Measurement of β-hydroxybutyrate in acute hyperketonemia in human brain

We report the measurement of D-beta-hydroxybutyrate (BHB) in the brains of six normal adult subjects during acute infusions of BHB. We used high field in vivo (1)H magnetic resonance (MR) spectroscopy in the occipital lobe in conjunction with an acute infusion protocol to elevate plasma BHB levels from overnight fasted levels (0.20 +/- 0.10 mM) to a steady state value of 2.12 +/- 0.30 mM. At this level of hyperketonemia, we determined a tissue BHB level of 0.24 +/- 0.04 mM. No increases in brain lactate levels were seen in these data. The concentrations of BHB and lactate were both considerably lower in comparison with previous data acquired in fasted adult subjects. This suggests that up-regulation of the monocarboxylic acid transporter occurs with fasting.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Some differences in intracellular distribution and properties of the chymotrypsin-like esterase activity of human and rabbit neutrophils

The intracellular localization and properties of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase acitivity) of the rabbit peritoneal neutrophil has been studied and shown to differ from that of the human neutrophil.The major portion of the esterase activity in the rabbit neutrophil is in the 100 000 × g supernatant fraction with distinctly less activity in the lysosomal fraction. The 100 000 × g supernatant contained the highest relative specific activity of any of the subcellular fractions. Rabbit peripheral blood neutrophils gave the same distribution.The 100 000 × g supernatant esterase is 95% esterase 1 and 5% esterase 3, whereas, the lysosomal esterase is 78% esterase 1, 10–16% esterase 2 and 9% esterase 3 as defined by their ability to be inhibited by p-nitrophenyllethyl-5-chloropentylphosphonate. The 100 000 × g supernatant The 100 000 × g supernatant and lysosomal esterase activities further differ in their susceptibility to other inhibitors, their pH optima, ease of elution from DEAE and isoelectric points. Two molecular weight species of 174 000 and 70 000 were found in the 100 000 × g supernatant fraction and extracts of the lysosomal fraction but usually in differing proportions.In confirmation of others, essentially all of the chymotrypsin-like esterase activity ($\text{N-}\text{acetyl}\text{-}\text{DL}\text{-}\text{phenlylalanine}\text{β-}\text{naphthyl}$ esterase activity) of the human neutrophil is in the lysosomal fraction, unlike the rabbit cell. The human neutrophil esterase was less susceptible to inhibition by p-nitrophenylethyl-5-chloropentylphosphonate and diisopropylphosphofluoridate but more susceptible to soybean trypsin inhibitor than rabbit esterase activity. The pH optimum of the human neutrophil esterase differed from either the rabbit lysosomal or 100 000 × g supernatant esterase, as did the isoelectric point and molecular weights.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.     

Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans

Pentoxifylline increases erythrocyte flexibility, reduces blood viscosity, and inhibits platelet aggregation and is thus used in the treatment of peripheral vascular disease. It is transformed into at least seven phase I metabolites, of which two, M1 and M5, are active. The reduction of the keto group of pentoxifylline to a secondary alcohol in M1 takes place chiefly in erythrocytes, is rapidly reversible, and creates a chiral center. The aims of this study were: to develop HPLC methods to separate the enantiomers of M1, to investigate the kinetics of the reversible biotransformation of pentoxifylline to (R)- and (S)-M1 in hemolysed erythrocyte suspension, and to quantify the formation of the enantiomers of M1 (as well as M4 and M5) after intravenous and oral administration of pentoxifylline to human volunteers. (R)- and (S)-M1 could be separated preparatively on a cellobiohydrolase column, while determination in blood or plasma was by HPLC after chiral derivatization with diacetyl-L-tartaric acid anhydride. The metabolism of pentoxifylline to (R)-M1 in suspensions of hemolysed erythrocytes followed simple Michaelis-Menten kinetics (K(m) = 11 mM), while that to (S)-M1 was best described by a two-enzyme model (K(m) = 1.1 and 132 mM). Studies with inhibitors indicated that the enzymes were of the carbonyl reductase type. At a therapeutic blood concentration of pentoxifylline, the calculated rate of formation of (S)-M1 is 15 times higher than that of the (R)-enantiomer. Back-conversion of M1 to pentoxifylline was 3-4 times faster for the (S)- than for the (R)-enantiomer. In vivo, the R:S plasma concentration ratio of M1 ranged from 0.010-0.025 after intravenous infusion of 300 or 600 mg of pentoxifylline, and from 0.019-0.037 after oral administration of 600 mg. The biotransformation of pentoxifylline to M1 was thus highly stereoselective in favor of the (S)-enantiomer both in vitro and in vivo.     

Pseudo-inhibitors of neutrophil superoxide production: evidence that soybean-derived polypeptides are superoxide dismutases

We have previously reported the purification of polypeptides from soybean which are potent inhibitors of superoxide production by human neutrophils. We now report that neither oxygen uptake nor hydrogen peroxide production by stimulated neutrophils is affected by these inhibitors. Furthermore, the E-1 and E-3 polypeptides inhibit ferricytochrome c reduction by a xanthine oxidase superoxide generation system. The inhibitory activity of E-3 in the model system is blocked by 1 mM KCN while E-1 is only slightly cyanide sensitive. Atomic absorption analysis of E-1 and E-3 polypeptides reveal copper in the latter and manganese in the former. Thus, E-3 is a copper-containing superoxide dismutase while E-1 appears to be a manganese-containing superoxide dismutase.     

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE₂, PGD₂, and PGF_2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A₂ (PLA₂) isozymes revealed that cytosolic PLA₂α, but not calcium-independent PLA₂s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.     

Structure-activity studies of cyclic ketone inhibitors of the serine protease plasmin: design, synthesis, and biological activity

Three series of cyclic ketone inhibitors were synthesized and evaluated against the serine protease plasmin. Peptide inhibitors that incorporated 3-oxotetrahydrofuran and 3-oxotetrahydrothiophene 1,1-dioxide groups had the highest activities. Alkylamino substituents, which were designed to bind in the S1 subsite of plasmin, were attached to the inhibitors. Compounds 5c and 5g, which incorporated 6-aminohexyl substituents, were found to be optimal and demonstrated IC₅₀ values in the low micromolar range. Incorporating conformationally constrained peptide segments into the inhibitors did not improve their activities.     

DDMP-conjugated Saponin (soyasaponin βg) Isolated from American Groundnut (Apios americana)

A DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one)-saponin, named soyasaponin βg, was isolated from rootstock of the American groundnut (Apios americana). The structure was identified by ¹H-NMR and ¹³C-NMR spectroscopy and by chemical techniques. The distribution of this DDMP-saponin in the rootstock was detected as the brown color produced by the reaction with FeCl₃. A high concentration of DDMP-saponin was observed around the cells in fibrovascular bundle connecting the stem to plumule.     

Hydrocarbon biosynthesis in Triatoma infestans eggs

Triatoma infestans eggs are shown to synthesize hydrocarbons. Radio-gas chromatography was used to demonstrate metabolism of [1-¹⁴C]propionate into precursor methyl-branched fatty acids and into methyl-branched hydrocarbons in T. infestans eggs. These reactions have not been demonstrated previously in insect eggs. An in vivo study showed that hydrocarbons are also transported to eggs by the hemolymph. Inhibition of hydrocarbon synthesis by sodium trichloroacetate (NaTCA) was correlated with reduced oviposition, reduced hatchability, and reduced insect survival. Scanning electron microscopy showed impoverishment of the eggs' epicuticular waxes following NaTCA treatment. © 1994 Wiley-Liss, Inc.