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P T Tran

Publications and source records attributed to P T Tran.

8 recordsLinked to original sources
PubMed1998

Birefringence of single and bundled microtubules.

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.

Animals↗
PubMed1997

A metastable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends.

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931-937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

Animals↗
PubMed1997

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28.

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3' end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.

Amino Acid Sequence↗
PubMed1997

How tubulin subunits are lost from the shortening ends of microtubules.

Microtubules exhibit dynamic instability, switching between persistent states of growth and shortening at their ends. The switch between growth and shortening has been proposed to depend on end conformation where growing ends have "straight" tubulin protofilaments stabilized by a terminal cap of GTP-tubulin, while-shortening ends have lost their GTP-tubulin cap, allowing terminal GDP-tubulin dimers to curve inside-out and peel rapidly away from the microtubule lattice. This "conformational cap" model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bonds between adjacent curved dimers. We have tested this model for the plus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Individual microtubule ends were imaged using video-enhanced differential interference contrast light microscopy. The rate of rapid shortening was systematically increased by isothermal dilution into assembly buffer containing various concentrations of Mg2+ or Ca2+ ions. At 1 mM Mg2+ and no Ca2+, shortening occurred at 20 (plus) and 45 (minus) microns/min. The ends appeared similar in contrast to growing ends and the core of the microtubule and the ends appeared blunt or slightly frayed by negative stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, microtubule shortening occurred at 60 (plus) and 115 (minus) microns/min or faster and "knobs" were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained constant during many micrometers of rapid shortening. Negative stain electron microscopy revealed that these knobs were "blossoms" of inside-out curved protofilaments, some extending for several helical turns (30 to 60 dimers in length) at constant curvature from the ends. At these high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylinder, suggesting that dimer curling and protofilament peeling is constrained to the tip by interactions between adjacent straight protofilaments. Depolymerization is produced by conformational changes in GDP-tubulin since microtubules assembled with a slowly hydrolizable analog of GTP, GMPCPP, are stable even at 20 mM Mg2+ or 5 mM Ca2+. Monte Carlo simulations show that the ratio of the peeling to breakage rate constants can control the steady-state average length of curved GDP-tubulin protofilaments at the depolymerizing end.

Animals↗
PubMed1996

Routine neonatal circumcision?

Routine neonatal circumcision is still a controversial procedure. This article attempts to clarify some of the advantages and disadvantages of neonatal circumcision. The increased rate of penile cancer among uncircumcised men appears to justify the procedure, but that alone is not sufficient justification. The final decision on neonatal circumcision should be made by parents with balanced counsel from attending physicians.

Circumcision, Male↗