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.

The degradation of fructosamines, formed from the non-enzymic glycation of proteins under physiological conditions, to advanced glycation end products was investigated by studying the model peptide fructosamine NE-(1-deoxy-D-fructos-1-y1)hippuryl-lysine (DHL). At pH 7.4 and 37°C in aerobic phosphate buffer, DHL degraded to form N,-carboxymethyl-hippuryl-lysine, and hippuryllysine over a 29-day incubation period. The expected NE-(3-1actato)hippuryl-lysine and 'hippuryllysylpyrraline' derivatives were not found. Superoxide radicals and hydrogen peroxide were formed during the degradation of DHL but were also both consumed during the degradation reaction. Reversal of the Amadori rearrangement was not a major fate of the fructosamine.The formation of N,-carboxymethyl-hippuryl-lysine was decreased by desferrioxamine, catalase, superoxide dismutase, catalase with superoxide dismutase, anaerobic conditions and aminoguanidine. The formation of hippuryl-lysine was decreased by desferrioxamine, catalase and catalase with superoxide dismutase, but was increased by the addition of aminoguanidine. N,-Carboxymethylserine and unmodified lysine residues are major peptide-based end products in the degradation of lysyl-fructosamine under physiological conditions. Oxygen, redox-active metal ions, catalase, superoxide dismutase and the pharmacological agent aminoguanidine are expected to be influential on the rate and fate of fructosamine degradation.Monosaccharides react non-enzymically with the N-terminal amino group and lysyl-side-chain amino groups of proteins (the Maillard reaction) under physiological conditions to form glycated proteins ( Led1 and Schleicher, 1990). Proteins known to undergo this reaction include haemoglobin, immunoglobulins, serum albumin, collagen and the lens crystallins (Bunn et al., 1978;Stevens et al., 1978;Day et al., 1979;Dolhofer et al., 1985;Bailey and Kent, 1989). The aldehyde or ketone carbonyl group of the acyclic conformer of the monosaccharide reacts with the amino group to form a Schiff s base or imine. This undergoes the Amadori rearrangement to form a ketoamine (Hodge, 1955). With glucose, the ketoamine formed is a NE-( 1 -deoxy-D-fructos-1 -yl) -amino-acid residue, a fructosamine (Mauron, 1981). The rate of formation and concentration of protein fructosamines are elevated in diabetes mellitus, due to the periodic hyperglycaemia in blood plasma and tissues associated with insulin-independent glucose uptake (lens fibre cells, capillary endothelial cells, peripheral neurones and red blood cells). The concentration of fructosamine in blood plasma and the concentration of glycated haemoglobin HbA1, are monitored clinically to assess the quality of glycaemic control in diabetic patients (Willms and Lehmann, 1990;Windeler and Kobberling, Correspondence to P. J. Thornalley,

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)–DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37°C than at 22°C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor–MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

The bioactive (B) and immunoreactive (I) pituitary contents/concentrations of FSH, together with the plasma concentrations of B-FSH, I-FSH and I-inhibin were determined in ovine fetuses at days 55, 75, 90 and 135 of gestation (day 145 = term). The pituitary contents and concentrations of B-FSH and I-FSH increased in both sexes with gestational age. The female fetuses had significantly (P < 0.01) higher pituitary contents/concentrations of B-FSH and I-FSH than the male fetuses at days 75 and 135. The pituitary B/I ratios of FSH were not significantly different with age or sex. The plasma concentrations of B-FSH remained relatively constant from days 75 to 135, with no significant differences between sexes or with age. In contrast, the plasma concentrations of I-FSH reached a peak at day 90 and then declined towards term in both sexes. At all gestational ages except day 55, the female fetuses had significantly (P < 0.05) higher plasma concentrations of I-FSH than the males. In both sexes, the plasma B/I ratios of FSH were lowest at day 90 and had increased again by day 135, with the male fetuses having significantly (P < 0.05) higher B/I ratios compared with the female group at days 75 and 135 but not at day 90. At all gestational ages, the plasma concentrations of I-inhibin declined throughout gestation in the female fetuses, whereas in the males they reached a nadir at day 75 and then increased towards term. The concentrations of I-inhibin were significantly (P < 0.01) higher in the male fetuses compared with the females.(ABSTRACT TRUNCATED AT 250 WORDS)

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.z 1998 Federation of European Biochemical Societies.