Patricia R. Smith

Anomalous Diffusion of Major Histocompatibility Complex Class I Molecules on HeLa Cells Determined by Single Particle Tracking

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

Blood residues on ancient tool surfaces: A cautionary note

Abstract Evidence is reviewed which suggests that proteins in general, and haemoglobins in particular, are unlikely to survive burial for many centuries in their native homogeneous state required for crystallization or for identification of species by isoelectric point determination. Crystal morphology is, in any case, an ambiguous way to determine the species of origin.

Detection of haemoglobin in human skeletal remains by ELISA

Abstract An ELISA, developed in order to detect and quantitate haemoglobin in skeletal remains from Roman and Medieval cemeteries, is described. It is important to establish that the assay detects only haemoglobin and not other bone proteins, therefore several techniques were used to establish the specificity of the antibody used in the ELISA. It is shown that the sensitivity of the ELISA is affected by the degraded nature of the haemoglobin. The findings that more haemoglobin is detected in the Roman remains than in the Medieval remains is discussed and explanations are proffered. The possible future applications of the techniques used in this study are discussed, with particular reference to the detection of antigens associated with disease in ancient human remains.

Mobility of cell surface receptors: a re-evaluation

It has long been known from fluorescence recovery after photobleaching experiments that the mobility of most cell surface receptors is much smaller than expected for free diffusion of proteins in a fluid lipid bilayer. Single‐particle tracking experiments are currently revealing the complexity of the constraints to free diffusion. Evidence has been obtained for several different processes: domain‐limited diffusion, temporary confinement and anomalous diffusion. The type of motion exhibited by a given receptor will profoundly influence the rate of any functional process which requires movement in the plane of the membrane. In particular, anomalous diffusion greatly reduces the distance travelled by a receptor on a time scale of minutes.

Biotechniques: Transfection of cells by immunoporation

Cell transfection is now a central technique in molecular biology and an essential prerequisite for gene therapy. Here we describe how beads coated with antibodies and bound to specific cell-surface transmembrane proteins can create holes in cells when the beads are removed, allowing transfection of the cells with DNA or other macromolecules. This unique targeted transfection of cells by immunoporation is very efficient and results in minimal cell death.

HLA-DR Genotyping and Mitochondrial DNA Analysis Reveal the Presence of Family Burials in a Fourth Century Romano-British Christian Cemetery

In Colchester, Britains oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the “vault complex” and demonstrate a continental European origin of the individuals investigated.