relative molecular mass 1,000K-2,000K) and subunit complexity. We now report evidence that the entire carboxy-terminal two-thirds of the 532K force-producing heavy chain subunit is required for ATP-binding activity. We further identify a microtubule-binding domain, which, surprisingly, lies well downstream of the entire ATPase region and is predicted to form a hairpin-like stalk. Direct ultrastructural analysis of a recombinant fragment confirms this model, and suggests that the mechanism for dynein force production differs substantially from that of other motor proteins.

Animals↗
PubMed1996

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

Amino Acid Sequence↗
PubMed1992

Using the discharge process as one method to capture adverse drug reaction reports.

Adverse drug reaction reporting programs must be constantly improved to remain successful. Using a combination of techniques that foster voluntary reporting from all disciplines--while providing routine feedback to the reporters--creates an environment that promotes better patient care through knowledge and education. The success of the ADR program at Mid-Maine Medical Center is based on this philosophy, and the program is really a system of programs. Capturing ADR reports through the discharge process, which is the cornerstone of the program, has proven very successful.

Adverse Drug Reaction Reporting Systems↗
PubMed1991

Tissue-specific and organ-specific expression of soybean auxin-responsive transcripts GH3 and SAURs.

We used in situ hybridization to localize two classes of auxin-regulated transcripts, GH3 and SAURs, within organs and tissues of soybean seedlings and flowers. GH3 transcripts occurred in the inner cortex and protoxylem ridges of roots and were expressed transiently during flower and pod development. SAUR transcripts were expressed in the epidermis, cortex, and starch sheath of epicotyls and immature hypocotyls. SAUR transcripts became more abundant on the bottom side of hypocotyls that were undergoing gravitropic curvature. SAURs were also expressed in developing xylem elements of the hypocotyl hook. When soybean organ sections were treated with 50 micromolar 2,4-dichlorophenoxyacetic acid (2,4-D), GH3 transcripts became more abundant in the vascular regions of all organs analyzed. High levels of GH3 transcripts were also found in developing palisade mesophyll cells of leaves, cotyledons, and flowers treated with 2,4-D. SAUR transcripts became more abundant in the epidermis, cortex, starch sheath, and pith of epicotyls and hypocotyls after 2,4-D treatment. Our results showed that a variety of tissues and cell types express auxin-responsive transcripts and that different tissues respond rapidly to exogenous auxin by expressing different hormone-responsive genes.

Gene Expression Regulation↗
PubMed1990

Induction and superinduction of auxin-responsive mRNAs with auxin and protein synthesis inhibitors.

We have identified a class of small mRNAs (approximately 0.5 kilobases), referred to as small auxin-up RNAs (SAURs), that increases in abundance within minutes after auxin application to excised elongating hypocotyl sections of soybean. In this study, we present evidence that SAURs accumulate in the absence of auxin when protein synthesis is inhibited. Superinduction of SAURs occurs if the synthetic auxin 2,4-dichlorophenoxyacetic acid is added under conditions where protein synthesis is inhibited. Transcription run-on experiments with isolated nuclei show that, unlike 2,4-dichlorophenoxyacetic acid, protein synthesis inhibitors do not activate transcription of the SAUR genes. These results suggest that protein synthesis inhibitors act by stabilizing SAURs and that some labile protein(s) are involved in the rapid turnover of SAURs. This stabilization is not observed with GH3, another auxin-inducible mRNA. Whether induced with 2,4-dichlorophenoxyacetic acid or cycloheximide, SAURs are primarily expressed in epidermal and cortical cells of elongating hypocotyl sections, with little or no expression in vascular tissue. Unlike 2,4-dichlorophenoxyacetic acid-induced SAUR accumulation, the increase in abundance of SAURs induced by cycloheximide is transient, with a peak approximately 1 h after inhibitor addition. Complete inhibition of protein synthesis is not required for SAUR accumulation in the presence of protein synthesis inhibitors.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1989

Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.

We have characterized a soybean gene cluster that encodes a group of auxin-regulated RNAs (small auxin up RNAs). DNA sequencing of a portion of the locus reveals five homologous genes, spaced at intervals of about 1.25 kilobases and transcribed in alternate directions. At least three of the genes are transcriptionally regulated by auxin. An increase in the rate of transcription is detected 10 min after application of auxin to soybean elongating hypocotyl sections. Each of the genes contains an open reading frame that could encode a protein of 9 kilodaltons to 10.5 kilodaltons. Sequence comparisons among the five genes reveal several areas of high homology. Two regions of high homology begin about 250 base pairs upstream of the open reading frames and two regions of homology have been identified in sequences downstream of the open reading frames. One of the latter sequences occurs in the 3'-untranslated region of the RNAs. The other occurs far downstream, 618 base pairs to 741 base pairs from the stop codon. Conservation of these sequences among the five different genes suggests that they may be important for the regulation of expression of the genes.

Amino Acid Sequence↗
PubMed1988

Evaluation of computer-assisted instruction versus traditional new-employee training.

Personnel time and costs associated with computer-assisted instruction (CAI) and traditional "one-on-one" instruction in basic computer drug-order entry procedures were compared. Both programs consisted of an introduction to the training session, familiarization of users with the computer hardware and keyboard, and step-by-step instructions in unit dose and intravenous admixture order-entry procedures. The CAI module was developed by a pharmacist with no prior programming experience. It was designed to be used side-by-side with the actual inpatient pharmacy computer system. Fifth-year pharmacy students in their clinical clerkship rotation served as the study population. The personnel (both subject and pharmacy supervisor) time and costs associated with each method were evaluated for significant differences by a two-tailed Student's t test. Of 21 subjects who participated in this study, 11 received CAI and 10 received traditional instruction. CAI was associated with significantly less mean total personnel time (99.64 +/- 18.78 minutes) than one-on-one instruction (133.60 +/- 22.89 minutes). Mean total salary expense was significantly lower for CAI ($13.03 +/- 2.15) than for one-on-one instruction ($28.79 +/- 4.91). Program development time for the CAI module was 18.5 hours longer than that for the one-on-one program. The CAI module cost $1577.77 more to develop than the one-on-one program; however, 24 new employees would be required to offset development costs if the hardware were already available and 100 new employees if hardware had to be purchased. Computer-assisted instruction of new employees in computer order-entry procedures can be a cost-effective alternative to traditional training methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer User Training↗