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Undifferentiated febrile illnesses present diagnostic and treatment challenges in the Firm Base, let alone in the deployed austere environment. We report a series of 14 cases from Operation TRENTON in South Sudan in 2017 that coincided with the rainy season, increased insect numbers and a Relief in Place. The majority of patients had headaches, myalgia, arthralgia and back pain, as well as leucopenia and thrombocytopenia. No diagnoses could be made in theatre, despite a sophisticated deployed laboratory being available, and further testing in the UK, including next-generation sequencing, was unable to establish an aetiology. Such illnesses are very likely to present in tropical environments, where increasing numbers of military personnel are being deployed, and clinicians must be aware of the non-specific presentation and treatment, as well as the availability of Military Infection Reachback services to assist in the management of these cases.

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.

Radioiodinated apolipoprotein A-I was separated into two pools by gel chromatography on Sephadex G-150 superfine resin or by high performance liquid chromatography using a TSK-2000 column. The first pool, pool 1, was indistinguishable from unlabeled apo-A-I as judged by sedimentation equilibrium, circular dichroic measurements, and radial immunodiffusion. This pool self-associated according to a monomer-dimer-tetramer-octamer scheme and underwent the expected conformation changes with dissociation as reported previously for unlabeled apo-A-I. The radioiodinated material in pool 2 had less secondary structure as compared to the unlabeled material and did not self-associate. Protein in this pool was immunochemically less reactive than unlabeled apo-A-I. Kinetic studies, in vivo, demonstrated that these two pools of radioiodinated apo-A-I are also distinguished metabolically. Discussion centers around possible mechanisms of formation of the incompetent protein in pool 2 and factors that should be taken into consideration when using unfractionated radioiodinated apo-A-I for experimental studies.

Quantitation and characterization of freely reversible ligand-receptor interactions in adherent cell lines grown in vitro have been technically difficult to perform. We report the growth of human fibroblasts on a microcarrier bead system which obviates the limitation of cell adherence in such determinations. This system has been applied to the reversible interaction of low density lipoproteins (LDL) with the fibroblast membrane receptors. Of the 125I-LDL specifically bound to filtered cells, 52.2 +/- 9.8% was reversibly bound. This reversible LDL-cell binding occurred in less than 1 h at 37 degrees C, was specific for LDL, and occurred without significant 125I-LDL degradation. Using these techniques, it is now possible to obtain direct measurement and characterization of lipoprotein-receptor interactions in adherent cell lines of normal subjects and patients with dyslipoproteinemia.

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.

Current studies on receptor-ligand interactions usually employ a "binding" step followed by extensive washing to remove free ligand. This procedure, by definition, removes reversibly bound ligand from the receptor; the equations used by most workers to analyze the data, however, require an equilibrium between free and bound ligand and are not applicable given the design of most binding assays. The assay, in fact, measures binding that is slowly reversible or irreversible. Assuming that ligand receptor interaction involves two stages with a reversible step followed by an "irreversible" event, the Ka for the reversible reaction may be obtained from the rate of the irreversible step. 125I-Choleragen binding to human fibroblasts was only slowly reversible at both 0 and 37 degrees C. The two-step model was, therefore, applied experimentally to determine the Ka for the reversible step in 125I-choleragen binding to human fibroblasts at 0 degrees C (Ka = 1.9 x 10(8) M-1) and at 37 degrees C (Ka = 3.6 x 10(8) M-1). As predicted by the two-step model for ligand binding, the addition of 50 micrograms/ml of unlabeled toxin enhanced the rate of release of radioactivity at 37 degrees C; the rate of radiolabel release remained low at 0 degrees C, even with unlabeled toxin present in the medium. The rate of release of previously incorporated 125I-toxin was accelerated by 50 micrograms/ml of toxin greater than 5 micrograms/ml of toxin. The two-step model for ligand binding appears to be applicable to the study of 125I-choleragen binding to fibroblasts and should be useful, in general, for the analysis of receptor-ligand interaction.

beta 2-Glycoprotein I (beta 2GI) has recently been identified as a component of circulating plasma lipoproteins. The metabolic role of this apolipoprotein is not known with certainty; it has been reported that beta 2GI has a high affinity for triglyceride-rich particles, causing their selective precipitation by detergents, and activates lipoprotein lipase in the in vitro hydrolysis of artificial lipid emulsions. In the present report, we have evaluated the secondary, tertiary, and quaternary structure of lipid-free beta 2GI. The weight average molecular weight of beta 2GI, as determined by sedimentation equilibrium measurements, was 43,000 in the presence and absence of denaturing agents. Thus, in contrast to other apolipoproteins, apolipoprotein H (apo-H) does not self-associate in aqueous solution. The circular dichroic spectra of apo-H is unusual in that there are no strong negative bands in the far-ultraviolet region of the spectrum; there is a weak positive maximum at 235 nm and a relatively weak negative maximum at 205 nm. Treatment with guanidinium chloride results in a loss of the positive band with only minor changes in the intensity of the band at 205 nm. Apolipoproteins A-I, A-II, C-I, and E, in contrast, have a secondary structure that contains a high percentage of residues in an alpha-helical configuration and undergo major changes in structure at low concentrations of guanidinium chloride. Highly flexible proteins, such as apolipoproteins A-I, A-II, and C-I, absorb rapidly and reversibly to air-water interfaces, whereas more rigid proteins, such as the classical globular proteins, interact with the interface more slowly and irreversibly. This difference is due to the loosely folded tertiary structure of apolipoproteins and the ease with which they can change structure to accommodate a given environment. The surface activity of beta 2GI at neutral pH resembles that of typical globular proteins. Treatment with acid or base, although causing only minor changes in the circular dichroic spectra, resulted in major increases in the rate of absorption to an air-water interface; under these conditions the rates of absorption were similar to that found for apolipoprotein A-I. These results are consistent with a more flexible structure for beta 2GI in acid or base that resembles other loosely folded apolipoproteins. beta 2GI associates with plasma lipoproteins and satisfies all of the criteria to be classified as an apolipoprotein. The secondary, tertiary, and quaternary structure of beta 2GI is, however, quite different from that of other well characterized apolipoproteins. This difference in structure would be expected to affect protein-lipid interactions; the relationship between apo-H and other apolipoproteins may be similar to that proposed for integral versus peripheral membrane proteins.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.

Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.

An alternative pathway C3 convertase is formed by the equilibrium association of Factor B with cobra venom factor (CVF) followed by the activation step catalyzed by Factor D. However, the association of Factor B with CVF has only occasionally been demonstrated and has not been quantitatively analyzed. Here we show that in the absence of metals the two proteins have significant affinity for each other and reversibly associate in a one-to-one stoichiometry with a dissociation constant of 11.6 microM. Upon the addition of metal ions, the complex is stabilized only 2- to 30-fold in the order Ni2+(Kd = 6.6 microM) less than Mg2+(Kd = 1.1 microM) less than Mn2+(Kd = 0.4 microM). These results suggest that metal ions may be less important in stabilizing the CVF.B complex and more important in promoting the subsequent equilibrium association of CVF.B with Factor D. The stability of the CVF.B complex is variously dependent on temperature in the range studied (14-21 degrees C) depending on the metal ion that is present. The complex formation was demonstrated in the analytical ultracentrifuge at sedimentation equilibrium employing a combination of single- and multiple-independent variable nonlinear least squares analytical techniques. Two different numerical approaches gave very similar results.

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the “high affinity” (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the “high” (KS) and “low affinity” (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or “ligand-free” state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.

A new, rapid method for purification of calmodulin-stimulated phosphodiesterase from bovine, ovine, and porcine brain using only DEAE-agarose and calmodulin-Sepharose chromatography is described. Purified enzymes from the three species each exhibited a single polypeptide of Mr approximately 59,000 on gel electrophoresis under denaturing conditions. Proteolysis of ovine and bovine enzymes with alpha-chymotrypsin, however, yielded different peptides, indicating that these proteins differ in primary sequence. Homogeneous preparations of bovine and ovine enzymes (purified approximately 5,000- and 2,000-fold, respectively) had different specific activities, although their substrate affinities and activation by calmodulin (8- to 14-fold activation, Kact approximately 1 nM) were very similar. The total amount in ovine was almost twice that in bovine brain. The hydrodynamic properties of bovine and ovine enzymes were indistinguishable with a Stokes radius of 4.35 nm and s20,w of 5.95 S. The calculated frictional ratios of 1.30 to 1.38 suggest a slightly asymmetric molecule. Equilibrium sedimentation data yielded apparent Mr approximately 57,000 in the presence of 6 M guanidine and 124,000 and 112,000 for the native bovine and ovine enzymes, respectively. In addition to the enzyme that was purified to homogeneity (pI approximately 5.6), a major fraction of calmodulin-activated phosphodiesterase with a lower isoelectric point was found in bovine and ovine brain. Whether these represent isozymes, perhaps localized in different types of cells, or whether one is a post-translationally modified form, remains to be determined. The existence of these two otherwise very similar forms of the enzyme has apparently not been previously recognized.

Ornithine transcarbamoylase (OTCase) has been purified in 100-mg quantities from a plasmid-containing, enzyme-overproducing strain of Saccharomyces cerevisiae. The specific activity of the homogeneous enzyme is 2.5-fold above that previously reported. The molecular weight and partial specific volume of OTCase were determined by sedimentation equilibrium in solutions containing H2O and D2O. Data from two rotor speeds were simultaneously fit using nonlinear least squares analysis with multiple independent variables giving a molecular weight of 110,000 +/- 2,200 and a partial specific volume of 0.732 +/- 0.006 ml g-1. The ultraviolet absorption spectrum of OTCase gives a specific absorbance at 280 nm of 0.36. This low value is consistent with a small number of aromatic residues. Amino acid analysis, fluorescence, and multicomponent analysis yield 1 tryptophan, 4 tyrosine, and 24 phenylalanine/polypeptide chain. From an analysis of the circular dichroic spectrum, it was determined that OTCase contained 22% alpha-helix, 43% beta-sheet, 8% beta-turn, and 27% random structure. The fluorescence of the single tryptophan/polypeptide chain has an emission maximum at 320 nm, indicating a hydrophobic environment.

Graded levels of free gossypol from cottonseed meats were added to corn-degossypolized cottonseed meal and corn-soybean meal diets for growing pigs. Solubility of the soybean nitrogen was reduced from 85% to 35% by autoclaving for 2 hours at 17 lb. of steam pressure. Solubility of cottonseed nitrogen was reduced from 87.4% to 79% by autoclaving for 30 minutes. Rate of gain of pigs fed the autoclaved meals was not significantly reduced. Addition of free gossypol at the levels of 0.03% and 0.036%, respectively, to both corn-cottonseed meal and corn-soybean meal diets resulted in depressed growth and death of the pigs.In a second study autoclaving degossypolized cottonseed meal for 3 hours at 17 lb. of pressure reduced growth of pigs from 1.26 lb. to 0.38 lb. per day. Addition of 0.2% of L-lysine resulted in growth almost equal to that of pigs receiving non-autoclaved meal (1.09 lb. per day). This was in the presence of 0.026% of added free gossypol. When the free gossypol level was increased to 0.036% of the diet, growth rate was reduced and four of six pigs died from gossypol toxicity. There was no evidence that addition of 0.2% of L-lysine to this high gossypol ration improved growth or reduced death losses.

Two experiments were conducted to compare xylose and glucose as sources of carbohydrate in the diets of young pigs.Experiment I consisted of a growth-performance study using 4 replications of pigs, which were hand fed three purified diets containing 37.4% of sugar, glucose, xylose, or a 50-50 mixture of the two. Experiment II included studies of nitrogen retention and sugar recovery as well as of growth-performance; data were obtained from 5 pairs of pigs, which were pair-fed comparative diets containing 56%, resspectively, of glucose or of xylose.Growth-performance and balance studies indicated that pigs were able to utilize glucose to a greater degree than xylose.Cataract formation resulted after feeding a diet containing xylose at the level of 56% of the dry matter.Voluntary activity was decreased, and the color of the hair coat was altered from deep red to yellowish-red in pigs fed xylose.