Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.

The nucleotide sequence of rabbit liver transferrin cDNA.

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 44.9 A, b = 57.0 A and c = 135.9 A, and diffract to beyond 2 A resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron.

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions.

Human serum transferrin (hTF) is a single-chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo-protein, binds strongly to transferrin receptors and is taken up by cells via receptor-mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant epsilon-[13C]Met-hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met-464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C-lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.

Zeta sedimentation ratio in rheumatic disease. Comparison of the zeta sedimentation ratio with the Wintrobe and Westergren sedimentation rates.

The zeta sedimentation ratios (ZSR) of 104 normal subjects and 106 rheumatology clinic patients were compared with their Westergren and Wintrobe erythrocyte sedimentation rates (ESR). The mean ZSR for normal subjects was 49.42 +/- 3.75%. The rheumatology clinic patients were separated into noninflammatory and inflammatory disease groups. The mean ZSR values for these groups were: noninflammatory, 51.60 +/- 5.33%; inflammatory, 59.43 +/- 7.32% (t = 5.5, p less than 0.001). The correlation between ZSR and ESR values was excellent. The ZSR determination has several advantages over the standard ESR in that it can be performed on blood anticoagulated with EDTA and need not be corrected for age, sex or packed cell volume. The ZSR is a rapid, reproducible test correlating well with rheumatic disease activity.

Effects of synergistic anions on the proton magnetic resonance spectra and pH titration of histidinyl side chains in the C-terminal half-molecule of ovotransferrin.

High-resolution proton magnetic resonance spectra of the C-terminal half-molecule of ovotransferrin (OTf/2C) clearly resolve the C(2)H resonances of the five histidinyl residues in the protein. Formation of the Ga(III)OTf/2C(anion) ternary complexes results in different chemical shift and titration behaviors for certain C(2)H resonances in the carbonato, oxalato, and malonato complexes. The pKa' of the imidazole group involved in a proton relay with the synergistic anion and a water of hydration appears to be anion independent. Thus the initial attack of a proton on the ternary complex appears to be at a ligand other than the anion or anion-binding imidazole group.

A pmr study of the effects of pH and anion and metal ion binding of the histidyl residues of ovotransferrin.

High resolution proton magnetic resonance studies of ovotransferrin show clear resolution of four groups of C(2)-H histidyl resonances to low field of the major aromatic envelope. Titrations of the protein in the absence and presence of synergistic anions, oxalic acid, malonic acid, and 2,6-dipicolinic acid, and anions plus metal ions reveal that six histidines are involved in the binding sites. These histidines, three in each binding site, are near to one another. In each binding site one histidine is involved in binding to anions and two are involved in binding to metal ions.

Specific membrane receptors for diferric-transferrin in cultured rat skeletal myocytes and chick-embryo cardiac myocytes.

A classical approach was used to determine whether myocyte cultures exhibited diferric-transferrin binding phenomena consistent with the presence of specific, high-affinity membrane receptors. Experiments were performed using a continuous cell line, designated L-6, derived from rat skeletal muscle, as well as primary cultures of chick-embryo cardiac myocytes. Rat transferrin isolated from pooled serum was used in experiments involving L-6 cells, and ovotransferrin isolated from hen's egg white was used with the chick-embryo cardiac myocytes. The data from equilibrium binding experiments, corrected for nonspecific binding, and analyzed by Scatchard analysis indicated that there were approximately 2 x 10(5) transferrin receptors per L-6 myocyte and 2 x 10(4) ovotransferrin receptors per cardiac myocyte present, under the conditions used for the equilibrium binding experiments. Whereas the L-6 myocytes grew exponentially under the assay conditions, the cardiac-myocyte cultures were in a non-dividing state. It is thought that the differences in receptor number per cell reflect changes arising form the differing ion demand made by the cells, under these two growth conditions. It is clear that myocytes acquire iron from diferric (ovo)transferrin in a process that involves high-affinity, specific binding to membrane receptors.

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.

Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin.

Seven high affinity antibodies to human serum transferrin which recognize at least four different epitopes are described. Apparent dissociation constants (Kd's) have been determined for the binding of the antibodies to human transferrin in the presence and absence of iron. Small differences in reactivity were found. Five of the antibodies bind to the isolated amino-terminal half-molecule of human transferrin. Two of the antibodies appear to be to the C-terminal lobe since they bind to holo-transferrin but do not recognize the N-terminal half-molecule. Immunoblotting shows that six of the antibodies recognize both reduced and nonreduced transferrin. In addition, all of the antibodies bind with sufficiently high avidity to transferrin to make them useful as probes in studies in which binding of transferrin to the specific transferrin receptor is examined.

Factors associated with the vaccination of international travelers: a multivariate analysis.

Travel medicine practice in the United States has not been extensively studied. This study included 1078 consecutive patients who presented to a university-based travel medicine clinic from 1990 through 1994. Analyses of patient demographics, clinic attendance, itineraries, and vaccinations were conducted. Mean patient age (+/- SD) was 37.4 +/- 16.2 years; 626 (58.1%) of the patients were male. Travel duration was 103.1 +/- 242.3 days (median, 21 days), and lead time (defined as the time between clinic presentation and departure for the purpose of this study) was 23.8 +/- 26.5 days (median, 16 days). Destination was the strongest independent factor affecting vaccination practices. A lead time of 31 or more days was associated with significantly elevated odds ratios for all immunobiologicals except immune globulin. These findings underscore the need to educate the traveling public, healthcare providers, and the travel industry about the benefits of seeking medical consultation at least 1 month prior to international travel.

Projected incidence of cardiovascular disease in male firefighters based on current risk factor prevalence.

The projected incidence of cardiovascular disease (CVD) in male firefighters was determined by the prevalence of current CVD risk factors and the use of the Framingham Study general cardiovascular risk profile in a probability sample of firefighters from two municipal fire departments. Hypercholesterolemia (60.9%) and obesity (56.0%) were the most prevalent risk factors. Significant age-related trends were observed for the prevalence of all CVD risk factors, except glucose intolerance (P = .21) and an abnormal resting electrocardiogram (P = .07). The projected incidence of CVD in firefighters did not differ from that of the general male population (relative risk, 1.0; 95% confidence interval, 0.7 to 1.4); similar risk estimates were observed in age-specific analyses. These findings are in accord with previous incidence and mortality studies that used circulatory diseases as an end point. The present method should be viewed primarily as a hypothesis-generating tool because of its limitations in assessing cause and effect.