Preparation and antitumor activity of nontoxic lipid A

Summary Highly refined, disaggregated endotoxic glycolipids (B5) from heptose-less (Re) mutant Salmonella typhimurium quantitatively converted to nontoxic (lethality for chick embryos) and nonpyrogenic (fever in rabbits) lipid A by treatment with boiling 0.1 N HCl (B5-HC1). Nontoxic B5-HCl, like toxic B5, caused regression of line-10 tumors and elimination of lymph node metastasis in 27 of 32 (84%) syngeneic strain 2 guinea pigs at a dosage of 150 g. At this dosage, toxic B5 led to a cure in 54 of 67 (81%) tumor-bearing animals. All cured animals rejected a second line-10 tumor cell transplant. This activity depended on combining the toxic or nontoxic endotoxins with mycobacterial trehalose mycolate (P3) and an essentially nontoxic peptide-containing side-fraction (ACP) recovered during the isolation of B5. In contrast to toxic B5 or endotoxins in general, nontoxic B5-HCl did not cause endotoxic shock when combined with adjuvant dipeptide (MDP) and injected IV into guinea pigs. Chemical analysis showed that the phosphate content of nontoxic B5-HCl was about one-half that observed in toxic B5 or in toxic KDO-free lipid A, which was obtained by treating toxic B5 with sodium acetate at pH 4.5 at 100° C (B5-pH 4.5). The molar ratio of glucosamine: phosphorus: fatty acids was 2:1:4 for nontoxic B5-HCl and was 2:2:4 for toxic B5-pH 4.5. These results demonstrate that endotoxic extracts could be selectively detoxified while retaining antitumor properties. Thus, nontoxic B5-HCl may be a potential candidate for immunotherapy of human cancer.Presented at the 72nd Annual Meeting of the American Association for Cancer Research, 1981, and abstract no. 1123 published in the Proceedings of the American Association for Cancer Research, Vol. 22, 1981 Abbreviations used in this paper: ACP, a nontoxic acetone-chloroform precipitated side-fraction of endotoxin that contains (an) ingredient(s) necessary for tumor regression of line-10 tumors in strain 2 guinea pigs; ReGl, endotoxic glycolipids from Re mutant gram-negative bacteria; ReGl-PCP, ReGl extracted with phenol-chloroform-petroleum ether (PCP); B5, refined endotoxin, free of phospholipids, divalent cations and disaggregated; B5-HCl, nontoxic lipid A prepared from B5 by treatment with hydrochloric acid; B5-pH 4.5, toxic lipid A prepared from B5 by treatment with sodium acetate at pH 4.5; lipid A, hydrochloric acid or sodium acetate hydrolysate of ReGl-PCP or B5; MDP, N-acetyl-muramyl-l-seryl-d-isoglutamine; KDO, keto-3-deoxyoctonate     

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.     

Sperm competition in fishes: the evolution of testis size and ejaculate characteristics

Fishes show one of the widest ranges of sperm competition intensity of any animal group. Here we present a comparative study whose aim is to investigate the effect of relative intensity of sperm competition on investment in spermatogenesis and the number and size of sperm produced. We find that both the gonadosomatic index (GSI = [gonad weight/body weight] x 100) and sperm numbers increase with intensity of sperm competition across species but that sperm length decreases. These new findings are consistent with a raffle-based mode of sperm competition in fishes. Most of these results (positive correlation of the GSI and sperm number with sperm competition intensity) concur with the predictions of current sperm competition theory. However, we also find that sperm longevity decreases with sperm length across species. Current models for continuous fertilization suggest that if length increases a sperm's speed but decreases its longevity, sperm length should increase with sperm competition intensity, whereas models for instant fertilization suggest that sperm length should remain constant. The negative relationship found between sperm competition and sperm length therefore does not fit predictions of either model.     

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.     

The effect of life cycle stage and genotype on desiccation tolerance in the colonizing parthenogenetic cockroach Pycnoscelus surinamensis and its sexual ancestor P. indicus

We compared the desiccation tolerance of nymphs of diploid and triploid clones of the colonizing parthenogenetic cockroach, Pycnoscelus surinamensis, and strains of its sexual ancestor, P. indicus, as a test of the general-purpose genotype hypothesis and the polyploidy hypothesis for geographic parthenogenesis in this species complex. Desiccation tolerance is strongly associated with nymphal size. Clones of P. surinamensis are highly variable in nymphal desiccation tolerance, adjusted for body weight by analysis of covariance. This heterogeneity is mirrored by significant differences among recently isolated sublines of a lab population of P. indicus. As a group, the clones are not more tolerant than the sexual strains. Likewise, the four triploid clones were not more resistant to desiccation than the four diploid clones tested. A second experiment revealed a negative association between adult and last instar desiccation tolerance, due to developmental factors not associated with size. These patterns of variation in the sexual and parthenogenetic forms are consistent with the conclusion that extensive genetic variation in desiccation tolerance in the sexual ancestor has been preserved in the clonal lineages, but that desiccation tolerance has not been selected on strongly during the dispersal of clones of P. surinamensis.     

Comparative imaging of Pseudomonas putida bacterial biofilms by scanning electron microscopy and both DC contact and AC non-contact atomic force microscopy

P.A.GUNNING, A.R.KIRBY, M.L.PARKER, A.P.GUNNING AND V.J.MORRIS. 1996. Both Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) have been used to visualize the morphology of Pseudomonas putida bacterial colonies isolated from model oil-in-water emulsions. A new method has been developed for growing flat homogeneous bacterial biofilms at planar oil-water interfaces. A marked increase in resolution has been achieved when these flat bacterial biofilms are imaged by both SEM and AFM methods. On flat bacterial biofilms AFM offers superior resolution with minimal sample preparation. High resolution DC contact mode AFM studies of the bacterial surfaces have revealed surface features comparable in size to large proteins. AC non-contact AFM methods have been used to image bacterial flagella trapped in the biofilm.     

Oxygen pulse in guinea pigs in hyperbaric helium and hydrogen

Kayar, Susan R., and Erich C. Parker. Oxygen pulse inguinea pigs in hyperbaric helium and hydrogen. J. Appl. Physiol. 82(3): 988-997, 1997.We analyzedO₂ pulse, the total volume of O₂ consumed per heart beat, inguinea pigs at pressures from 10 to 60 atmospheres. Animals were placedin a hyperbaric chamber and breathed 2%O₂ in either helium (heliox) orhydrogen (hydrox). Oxygen consumption rate(O₂) was measured by gaschromatographic analysis. Core temperature and heart rate were measuredby using surgically implanted radiotelemeters. TheO₂ was modulated over afourfold range by varying chamber temperature from 25 to 36°C. There was a direct correlation betweenO₂ and heartrate, which was significantly different for animals in heliox vs.hydrox (P = 0.003). By usingmultivariate regression analysis, we identified variables that weresignificant to O₂ pulse: bodysurface area, chamber temperature, core temperature, and pressure.After normalizing for all nonpressure variables, the residualO₂ pulse was found to decreasesignificantly (P = 0.02) with pressurefor animals in heliox but did not decrease significantly(P = 0.38) with pressure for animalsin hydrox over the range of pressures studied. This amounted to aroughly 25% lower O₂ pulse fornormothermic animals in 60 atmospheres heliox vs. hydrox. These resultssuggest that reduction of cardiovascular efficiency in a hyperbaricenvironment can be mitigated by the choice of breathing gas.

     

Contractile responsiveness of coronary arteries from exercise-trained rats

Parker, Janet L., Mildred L. Mattox, and M. Harold Laughlin.Contractile responsiveness of coronary arteries from exercise trained rats. J. Appl. Physiol. 83(2):434-443, 1997.The purpose of this study was to determine whetherexercise training alters vasomotor reactivity of rat coronary arteries.In vitro isometric microvessel techniques were used to evaluatevasomotor properties of proximal left anterior artery rings (1 ring peranimal) from exercise-trained rats (ET;n = 10) subjected to a 12-wk treadmill training protocol (32 m/min, 15% incline, 1 h/day, 5 days/wk) andcontrol rats (C; n = 6) restricted tocage activity. No differences in passive length-tension characteristicsor internal diameter (158 ± 9 and 166 ± 9 µm) were observedbetween vessesls of C and ET rats. Concentration-response curves toK⁺ (5-100 mM), prostaglandinF₂(10⁸-10⁴M), and norepinephrine(10⁸-10⁴)were unaltered (P > 0.05) incoronary rings from ET rats compared with C rats; however, lower valuesof the concentration producing 50% of the maximal contractile responsein rings from ET rats (P = 0.05)suggest that contractile sensitivity to norepinephrine wasenhanced. Vasorelaxation responses to sodium nitroprusside (10⁹-10⁴M) and adenosine(10⁹-10⁴M) were not different (P > 0.05)between vessels of C and ET rats. However, relaxation responses to theendothelium-dependent vasodilator acetylcholine (ACh;10¹⁰-10⁴M) were significantly blunted (P < 0.001) in coronary rings from ET animals; maximal ACh relaxationaveraged 90 ± 5 and 46 ± 12%, respectively, in vessels of Cand ET groups. In additional experiments, two coronary rings (proximaland distal) were isolated from each C(n = 7) and ET(n = 7) animal. Proximal coronaryartery rings from ET animals demonstrated decreased relaxationresponses to ACh; however, ACh-mediated relaxation of distal coronaryrings was not different between C and ET groups.N^G-monomethyl-L-arginine(inhibitor of nitric oxide synthase) blocked ACh relaxation of allrings. L-Arginine (substrate fornitric oxide synthase) did not improve the blunted ACh relaxation in proximal coronary artery rings from ET rats. These studies suggest thatexercise-training selectively decreases endothelium-dependent (ACh) butnot endothelium-independent (sodium nitroprusside) relaxation responsesof rat proximal coronary arteries; endothelium-dependent relaxation ofdistal coronary arteries is unaltered by training.

     

Polygynandry inPseudotropheus zebra, a cichlid fish from Lake Malawi

Synopsis Parental care in the Malawian cichlid fishPseudotropheus zebra BB is extensive and exclusively maternal; males contribute only genetic material. The costs of searching for multiple mates (in this case risk of predation on orally incubated eggs) suggested that females should be monandrous; microsatellite genetypes of seven brooding females and their young, however, reveal extensive multiple paternity in this species, with a mean of 3.8 paternal individuals per brood. Polygynandry inP. zebra is probably not maintained by selection for genetically diverse offspring; potential explanations include avoidance of inbreeding, and bet-hedging on other male characteristics that females are unable to evaluate when selecting a mate. The observed degree of multiple paternity strongly suggests that females are free to choose mates as they will, a prerequisite of many theories positing sexual selection as a key element in Malawi chichlid evolution. It should also result in elevation of effective population sizes, and thus be antagonistic to runaway evolution of male secondary sexual characteristics, but not necessarily to other modes of sexual selection.