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A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.

The in vitro effects of the water-soluble fraction of whole cigarette smoke (WSF) and two alpha, beta-unsaturated aldehydes of cigarette smoke (acrolein and crotonaldehyde) on polymorphonuclear leukocyte (PMNL) adherence were determined with nylon fiber columns. Each of these cigarette smoke constituents caused a dose-dependent inhibition of PMNL adherence. However, at least fivefold higher concentrations of these agents were necessary to inhibit adherence as compared with those necessary to achieve the same level of inhibition of PMNL chemotaxis. Furthermore, inhibition of adherence by WSF could be differentiated from its effects on chemotaxis in that reduced glutathione completely protected chemotaxis from the effects of WSF but only afforded partial protection to PMNL adherence. These data suggest that the inhibitory effects of WSF, acrolein, and crotonaldehyde on PMNL chemotaxis are not due to their inhibition of adherence. Finally, although PMNL adherence is considered to be an integral part of the chemotactic mechanism, differentiation between these two PMNL functions may be possible, since some inhibitors of chemotaxis do not have corresponding inhibitory effects on adherence.

The effect of tobacco smoke on in vitro chemotaxis of human polymorphonuclear leukocytes (PMN) was determined. Whole tobacco smoke, gas phase of smoke, and water-soluble fraction were potent inhibitors of PMN chemotaxis. The results indicated that PMN chemotaxis was inhibited in a dose-dependent manner by water-soluble fraction and that this suppression was not a result of cytotoxicity. In an attempt to determine the mechanism of chemotaxis inhibition, the effect of tobacco smoke on glucose metabolism of PMN was studied. Exposure of PMN to whole smoke, gas phase, or water-soluble fraction resulted in an increase (twofold) in glucose catabolism via both glycolysis and the hexose monophosphate shunt, with no apparent effects on the metabolism of glucose via the tricarboxylic acid cycle. These results suggest that the inhibitory effects of tobacco smoke on PMN chemotaxis were not directly attributable to effects on glucose metabolism of these cells. Further, the inhibitory effects of water-soluble fraction on PMN chemotaxis were shown to be largely irreversible and preventable in the presence of cysteine. Thus, the major inhibitory effects of tobacco smoke probably result from the direct action of oxidants and/or thiol-reactive substances on PMN.

Lung infections are a common cause of morbidity and mortality in neonates. To evaluate neonatal lung defenses against pneumococci, we challenged rats with aerosols of encapsulated pneumococci in an airborne infection apparatus. Whereas adult rats cleared greater than 95% of inhaled type 1 or type 25 pneumococci within 4 h, pneumococci proliferated in the lungs of newborn rats and reached 200-600% of the baseline value by 4 h and 1000-1700% by 24 h. As neonatal rats matured, their ability to clear inhaled pneumococci improved, but compared with adults some impairment in clearance was present until approximately 4 wk of age. Newborn rats had significantly fewer resident alveolar macrophages per g of lung tissue than did adults (p less than 0.001). Although the number of resident macrophages increased with time, a significant deficit in alveolar macrophages persisted for the first 3 wk of life (p less than 0.01). Aerosols of pneumococci caused an influx of granulocytes into the lungs of adult rats within 4 h, compared with 24 h for neonatal rats. Even at 24 h after pneumococcal challenge, newborn rats had significantly fewer granulocytes per g of lung tissue (p less than 0.05) than did adults, although 7-day-old rats had reached an adult level by this time. Significant (p less than 0.05) increases in granulocyte chemotactic activity were observed in lavage fluids of adult, but not newborn, rats after pneumococcal challenge. Thus, impaired clearance of pneumococcal aerosols by neonatal rats was associated with an age-dependent deficiency in numbers of resident alveolar macrophages and impaired generation of chemotactic activity and recruitment of granulocytes to the lung.

Some ciliostatic components of cigarette smoke were studied as inhibitors of in vitro chemotaxis of human polymorphonuclear leukocytes (PMNs). In comparison to their concentration in an inhibitory level of cigarette smoke, the unsaturated aldehydes acrolein and crotonaldehyde were the most potent inhibitors, whereas nicotine, cyanide, acetaldehyde, and furfural were the next strongest inhibitors. In contrast, sulfide, propionaldehyde, butyraldehyde, and the phenols (phenol and o-, m-, and p-cresol) were relatively weak inhibitors of PMN chemotaxis. Acrolein and crotonaldehyde mimicked whole cigarette smoke in their effects on PMNs by not causing loss of PMN viability, yet their effects were prevented by the addition of cysteine. On the other hand, addition of nicotine, cyanide, acetaldehyde, and furfural to PMN suspensions resulted in a limited loss of cellular viabilities, and their effects on PMNs were not prevented by cysteine. Of the tested components, only cyanide significantly altered PMN glucose metabolism by increasing carbon flow via the glycolytic and hexose monophosphate pathways in a manner similar to that observed with whole cigarette smoke. The results of this study suggest that the unsaturated aldehydes, including acrolein and crotonaldehyde, are major contributors to the inhibitory properties of cigarette smoke. The inhibitory effects of these unsaturated aldehydes are probably due to a direct interaction of these oxidants and/or thiol-alkylating agents with PMNs, yet the glucose metabolism of these cells is unaffected. One interpretation of these data is that PMN chemotaxis is dependent upon particular cellular proteins containing one or more essential thiol group(s) but that these proteins are unrelated to glucose metabolism.

The effects of cigarette smoking fractions on polymorphonuclear leukocyte (PMN) chemotaxis were determined using the 51Cr-assay. Water-soluble fractions (WSF) of cigarette smoke produced from several tobacco types differed in inhibitory potencies (i.e., flue cured greater than or equal to Maryland greater than or equal to blended greater than Burley greater than or equal to Turkish) corresponding to the respective unsaturated aldehyde content of the smoke from these tobaccos. Fractionation of cigarette smoke condensate (CSC) demonstrated that the more polar fractions were potent inhibitors of chemotaxis whereas those containing nicotine and the polycyclic hydrocarbons were weak inhibitors of chemotaxis. Unlike the inhibitory effects of WSFs, CSC fractions did not inhibit random migration and their inhibition of chemotaxis could not be completely prevented by reduced glutathione. These data suggest that while the alpha, beta-unsaturated aldehydes present in the vapor phase of smoke are among the most potent inhibitors of in vitro PMN chemotaxis, other polar, nonvolatile constituents of cigarette smoke also inhibit chemotaxis and by a mechanism which differs from that of the unsaturated aldehydes.