Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant.

Uptake of oxidatively modified low-density lipoprotein (LDL) by cells in the arterial wall is believed to be an important early event in the development of atherosclerosis. Because vitamin E is the major antioxidant present in human lipoproteins, it has received much attention as a suppressor of LDL lipid oxidation and as an epidemiological marker for ischaemic heart disease. However, a careful examination of lipid peroxidation in LDL induced by a steady flux of aqueous peroxyl radicals has demonstrated that, following consumption of endogenous ubiquinol-10, the rate of peroxidation (i) declines as vitamin E is consumed, (ii) is faster in the presence of vitamin E than following its complete consumption, (iii) is substantially accelerated by enrichment of the vitamin in LDL, either in vitro or by diet, and (iv) is virtually independent of the applied radical flux. We propose that perodixation is propagated within lipoprotein particles by reaction of the vitamin E radical (i.e. alpha-tocopheroxyl radical) with polyunsaturated fatty acid moieties in the lipid. This lipid peroxidation mechanism, which can readily be rationalized by the known chemistry of the alpha-tocopheroxyl radical and by the radical-isolating properties of fine emulsions such as LDL, explains how reagents which reduce the alpha-tocopheroxyl radical (i.e. vitamin C and ubiquinol-10) strongly inhibit lipid peroxidation in vitamin E-containing LDL.


Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol.

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.


Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.

Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.


Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex.

The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo.


A competitive inhibitor of phospholipase A2 decreases surfactant phosphatidylcholine degradation by the rat lung.

We have shown previously that radiolabelled phosphatidylcholine (PC) in liposomes or natural surfactant is removed from the alveolar space and metabolically recycled in a process that is stimulated by cyclic AMP (cAMP). In this study, we evaluated the effect of a transition-state phospholipid analogue (MJ33; 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol) that competitively inhibited acidic phospholipase A2 (PLA2) activity (pH 4.0) of lung homogenate by more than 97%, but had no effect on PLA2 activity at pH 8.5. MJ33 incorporated into unilamellar liposomes (dipalmitoyl PC/egg PC/cholesterol/phosphatidylglycerol, molar proportions 10:5:3:2) or co-sonicated with biosynthesized natural surfactant was instilled into the trachea of the anaesthetized rat; lungs were then removed for 2 h perfusion in the absence or presence of 0.1 mM-8-bromo cAMP. Total uptake for phospholipid was unchanged in the presence of the inhibitor MJ33. Degradation of labelled PC during 2 h perfusion in the absence of MJ33 was approx. 26% of that instilled for choline-labelled liposomal PC, 16% for liposomal PC labelled in the second fatty-acyl position, and 33% for choline-labelled natural surfactant. Degradation of PC was decreased by approx. 25-40% for each substrate in the presence of MJ33. Inhibition of lipid degradation depended on the mole fraction of MJ33 in the liposomes and was maximal at 1 mol%. These studies demonstrate a significant role for acidic Ca(2+)-independent PLA2 in the degradation of internalized alveolar PC, but further indicate that this enzyme accounts for a minor fraction of total lung PC metabolism.


Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.


Protein stability and interaction of the nicotinic acetylcholine receptor with cholinergic ligands studied by Fourier-transform infrared spectroscopy.

Based on the conformational dependence of the amide-I i.r. band, this paper explores the use of Fourier-transform i.r. spectroscopy methods to probe structural features of proteins present in native membranes from Torpedo highly enriched in acetylcholine receptor (AcChR). The interference of water absorbance on the amide-I spectral region has been eliminated through isotopic exchange by freeze-drying the membranes in the presence of trehalose to avoid protein denaturation induced by drying, followed by resuspension in deuterated water. AcChR-rich membrane samples prepared in such a way maintained an ability to undergo affinity-state transitions and to promote cation translocation in response to cholinergic agonists, which are functional characteristics of native untreated samples. The temperature-dependence of the i.r. spectrum indicates a massive loss of ordered protein structure, occurring at temperatures similar to those reported for thermal denaturation of the AcChR by differential scanning calorimetry and by thermal inactivation of alpha-bungarotoxin-binding sites on the AcChR [Artigues, Villar, Ferragut & Gonzalez-Ros (1987) Arch. Biochem. Biophys. 258, 33-41], thus suggesting that the observed i.r. spectral changes correspond to alterations in the structure of the AcChR protein. Furthermore, the presence of detergents as well as cholinergic agonists and antagonists produces spectral changes that are also consistent with the alterations in AcChR protein structure expected from previous calorimetric studies. In contrast with the information obtained by calorimetry, i.r. spectroscopy allows the contribution of secondary structural changes to be distinguished from the overall change in protein structure. Thus prolonged exposure to cholinergic agonists, which drives the AcChR protein into the desensitized state, produces only negligible alterations in the amide-I band shape, but increases substantially the thermal stability of the protein. This suggests that rearrangements in the tertiary or quaternary structure of the protein are more likely to occur than extensive changes in secondary structure as a consequence of AcChR desensitization.


Dissociated effects of 2-deoxy-D-glucose on D-[2-3H]glucose and D-[5-3H]glucose conversion into 3HOH in rat erythrocytes.

When rat erythrocytes were preincubated with 2-deoxy-D-glucose, the generation of both 3H-labelled acidic metabolites and 3HOH from D-[5-3H]glucose, the total production of L-lactate, and the generation of 14CO2, 14C-labelled acidic metabolites and 14C-labelled lactate from D-[1-14C]glucose or D-[U-14C]glucose were all lower than in erythrocytes preincubated in the absence of a hexose or in the presence of 3-O-methyl-D-glucose. However, preincubation with 2-deoxy-D-glucose failed to decrease the generation of 3H-labelled acidic metabolites and L-[3-3H]lactate from D-[2-3H]glucose, while decreasing the production of 3HOH more severely from D-[2-3H]glucose than from D-[5-3H]glucose. This may be attributable not solely to inhibition of D-glucose phosphorylation by 2-deoxy-D-glucose and 2-deoxy-D-glucose 6-phosphate, but also to inhibition by 2-deoxy-D-glucose 6-phosphate of hexose 6-phosphate interconversion in the reaction catalysed by phosphoglucoisomerase, as also observed with the purified enzyme. The generation of 3HOH from D-[2-3H]glucose should therefore be considered as a tool to assess the efficiency of interconversion of hexose 6-phosphates in the reaction catalysed by phosphoglucoisomerase, rather than to estimate D-glucose phosphorylation rate.


Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode.

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of nigrostriatal dopaminergic neurons in primates, giving rise to a condition resembling Parkinson's disease. The toxicity of MPTP is believed to be due to its metabolite 1-methyl-4-phenylpyridinium (MPP+). MPP+ is an inhibitor of mitochondrial respiration at the NADH-ubiquinone oxidoreductase site and this, together with its selective transport into dopaminergic nerve terminals, accounts for its neurotoxicity. In this paper an electrode selective for MPP+ was developed and used to measure the rate of uptake and the steady-state accumulation of MPP+ in rat liver mitochondria. The initial rates of MPP+ uptake were not saturable, confirming previous work that the transport of MPP+ is not carrier-mediated. The membrane potential of mitochondria respiring on succinate was decreased by MPP+ and the steady-state accumulation ratio of MPP+ did not come to equilibrium with the mitochondrial transmembrane potential gradient (delta psi). The effect of the cation exchanger tetraphenylboron (5 microM) was to increase the initial rate of MPP+ uptake by about 20-fold and the steady-state accumulation by about 2-fold. This suggests that there may be a mechanism of efflux of MPP+ from mitochondria which allows MPP+ to cycle across the membrane and thus decrease delta psi. These data indicate that MPP+ interacts with mitochondria independently of its inhibition of NADH-ubiquinone oxidoreductase, and these alternative interactions may be of relevance for its mechanism of neurotoxicity.


Ethanol and glycogen synthesis in cardiothoracic and skeletal muscles following glucose re-feeding after starvation in the rat.

The pattern of glycogen deposition in individual cardiothoracic and skeletal muscles in response to oral and intraperitoneal glucose administration was examined in 40 h-starved rats. Rates of glycogen synthesis were consistently higher in oxidative muscles than in non-oxidative muscles. Intragastric ethanol administration was associated with an impaired glycaemic response and the almost total abolition of glycogen deposition in oxidative muscles in response to oral or intraperitoneal glucose re-feeding. This effect was dose-dependent and differential, in that ethanol produced no equivalent impairment in glycogen deposition in non-oxidative muscles. Ethanol treatment also selectively promoted glycogenolysis in oxidative muscles in the starved state. There was positive correlation (P < 0.001) between the decrease in glycogen levels in soleus and diaphragm muscles in response to increasing ethanol doses and blood glucose and lactate concentrations after intraperitoneal glucose administration, implying that the basis for the impairment in glycogen synthesis may be diminished glucose availability. The mechanism whereby ethanol may differentially compromise carbohydrate metabolism in oxidative muscles is discussed.


A non-glycosylated extracellular superoxide dismutase variant.

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.


Ca2+ influx induced by the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone and thapsigargin in bovine adrenal chromaffin cells.

We have characterized the effect of the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) and thapsigargin on the concentration of cytosolic Ca2+ in single bovine adrenal chromaffin cells by video-imaging of fura-2-loaded cells. Addition of either inhibitor released Ca2+ from internal stores in the absence of external Ca2+. tBHQ was unable to stimulate further Ca2+ release after addition of thapsigargin, but thapsigargin could do so after release by tBHQ, indicating that the tBHQ-sensitive stores are a sub-set of those sensitive to thapsigargin. Angiotensin II was able to elicit Ca2+ release after application of tBHQ, indicating that at least part of the tBHQ-sensitive stores were distinct from those discharged by Ins(1,4,5)P3. In the presence of external Ca2+, both Ca(2+)-ATPase inhibitors produced a more prolonged rise in cytosolic Ca2+ consistent with stimulated Ca2+ entry. The ability of the inhibitors to activate a Ca(2+)-entry pathway was confirmed by monitoring quenching of fura-2 after stimulated entry of the Ca2+ surrogate Mn2+. These findings indicate that bovine adrenal chromaffin cells possess a mechanism by which Ca2+ entry can be activated, following emptying of certain internal stores, independently of receptor occupation.


Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain. Evidence for constitutive presence in rat and human brain.

Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).


D-myo-inositol 1,3,4,5-tetrakisphosphate releases Ca2+ from crude microsomes and enriched vesicular plasma membranes, but not from intracellular stores of permeabilized T-lymphocytes and monocytes.

In the human T-lymphocyte cell lines Jurkat and HPB.ALL and the human monocytoid cell line U937, Ins(1,3,4,5)P4 triggers a dose-dependent release of Ca2+ from crude microsomal preparations, with a half-maximal effective concentration (EC50) of 1.2-2.3 microM. Similar results were obtained with enriched vesicular plasma membranes from U937 cells. However, in permeabilized preparations of the same cell types only Ins(1,4,5)P3 was able to release Ca2+ from intracellular stores, with EC50 values in the range 0.11-0.84 microM. In crude microsomes the effects of Ins(1,3,4,5)P4 and Ins(2,4,5)P3, a non-metabolizable InsP3 isomer, occurred independently of each other, indicating subpopulations of Ins(1,3,4,5)P4- and Ins(1,4,5)P3-sensitive vesicles. The Ins(1,3,4,5)P4 preparation used for the Ca(2+)-release experiments contains neither Ca2+ nor contaminating Ins(1,4,5)P3 and was not metabolized to Ins(1,4,5)P3 during the Ca(2+)-release experiments. We conclude that Ins(1,3,4,5)P4 independently of Ins(1,4,5)P3 induces a Ca2+ flux via a membrane compartment, most likely the plasma membrane, that is functionally destroyed during the permeabilization of the cells.


Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content.

The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5' end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.


Control of plasma-membrane Ca2+ entry by the intracellular Ca2+ stores. Kinetic evidence for a short-lived mediator.

We have studied the correlation between the degree of filling of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, a Ca2+ surrogate for plasma-membrane Ca2+ channels, in human neutrophils loaded with fura-2. Refilling of the stores of cells previously depleted of Ca2+ decreased the entry of Mn2+, but the magnitude of this effect depended on the refilling protocol. When refilling was allowed to proceed to steady-state levels by a 3 min incubation with different external Ca2+ concentrations (0.05-1 mM), almost complete inhibition of Mn2+ entry was observed at 40% of maximum refilling. In contrast, when different degrees of store refilling were attained by incubation with 1 mM-Ca2+ for short periods (10-40 s), inhibition of Mn2+ entry was smaller at comparable degrees of refilling. When quick refilling was allowed to proceed up to 40% (about 20 s at 37 degrees C) and then stopped at this level by removal of external Ca2+, the rate of Mn2+ uptake was high just after refilling and then decreased with time within the next few seconds (half-times approximately 7 s at 37 degrees C and approximately 20 s at 25 degrees C). We have proposed previously that the Ca2+ stores, when emptied of Ca2+, may generate a second messenger able to open the plasma-membrane Ca2+ channels by a mechanism involving cytochrome P-450. The results here are consistent with the existence of such a messenger and suggest that it is cleared from the cytoplasm with a half-time of about 7 s at 37 degrees C. In addition, inhibition of Mn2+ entry in cells with empty Ca2+ stores by cytochrome P-450 inhibitors showed a time lag consistent with the clearance kinetics proposed above.


Analysis of algebraic weighted least-squares estimators for enzyme parameters.

An algorithm for the least-squares estimation of enzyme parameters Km and Vmax. is proposed and its performance analysed. The problem is non-linear, but the algorithm is algebraic and does not require initial parameter estimates. On a spreadsheet program such as MINITAB, it may be coded in as few as ten instructions. The algorithm derives an intermediate estimate of Km and Vmax. appropriate to data with a constant coefficient of variation and then applies a single reweighting. Its performance using simulated data with a variety of error structures is compared with that of the classical reciprocal transforms and to both appropriately and inappropriately weighted direct least-squares estimators. Three approaches to estimating the standard errors of the parameter estimates are discussed, and one suitable for spreadsheet implementation is illustrated.


A cDNA clone for human glucosamine-6-sulphatase reveals differences between arylsulphatases and non-arylsulphatases.

Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal degradation of heparan sulphate and keratan sulphate. Deficiency of glucosamine-6-sulphatase activity leads to the lysosomal storage of the glycosaminoglycan, heparan sulphate and the monosaccharide sulphate N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a non-arylsulphatase since, relative to arylsulphatase B, it shows negligible activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones and derived amino acid sequence coding for the entire glucosamine-6-sulphatase protein. The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, of which it is likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence similarity to other sulphatases such as the family of arylsulphatases, although the degree of similarity is not as high as that between members of the arylsulphatase family. This pattern of inter- and intra-family similarity delineates regions and amino acid residues that may be critical for sulphatase function and substrate specificity.


Erythroid expression and DNAaseI-hypersensitive sites of the carbonic anhydrase 1 gene.

The carbonic anhydrase 1 gene is expressed in adult human and mouse erythroid cells and colon epithelia from two distinct promoters. We have explored the erythroid promoter for cis-acting sequences involved in transcription using DNAaseI as a probe. Two DNAaseI-hypersensitive sites (DHS-1 and DHS-2) have been identified in the distal erythroid promoter in CA1-expressing erythroleukaemic cells. These sites are present at low levels in K562 cells, which have a foetal/embryonic phenotype and do not express CA1. DHS-1 and DHS-2 are not present in non-erythroid cells, including colon cells, which express CA1 from the proximal colon promoter. DHS-1 and DHS-2 were also generated in an heterologous CA1 gene containing 5 kb of erythroid promoter sequence after transfection into erythroid cells, including K562 cells. These transfection studies showed that both this fragment, and an abbreviated 817 bp promoter fragment which contains only DHS-1, were sufficient to confer erythroid-specific expression to a reporter gene. These promoters were active in cell lines expressing CA1 and in K562 cells. This latter observation implies that a developmental repressor factor is both present in K562 cells and binds to a cis-acting sequence that is absent from the sequence 5 kb upstream of the erythroid transcription start site.


Characterization of a human seminal plasma glycosaminoglycan-bearing polypeptide.

A glycosaminoglycan-bearing polypeptide (S.GP), present in human seminal plasma, was purified to homogeneity by a combination of CsCl density-gradient centrifugation, f.p.l.c. ion-exchange chromatography on a Mono Q HR column and Superose 6 gel filtration. The observed polydispersity of S.GP was attributed to the heterogeneity of its glycosaminoglycan content. Enzymic deglycosylation experiments and N-terminal amino-acid sequence determination indicate that it consists of a polypeptide (apparent molecular mass approx. 18 kDa) bearing both chondroitin and heparan sulphate chains. Evidence is given that S.GP contains a glycosaminoglycan-linkage domain of a so far uncharacterized gene product, proteolytically processed in the genital tract.


Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis.

Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.


Synthesis of 35S-labelled macromolecules by polymorphonuclear neutrophils. Evidence for the production of [35S]sulphite which can modify both endogenous and exogenous proteins.

The incorporation of [35S]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of 35S-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80% of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that 35S-labelled macromolecules isolated from the cell extract migrated with Kav. of 0.36, while corresponding material isolated from the medium migrated with Kav. of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise 35S-labelled proteins of various molecular masses. The 35S label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [35S]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [35S]sulphite from [35S]sulphate. The release of [35S]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [35S]sulphite reacted with these proteins to form a stable 35S-labelled conjugate.


Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation.

Ligation of the common bile duct (BDL) in the male rabbit resulted in increased gall-bladder microsomal total cyclo-oxygenase activity with prostaglandin E2 (PGE2) and 6-oxoprostaglandin F1 alpha [6-oxo-PGF1 alpha, stable metabolite of prostaglandin I2 (PGI2; prostacyclin)] as the major prostanoids synthesized after 24 and 72 h. Kinetic analysis of gallbladder microsomal membrane fractions incubated with increasing levels of [14C]arachidonic acid indicated that BDL for 24 and 72 h did not change substrate affinity (apparent Km) but markedly increased the rate of conversion (apparent Vmax.) suggesting the presence of more total enzyme responsible for synthesis of 6-oxo-PGF1 alpha and PGE2. BDL for 24 and 72 h significantly increased gall-bladder tissue slice basal release of 6-oxo-PGF1 alpha, but not PGE2, when compared with the controls. Gall-bladder slice release of PGE2 was 3-fold less than 6-oxo-PGF1 alpha in the control gall-bladder slices. Immunoblot analysis of 72 h BDL gall-bladder microsomal membrane fractions showed a slight increase in cyclo-oxygenase content and a 5-fold increase in the content of prostacyclin synthase as compared with the control. These data suggest that the BDL-stimulated total gall-bladder cyclo-oxygenase activity was the result of an increase in the level of specific prostaglandin-synthetic enzymes, in particular prostacyclin synthase, and not from a change in enzyme affinity.


Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin.

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.


Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases.

The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely.


Demonstration of cathepsins B, H and L in xenografts of normal and Duchenne-muscular-dystrophy muscles transplanted into nude mice.

The activities and contents of the lysosomal cysteine proteinases cathepsins B, H and L were examined in xenografts of biopsied muscles transplanted from age-matched normal subjects and Duchenne-muscular-dystrophy (DMD) patients into nude mice. The activity of cathepsin B increased 9-fold and that of B-plus-L increased 24-fold in the first week after transplantation in normal muscle xenografts. By the third week, the activity of cathepsin B increased a total of 20-fold and B-plus-L increased to 36-fold the original level. The activity levels of cathepsin B, B-plus-L, H and D, and acid phosphatase in normal and DMD xenografts were not significantly different when compared 2 weeks after transplantation. However, the protein content of cathepsin B in DMD muscle xenografts was more than 3-fold that of normal xenografts at 2 weeks. The profile of cathepsin H activity in normal muscle xenografts was different than those of cathepsins B and B-plus-L. In the first week, the cathepsin H diminished sharply to about one-third of the biopsied muscle level and then, by 3 weeks after transplantation, it had increased slightly to about half the original level. The amount of endogenous cysteine-proteinase inhibitor changed in parallel with the activity of cathepsins B and B-plus-L. Cathepsins B and H, but not cathepsin L, were found immunohistochemically in regenerating muscle fibres of normal and DMD xenografts 2 weeks after transplantation. Staining of cathepsin B in DMD xenografts was slightly stronger than that in normal subjects. There was no immunostaining in degenerating or necrotic muscle fibres 2 weeks after transplantation. Western-blot analysis revealed that the cathepsin B band at 29 kDa was increased in normal xenografts 2 and 3 weeks after transplantation. Also, 2 weeks after transplantation the staining intensity of this band was slightly stronger in DMD xenografts than in normal xenografts. These results suggest that cathepsin B participates in the regeneration of transplanted muscle, both normal and DMD, and in the DMD muscle fibre-wasting processes, during regeneration.


Caeruloplasmin biosynthesis by the human uterus.

The blue copper protein caeruloplasmin is synthesized mainly by hepatocytes. An alternative transcript for caeruloplasmin produced in certain extrahepatic tissues, CP-1, contains an additional 12 nucleotides encoding 4 amino acids not present in the hepatic transcript, CP-2 [Yang, Friedrichs, Cupples, Bonifacio, S Sanford, Horton & Bowman (1990) J. Biol. Chem. 265, 10780-10785]. We have demonstrated transcription of caeruloplasmin mRNA by a well-differentiated human uterine epithelial adenocarcinoma cell line, Ishikawa, and by human uterine endometrium and purified endometrial glands. Identical CP-2 nucleotide sequences were obtained for partial caeruloplasmin transcripts from human liver and Ishikawa cells, indicating that CP-2 transcripts are produced by uterine epithelial lining cells. The synthesis of caeruloplasmin protein was demonstrated for Ishikawa cells and another uterine adenocarcinoma cell line, ECC1. Peptide-mapping analysis indicated that caeruloplasmin secreted by Ishikawa cells was structurally identical with the protein synthesized by the human hepatoblastoma cell line HepG2. The secretion of a 135,000-M(r) caeruloplasmin by Ishikawa and ECC1 cells, comparable with that of the human hepatoblastoma cell line, HepG2, indicated similar processing of uterine and hepatic caeruloplasmin. Incorporation of 67Cu into caeruloplasmin was demonstrated for Ishikawa and ECC1 cells, suggesting that the human uterus produces a bioactive form of caeruloplasmin and possesses the necessary metal transporters and intracellular machinery for copper incorporation into this protein.


Possible multifunction of glucose transporter. Transport of nicotinamide by reconstituted liposomes.

A kinetic study of the uptake of nicotinamide by reconstituted liposomes containing the human erythrocyte glucose transporter, compared with that of D-glucose, demonstrated that the Km and Vmax. values were almost the same for each compound, and that the uptake of D-glucose was competitively inhibited by nicotinamide. At 20 mM concentration, 2-deoxy-D-glucose, 3-O-methyl-D-glucose and 4,6-O-ethylidene-D-glucose all caused 50% inhibition of nicotinamide uptake, but L-glucose and nicotinic acid were not inhibitory. Similar results were obtained for the uptake of D-glucose. Cytochalasin B binding to the liposomes was inhibited in a dose-dependent manner by either nicotinamide or D-glucose. Antibody for glucose transporter detected in band 4.5 by SDS/PAGE inhibited the uptake of D-glucose and nicotinamide. A possible uptake of nicotinamide by nucleoside transporter was excluded. In human erythrocytes, cytochalasin B binding was inhibited dose-dependently by either nicotinamide or D-glucose, and cytochalasin B depressed the uptake of both nicotinamide and 2-deoxy-D-glucose. These findings were well reproduced in the reconstituted liposomes. The very close similarities between uptake of nicotinamide and D-glucose suggest that the glucose transporter plays a direct role in transport of nicotinamide, which is structurally quite different from monosaccharides, and thus that the transporter is probably multifunctional.


Evidence that GLUT-2 mRNA and protein concentrations are decreased by hyperinsulinaemia and increased by hyperglycaemia in liver of diabetic rats.

GLUT-2, glucokinase (GK) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was studied in the liver of chronically catheterized diabetic rats during the 3 days after an intravenous injection of 65 mg of streptozotocin (STZ)/kg. At 6 h after the STZ injection, portal plasma insulin levels were 270 +/- 32 mu-units/ml and blood glucose was 1.4 +/- 0.4 mmol/l, owing to pancreatic beta-cell destruction. GLUT-2 and PEPCK mRNA concentrations were rapidly and dramatically decreased (> 90%), whereas GK mRNA was increased. After 30 h, plasma insulin concentrations were lower than 5 mu-units/ml and blood glucose was > 20 mmol/l. GLUT-2 and PEPCK mRNA concentrations increased 2-fold and GK mRNA disappeared progressively. In order to assess the relative roles of hyperglycaemia and insulinopenia, blood glucose was clamped at 6.4 +/- 0.5 mmol/l from 18 to 72 h after STZ injection by phlorizin infusion (0.5-2 g/day per kg) or at 6.6 +/- 0.3 mmol/l from 18 to 48 h after STZ injection by insulin infusion (0.25 unit/min per kg). GLUT-2 mRNA concentrations were 50% lower in phlorizin-infused than in untreated diabetic rats. The low levels of GK mRNA and the high levels of PEPCK mRNA were unaffected by normalization of hyperglycaemia in phlorizin-infused diabetic rats. In insulin-infused rats (portal plasma insulin levels of 40 mu-units/ml) GLUT-2 mRNA levels were 25% of those in untreated diabetic rats, and they increased rapidly 6 h after insulin infusion was stopped. Liver GLUT-2 protein concentration showed similar changes in response to STZ injection and to phlorizin or insulin treatment, but after a delay of several hours. From this work we conclude that GLUT-2 gene expression is dramatically and rapidly (< 6 h) decreased by portal hyperinsulinaemia and increased by hyperglycaemia.


Cell volume and bile acid excretion.

The interaction between cell volume and taurocholate excretion into bile was studied in isolated perfused rat liver. Cell swelling due to hypo-osmotic exposure, addition of amino acids or insulin stimulated taurocholate excretion into bile and bile flow, whereas hyperosmotic cell shrinkage inhibited these. These effects were explained by changes in Vmax of taurocholate excretion into bile: Vmax. increased from about 300 to 700 nmol/min per g after cell swelling by 12-15% caused by either hypo-osmotic exposure or addition of amino acids under normo-osmotic conditions. Steady-state taurocholate excretion into bile was not affected when the influent K+ concentration was increased from 6 to 46 mM or decreased to 1 mM with iso-osmoticity being maintained by corresponding changes in the influent Na+ concentration. Replacement of 40 mM-NaCl by 80 mM-sucrose decreased taurocholate excretion into bile by about 70%; subsequent hypo-osmotic exposure by omission of sucrose increased taurocholate excretion to 160%. Only minor, statistically insignificant, effects of aniso-osmotic cell volume changes on the appearance of bolus-injected horseradish peroxidase in bile were observed. Taurocholate (400 microM) exhibited a cholestatic effect during hyperosmotic cell shrinkage, but not during hypo-osmotic cell swelling. Both taurocholate and tauroursodeoxycholate increased liver cell volume. Tauroursodeoxycholate stimulated taurocholate (100 microM) excretion into bile. This stimulatory effect was strongly dependent on the extent of tauroursodeoxycholate-induced cell swelling. During continuous infusion of taurocholate (100 microM) further addition of tauroursodeoxycholate at concentrations of 20, 50 and 100 microM increased cell volume by 10, 8 and 2% respectively, in parallel with a stimulation of taurocholate excretion into bile by 29, 27 and 9% respectively. There was a close relationship between the extent of cell volume changes and taurocholate excretion into bile, regardless of whether cell volume was modified by tauroursodeoxycholate, amino acids or aniso-osmotic exposure. The data suggest that: (i) liver cell volume is one important factor determining bile flow and biliary taurocholate excretion; (ii) swelling-induced stimulation of taurocholate excretion into bile is probably not explained by alterations of the membrane potential; (iii) bile acids modulate liver cell volume; (iv) taurocholate-induced cholestasis may depend on cell volume; (v) stimulation of taurocholate excretion into bile by tauroursodeoxycholate can largely be explained by tauroursodeoxycholate-induced cell swelling.


Escherichia coli dihydrodipicolinate synthase. Identification of the active site and crystallization.

Escherichia coli dihydrodipicolinate synthase (DHDPS) (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis, catalyses the condensation of pyruvate and aspartate beta-semialdehyde (ASA) by a ping-pong mechanism. Pyruvate binds first to the enzyme, forming a Schiff base with the epsilon-amino group of Lys-161, followed by binding of ASA. Km values of 0.57 and 0.55 mM were determined for pyruvate and DL-ASA respectively. 3-Bromopyruvate inhibits DHDPS with a Ki of 1.6 mM. DHDPS is 50% inhibited by 1.0 mM-L-lysine, 1.2 mM-sodium dipicolinate or 4.6 mM-S-2-aminoethyl-L-cysteine. Crystals of DHDPS diffracting to beyond a resolution of 0.24 nm (2.4 A) were obtained under several experimental conditions. Diffraction patterns were compatible with trigonal space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 12.26 nm and c = 11.19 nm. The density of the crystals indicates the presence of a dimer of DHDPS subunits per asymmetric unit.


NIH conference. Gastrointestinal infections in AIDS.

As the largest lymphoid organ in the body, the gastrointestinal tract is a potential reservoir for human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), and it is an important site for HIV-induced immunodeficiency. The resulting defects in cellular and humoral defense mechanisms predispose the gastrointestinal tract to a spectrum of viral, fungal, bacterial, and protozoan pathogens that cause relentless morbidity and, in some cases, death. With a thorough diagnostic evaluation, physicians can identify one or more of these pathogens in a majority of patients with AIDS who have gastrointestinal symptoms. The identification of enteric pathogens in patients with AIDS is important because an increasing array of therapeutic regimens is becoming available to treat many of these infections.


Pigmented villonodular synovitis of the knee. The results of total arthroscopic synovectomy, partial, arthroscopic synovectomy, and arthroscopic local excision.

Twenty-five patients who had had a diagnosis of pigmented villonodular synovitis of the knee were followed for an average of four and one-half years (range, two to ten years) after arthroscopic treatment. Five patients had had localized lesions and had been managed with local resection; all five had improvement, with no apparent recurrence. The remaining twenty patients had had diffuse disease. Of these twenty, eleven had had a complete arthroscopic synovectomy. All eleven had definite improvement in pain and function, and almost all had a decrease in synovitis and an increase in the range of motion of the knee; the disease recurred in only one. The other nine patients had had a partial arthroscopic synovectomy. Although most had some improvement in function and range of motion and a decrease in pain and synovitis, the disease recurred in five of the nine. Thus, in the patients who had had diffuse pigmented villonodular synovitis, the rate of recurrence was lower in those who had had a complete arthroscopic synovectomy than in those who had had a partial arthroscopic synovectomy (p = 0.01).


Barriers to pediatric referral in managed care systems.

Anecdotal evidence suggests that children's access to pediatric subspecialty and inpatient care is hampered by referral barriers imposed by managed care systems. To identify such barriers and determine how they affect the referral process, a sample of American Academy of Pediatrics Fellows (n = 1598) was surveyed. The response rate was 79.1% (n = 1264). Of those pediatricians in direct patient care (n = 956), 71.4% participated in a managed care plan. Pediatricians referred patients in managed care systems somewhat less frequently than in traditional pay systems: 8.7% and 6.9% referred managed care patients to subspecialists and inpatient care, respectively, less often. More than 20% and 10% of pediatricians with patients in managed care systems had at least one referral to subspecialist care and inpatient care, respectively, denied in the previous year. Pediatricians experienced more barriers in preferred provider organizations than in health maintenance organizations. These data suggest that utilization management programs, such as those used in managed care systems, may limit necessary access to pediatric subspecialty and inpatient care.


The case for using waist to hip ratio measurements in routine medical checks.

To provide a rationale for using waist:hip ratio (WHR) measurements in clinical practice. The article reviews the literature on body fat distribution back to the mid 1950s. Studies are reviewed which show a clear association between abdominal obesity and a range of ailments including coronary events, hypertension, blood lipid levels, cholecystectomy, diabetes and gallbladder disease. Key data on the correlation of body fat distribution and health risks are summarised. Abdominal fat measured by a WHR may be a better single predictor of many diseases than other risk factors such as overall obesity, hypertension, smoking, or hypercholesterolaemia. The association between WHR and risk indicators appears to be "dose" related, and independent of sex, race and age. High WHRs, however, are more characteristic of men with lower socioeconomic status, whereas weight control programs are more commonly developed for women. A reorientation of weight control initiatives based on health rather than aesthetic priorities is needed. Measurement of WHR should be a routine part of clinical assessments. The predictability of the measure can be improved by combining it with a measure of body mass.


The appropriate goals of antihypertensive therapy: neither too much nor too little.

Although the treatment of hypertension has increased markedly during the last decade, many patients have been left undertreated, including many of the disadvantaged, the elderly, and those at relatively high overall risk for cardiovascular disease. A rapidly growing number of patients, however, are being exposed to overtreatment with resultant interference with their quality of life and potential hazards to their health. These include patients who are diagnosed and treated without adequate documentation of the presence of persistent hypertension, patients who are not appropriately managed with nondrug therapies, and patients who are given inappropriate and overly aggressive drug therapies. Better recognition of the frequency and potential hazards of overtreatment is needed so that more appropriate goals of antihypertensive therapy can be established and maintained.


Genomic structure and mapping of the chromosomal gene for transcobalamin I (TCN1): comparison to human intrinsic factor.

Transcobalamin I (TCI) is a vitamin B12 binding protein that is found in the secondary granules of mature neutrophils. The expression of the gene for TCI (TCN1) within neutrophils has been shown to be restricted to the later stages of myeloid development and can therefore be used as a marker for granulocyte differentiation. To study transcriptional control regions important in late stage myeloid gene regulation the genomic sequence for TCN1 has been cloned. Clones were isolated from a genomic library constructed in Charon 4A using homologous full-length cDNA probes. Southern blot analysis showed the gene to reside on five EcoRI fragments totaling 14 kb in length. Two overlapping phage clones, containing the entire 14 kb, were isolated and the introns and exons were mapped using Southern blotting and dideoxy sequencing of subclones. The cDNA is represented by nine exons contained within 12 kb of genomic DNA. Comparison of the genomic structure to gastric intrinsic factor (GIF), another vitamin B12 binding protein, revealed a strikingly similar intron/exon structure, with several positionally conserved splice sites. The gene was localized to chromosome 11 using in situ hybridization.


Crystalline ribonuclease A loses function below the dynamical transition at 220 K.

When the dynamic properties of many different proteins are plotted as a function of temperature, biphasic behaviour is observed, with a broad transition centred around 220 K. Atomic mean-square displacements from X-ray crystallography and Mössbauer scattering show this behaviour, as do electron transfer rates and dynamic information from inelastic neutron scattering. Molecular dynamics simulations over a range of temperatures also exhibit a transition at about 220 K: high-temperature atomic fluctuations are dominated by anharmonic collective motions of bonded and nonbonded groups of atoms, but below 220 K the predominant dynamic behaviour is harmonic vibration of individual atoms. Here we show by high-resolution X-ray diffraction that crystalline ribonuclease A does not bind substrate or inhibitor at 212 K but will bind either rapidly at 228 K. Once bound at the higher temperature, inhibitor cannot be washed off after the enzyme is cooled to below the transition temperature. These results suggest that enzyme flexibility is required for catalytic function.


HTLV-I infection in selected populations in Australia and the western Pacific region.

The prevalence of infection with human T lymphotropic virus type I (HTLV-I) in 19,975 blood samples from Australia and the western Pacific was determined by measuring the presence of specific antibody (anti-HTLV-I) by enzyme-linked immunosorbent assay (ELISA) with confirmation by western blot and/or radioimmunoprecipitation techniques. In Australia no evidence of HTLV-I infection was found in injecting drug users, patients with the acquired immunodeficiency syndrome (AIDS), subjects attending a sexually transmitted diseases clinic, female prostitutes, or transfusion recipients. A low prevalence of infection was detected in people with haemophilia (0.5%) and in male homosexuals (0.5%-1%). No antibody was detected in sera from Vanuatu, Kiribati, American Samoa, the Cook Islands, New Caledonia, the Federated States of Micronesia, French Polynesia and Fiji, but a low frequency of anti-HTLV-I was detected in sera from the Solomon Islands (1.2%) and Nauru (0.6%).


Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase.

Superoxide dismutase enzymes protect aerobic organisms from oxygen-mediated free-radical damage. Crystallographic structures of recombinant human Cu,Zn superoxide dismutase have been determined, refined, and analyzed at 2.5 A resolution for wild-type and a designed thermostable double-mutant enzyme (Cys-6----Ala, Cys-111----Ser). The 10 subunits (five dimers) in the crystallographic asymmetric unit form an unusual stable open lattice with 80-A-diameter channels. The 10 independently fit and refined subunits provide high accuracy, error analysis, and insights on loop conformations. There is a helix dipole interaction with the Zn site, and 14 residues form two or more structurally conserved side-chain to main-chain hydrogen bonds that appear critical to active-site architecture, loop conformation, and the increased stability resulting from the Cys-111----Ser mutation.