Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation.

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin.

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.

Structural perturbation of alpha-crystallin and its chaperone-like activity.

alpha-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of alpha-crystallin towards photo-induced aggregation of gamma-crystallin, aggregation of insulin and on the refolding induced aggregation of beta- and gamma-crystallins. We observed that alpha-crystallin could prevent photo-aggregation of gamma-crystallin and this chaperone-like activity of alpha-crystallin is enhanced several fold at temperatures above 30 degrees C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or re-organized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to alpha-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.

Fibronectin enhances in vitro lipopolysaccharide priming of polymorphonuclear leukocytes.

We investigated the role of humoral factors in lipopolysaccharide (LPS) priming of polymorphonuclear leukocytes (PMN) using cells isolated from adults and from neonates. Plasma from newborn infants had decreased priming activity of adult plasma when mixed with LPS in studies measuring oxidative radical production of PMN after stimulation with a formyl bacterial oligopeptide (fMLP). This marked difference was not caused by LPS binding protein (LBP) because the LBP concentration in newborn and adult plasma were similar (138.4 +/- 12.9 U for adults, and 126.9 +/- 12.1 U for neonates, P = .53). Therefore, we attempted to identify other plasma factors that may contribute to LPS priming of PMN. We identified an LPS priming factor for PMN that is present in plasma, heat stable (56 degrees C for 30 minutes), enhanced by heparin, and concentrated in cold precipitates of plasma. Because these properties resemble those of plasma fibronectin, we assessed the role of fibronectin in LPS priming of PMN. Although fibronectin in phosphate-buffered saline (PBS) had little effect on LPS priming of PMN, fibronectin in combination with other plasma factors appeared to play a role in LPS priming of PMN because (1) removing fibronectin from adult plasma dramatically decreased LPS priming activity from plasma (P < .005), (2) addition of fibronectin to fibronectin-depleted plasma restored its LPS plasma priming activity (P < .05), and (3) neutralizing fibronectin with antibody decreased the LPS priming activity of plasma (60.3 +/- 1.3 v 30.2 +/- 2.2, P < .01). Thus, plasma fibronectin plays a role in LPS priming of PMN in the presence of other factors in plasma.

Molten-globule state of carbonic anhydrase binds to the chaperone-like alpha-crystallin.

alpha-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of alpha-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60 degrees C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn2+, by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. alpha-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the alpha-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins.

Deficient tumor necrosis factor secretion by cord blood mononuclear cells upon in vitro stimulation with Listeria monocytogenes.

We examined the production of tumor necrosis factor (TNF) by mononuclear cells (MNC) after incubating adult or cord blood MNC with Listeria monocytogenes in vitro. With adult MNC cultures, we found that TNF activity reached a peak at 6 h (606 +/- 120 x 10(3) units/liter) and declined to the baseline by day 3. In contrast, using cord blood MNC, we found that TNF activity increased gradually reaching a peak at 24 h. In addition, the peak TNF activity using newborn MNC (189 +/- 26 x 10(3) U/liter) at 24 h was still lower than the peak using adult MNC at 6 h (p < 0.0002). In seeking an explanation for the decreased TNF secretion from newborn MNC, we examined the possibility that newborn cells produce TNF but failed to secrete it. However, lysates of newborn cells contained functionally and antigenically less TNF than adult cells. Based on these observations, we conclude that the overall TNF production by newborn cells incubated with L monocytogenes is decreased compared with similarly stimulated adult cells.

Finite time analysis of the pursuit algorithm for learning automata.

The problem of analyzing the finite time behavior of learning automata is considered. This problem involves the finite time analysis of the learning algorithm used by the learning automaton and is important in determining the rate of convergence of the automaton. In this paper, a general framework for analyzing the finite time behavior of the automaton learning algorithms is proposed. Using this framework, the finite time analysis of the Pursuit Algorithm is presented. We have considered both continuous and discretized forms of the pursuit algorithm. Based on the results of the analysis, we compare the rates of convergence of these two versions of the pursuit algorithm. At the end of the paper, we also compare our framework with that of Probably Approximately Correct (PAC) learning.

Diminished priming of neonatal polymorphonuclear leukocytes by lipopolysaccharide is associated with reduced CD14 expression.

Previous research in our laboratory has shown that polymorphonuclear leukocytes (PMN) from neonates are not primed effectively in vitro with lipopolysaccharide (LPS) (from Escherichia coli 0111:B4) compared with priming of adult PMN. This finding led us to speculate that differences between neonatal and adult LPS receptors may account for the lower response by neonatal PMN to LPS. In these experiments, we investigated if CD14 or other LPS receptors contribute to the priming activity of PMN by LPS. We found that unprimed neonatal and adult PMN expressed similar numbers of CD14 (11.6 +/- 9.2 versus 18.6 +/- 2.7 fluorescence units [FlU]; P > 0.05) and LPS-binding sites (2.94 +/- 1.4 versus 4.94 +/- 0.79 FlU; P > 0.05). Monoclonal antibody against CD14 (MY4) did not significantly change the binding of LPS to adult unprimed PMN, suggesting that LPS receptors other than CD14 receptors are predominant on PMN. However, when PMN were pretreated with LPS (10 ng/ml) for 45 min at 37 degrees C, expression of CD14 on adult PMN increased to 33.8 +/- 4.9 FlU (P < 0.05 versus unprimed adult PMN) while that on neonatal PMN showed little change, increasing to 17.2 +/- 10.3 FlU (P > 0.05 versus unprimed neonatal PMN; P < 0.05 versus primed adult PMN). Furthermore, MY4 specifically blocked oxidative-radical production from PMN primed with LPS (10 ng/ml) compared with that from control PMN (P < 0.01). These studies suggest that LPS primes PMN by activating CD14 expression. We conclude that lower expression of CD14 or failure to up-regulate CD14 after LPS pretreatment contributes to the inability of neonatal PMN to be primed by LPS.

Deficient priming activity of newborn cord blood-derived polymorphonuclear neutrophilic granulocytes with lipopolysaccharide and tumor necrosis factor-alpha triggered with formyl-methionyl-leucyl-phenylalanine.

Newborn infants are more susceptible to bacterial infections than adults. This susceptibility has been attributed to defects in humoral and cellular activity. Host cellular activity can be modified by factors produced by bacteria or the host in response to infection. We assessed the effect of two factors associated with gram-negative bacterial infection, lipopolysaccharide (LPS) and TNF-alpha, on polymorphonuclear neutrophilic granulocytes (PMN) obtained from adult or newborns (umbilical cord blood). PMN were primed in vitro with LPS (10 micrograms/L) or TNF-alpha (10(-9) M) for 45 min and then assessed, using a chemiluminescence (CL) assay as an indicator of oxidative radical production with formyl-methionyl-leucyl-phenylalanine as the trigger for CL initiation. CL activity of unprimed PMN was similar for adults and newborns (13.3 and 13.7 CL units, respectively). After priming with LPS, CL activity was increased to 43.4 CL units for PMN from adults but to only 17.6 CL units for PMN from newborns (p < 0.001, adults versus newborn increment). Priming of PMN with LPS was most effective when autologous plasma was present. Using FITC-conjugated LPS and a flow cytometry assay, we could demonstrate no difference between the binding affinity of LPS for adult and newborn PMN. However, formyl-methionyl-leucyl-phenylalanine binding studies indicated that adult PMN had a higher number of binding sites. TNF-alpha priming of newborn PMN was also ineffective. Adult PMN increased CL activity by 3.9-fold when primed with TNF-alpha, whereas newborn PMN increased by only 1.75-fold (p < 0.005). This priming deficiency was not attributable to TNF-alpha receptors because phycoerythrin-conjugated TNF-alpha was associated with PMN from adults and newborns equally. Thus, PMN from newborns are not primed effectively in vitro with LPS or TNF-alpha. This defect may contribute to neonatal susceptibility to bacterial infection.

Production of interleukin 8 (IL-8) by cord blood mononuclear cells induced by Listeria monocytogenes.

The defective ability of human newborns to mobilize phagocytes to the site of infection led us to examine the ability of cord blood mononuclear cells to secrete interleukin-8, a major neutrophil chemotactic factor, in response to stimulation with Listeria monocytogenes. Adult or cord blood mononuclear cells were incubated with L. monocytogenes for varying lengths of time, and IL-8 was measured in the culture supernatants by the enzyme-linked immunosorbent assay (ELISA). Spontaneous IL-8 secretion by unstimulated cells was undetectable or at the minimal detection limit of the assay. By 24 h of cell incubation with L. monocytogenes, newborn cells produced as much IL-8 as adult cells did (300 +/- 113 versus 269 +/- 189 ng/ml, respectively). Over the next 2-4 days, IL-8 output by adult cells was slightly higher than that by newborn cells, but the difference was not statistically significant. The in vitro results suggested that newborns are as able as adults to produce IL-8, although they are defective in mobilizing neutrophils, the IL-8 target cells, to the site of infection.

Role of tumor necrosis factor-alpha and interferon-gamma in newborn host defense against Listeria monocytogenes infection.

The fetus and newborn are particularly susceptible to Listeria monocytogenes infection. We used a newborn rat animal model to investigate neonatal host defense against Listeria. In this animal model, newborn (3-d-old) rats are more susceptible to L. monocytogenes than older animals. Juvenile (23-d-old) L. monocytogenes-infected rats pretreated with lipopolysaccharide (LPS) had a lower bacterial load in blood than control animals, whereas LPS pretreated newborn rats had a higher bacterial load. Because LPS is a potent inducer of tumor necrosis factor (TNF) and TNF enhances host defense against this organism in adult animals, we assessed TNF content in splenic homogenates for animals of different ages. The age at which TNF was detectable in L. monocytogenes-Infected rats corresponded to the age at which LPS became active in preventing severe bacteremia. TNF was less than 1 unit/mL in splenic homogenates taken from rats less than 8 d of age, whereas 16-d-old rats infected with L. monocytogenes 1 d earlier had greater than 80 units/mL (p less than 0.0001 for 3-d-old versus 16-d-old rats). We also assessed the responsiveness of rats to exogenous TNF-alpha. Juvenile rats pretreated with TNF-alpha before L. monocytogenes infection had decreased bacterial load in spleen (p less than 0.02 versus controls) and better survival at 7 d (p less than 0.05 versus controls), whereas newborn rats did not improve with TNF-alpha pretreatment (p greater than 0.05 treated versus controls for splenic bacterial load and 7-d survival).(ABSTRACT TRUNCATED AT 250 WORDS)

The cellular basis for differential lymphokine responses to mitogen stimulation.

Human mononuclear cells from some individuals produce macrophage migration inhibition factor (MIF) when stimulated with Con A while those of others produce migration stimulation factor (MStF). T cells were responsible for these different responses but T4 cells produced MIF and T8 cells produced MStF regardless of the global response which was not explained by the individual T4:T8 ratios. Admixing the T-cell subpopulations in vitro revealed that MIF responses switched to MStF responses between T4:T8 ratios of 75:25 and 50:50 with MStF responders switching at higher ratios than MIF responders. Pulse exposure to supernatants from Con A-stimulated T4-enriched cells significantly reduced migration indices resulting from stimulation of fresh cells, promoting MIF responses regardless of the responder status of the supernatant donor. In contrast, supernatants from T8-enriched cells, when obtained from MStF responders, significantly increased migration indices while there was no effect when the supernatants were obtained from MIF responders. These results suggest that soluble factors from T8 cells are primarily responsible for determining whether an individual mounts a MIF or MStF response to Con A stimulation.

Lymphokine responses to concanavalin A stimulation: association with HLA DR antigens.

Stimulation of human lymphocytes with concanavalin A (Con A) resulted in variable lymphokine responses as indicated by factors inhibiting macrophage migration (MIF) or stimulating macrophage migration (MStF), or resulted in negligible responses. These responses were consistent for a given individual when repeated after several months. MIF responses were observed more frequently than MStF responses in patients with renal failure who had demonstrable alloantibodies. MStF responses were statistically associated with the presence of HLA DR1 antigens in patients with renal failure and two separate groups of healthy individuals, while MIF responses were associated with DR7 in the three groups studied. There was no correlation between immunoglobulin allotypes and lymphokine responses. These results suggest that lymphokine responses to Con A are indicators of nonspecific immunological responsiveness and are influenced by genes associated with the major histocompatibility complex.

Lymphokine responses to mitogenic and antigenic stimulation. Predictive value in renal transplantation.

Immune responsiveness of recipients of renal transplants from cadaver donors was monitored by measuring lymphokine responses to specific (donor antigen) and nonspecific (concanavalin A (Con A)) stimuli. Production of migration inhibition factor (MIF) in response to stimulation by Con A before and 3 days after transplantation correlated strongly with graft rejection and production of migration stimulation factor (MSTF) with graft acceptance. This pattern continued at later time intervals but was more variable. An initial vigorous MIF response to donor cell extract was associated with rejection but subsequent responses to donor extract in the 1st month after transplantation were variable and not predictive of the outcome. In some cases, there was clearly a dissociation of lymphokine responses to donor-specific and nonspecific stimuli.

Macrophage migration inhibition factor (MIF): reducing the variables.

While the phenomenon of macrophage migration inhibition is a useful indicator of lymphokine release, it may be caused by other substances, it is subject to considerable variability, and it may be masked by the concomitant presence of substances stimulating migration. We have investigated certain aspects of the lymphokine macrophage interaction in order to circumvent these problems. Cells from the murine macrophage cell line RAW 264-7 migrated with less variability than fresh guinea pig peritoneal macrophages and were more sensitive target cells for human macrophage migration inhibition factor (MIF). Assays were performed with serum-free and endotoxin-free medium and in all cases in the presence and absence of L-fucose. This added specificity to the assay in that biological MIF activity was invariably blocked by L-fucose whereas migration inhibition produced by antigen complexed antibody, endotoxin, and periodate was not affected by L-fucose. It was also possible to demonstrate MIF activity in mixtures of MIF and migration stimulation factor by using L-fucose. We suggest that MIF activity is determined with less variability by using a macrophage cell line as indicator cells and performing the assays in the presence and absence of L-fucose.