Biomedical subjects

R D Mullins

Publications and source records attributed to R D Mullins.

23 records · Page 2Linked to original sources

PubMed1997

Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba.

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.

Acanthamoeba↗

PubMed1996

Actin' like actin?

The most biologically significant property of actin is its ability to self-associate and form two-stranded polymeric microfilaments. In living cells, these micro filaments form the actin cytoskeleton, essential for maintenance of the shape, passive mechanical properties and active motility of eukaryotic cells. Recently discovered actin-related proteins (ARPs) appear to share a common ancestor with conventional actin. At present, six classes of ARPs have been discovered, three of which have representatives in diverse species across eukaryotic phyla and may share functional characteristics with conventional actin. The three most ubiquitous ARPs are predicted to share a common core structure with actin and contain all the residues required for ATP binding. Surface residues involved in protein protein interactions, however, have diverged. Models of these proteins based on the atomic structure of actin provide some clues about how ARPs interact with each other, with conventional actin and with conventional actin-binding proteins.

Journal Article↗

PubMed1993

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths.

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (microT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4-100 Hz and magnetic flux density range of 0.005-0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4 degrees C by constant perfusion of warmed culture medium through the sample chamber.

Cells, Cultured↗

PubMed1993

Altered calcium regulation in SV40-transformed Swiss 3T3 fibroblasts.

Calcium homeostasis has long been thought to be altered in transformed cells but mechanisms have not been established. In this study, the photoprotein, aequorin, was used to examine calcium regulation in 3T3 and SV40-transformed 3T3 cells. It was found that calcium transients induced by bradykinin or serum in serum-starved cells are lower and delayed in the transformed cells and decay kinetics are altered. These changes are not related to differences in cell cycle distribution. Though the serum transient is insensitive to nifedipine, verapamil, or lanthanum, removal of extracellular calcium accelerates transient decay in both cell types. Treatment of unstimulated cells with the ER Ca(2+)-ATPase inhibitor, thapsigargin, causes a 4-5-fold greater increase in [Ca2+]i in the transformed than in the nontransformed cells. Following serum stimulation, transformed cells still exhibit a large thapsigargin-induced increase in [Ca2+]i whereas the response in nontransformed cells is nearly abolished. When the 3T3 or SV3T3 cells are exposed to serum or thapsigargin in the absence of extracellular calcium and subsequently exposed to 11.8 mM Ca2+, a much greater influx of calcium again occurs in the SV3T3 cells. The observed changes in SV3T3 cells are most likely due to an alteration in a capacitative mechanism which regulates influx of calcium through the plasma membrane.

3T3 Cells↗

PubMedDate not supplied

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts.

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.

Adult↗