Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k_cat/K_m). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively “inhibitor susceptible” (K_i of 14 ± 1 μM for clavulanic acid versus K_i of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K_m values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the “relatively” clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.     

Crystal structures of KPC-2 β-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K_m value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K_m value of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (k_inact) was 0.034 ± 0.003 s⁻¹. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.     

Formation of 7-carboxyheptyl radical induced by singlet oxygen in the reaction mixture of oleic acid, riboflavin and ferrous ion under the UVA irradiation

Identification of the radicals was performed for the standard reaction mixtures, which contained 4.3 mM oleic acid, 25 µM riboflavin, 1 mM FeSO₄(NH₄)₂SO₄, 10 mM cholic acid, 40 mM phosphate buffer (pH 7.4) and 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone under the UVA irradiation (365 nm), using an electron spin resonance, an high performance liquid chromatography-electron spin resonance and an high performance liquid chromatography-electron spin resonance-mass spectrometry. The electron spin resonance and high performance liquid chromatography-electron spin resonance measurements of the standard reaction mixtures showed prominent signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) and peaks 1 and 3 (retention times, 37.0 min and 49.0 min). Since the peak 3 was not observed for the standard reaction mixture without oleic acid, the radical of the peak 3 seems to be derived from oleic acid. Singlet oxygens seem to participate in the formation of the oleic acid-derived radicals because the peak height of the peak 3 observed in the standard reaction mixture of D₂O increased to 308 ± 72% of the control. The high performance liquid chromatography-electron spin resonance-mass spectrometry analysis showed that 7-carboxyheptyl radical forms in the standard reaction mixture.     

Vitamin B12 uptake by intestinal microorganisms: mechanism and relevance to syndromes of intestinal bacterial overgrowth

The mechanism of bacterial uptake of vitamin B₁₂, the spectrum of microorganisms capable of such uptake, and the factors involved were the subject of this study. Bacterial uptake of vitamin B₁₂ was found to be at least a two stage process. A primary uptake phase which was rapid (1 min or less), pH dependent, nontemperature dependent, did not require viable organisms and was insensitive to either the metabolic inhibitor dinitrophenol or to the sulfhydryl inhibitor N-ethyl-maleimide. Protein denaturation (formalin treatment or autoclaving) abolished all B₁₂ uptake. This primary uptake phase is thought to represent adsorption to binding or “receptor” sites on the cell wall. Second stage uptake was slower, pH and temperature dependent, required living bacteria, and was abolished by either dinitrophenol or N-ethyl-maleimide. This phase is dependent upon metabolic processes and may reflect transfer of B₁₂ from surface “receptor” sites into the bacterial cell. Although differences among organisms were observed in total 1 hr uptake, number of surface “receptor” sites, and relative avidities for B₁₂, all organisms except Streptococcus fecalis shared the two stage mechanism. Two Gram-positive organisms. Bacillus subtilis and Group A streptococcus, demonstrated the highest 1 hr vitamin B₁₂ uptake values; Gram-negative bacteria required 2,000-10,000 the number of organisms for comparable uptake. Binding constants (K_m) varied from 5.05 ±1.67 × 10^-10M for B. subtilis to 6.18 ±3.08 × 10^-9M for Klebsiella pneumoniae which approximate the Km for human intrinsic factor (0.38 × 10^-10M). Competition between bacteria and intrinsic factor for vitamin B₁₂ may be inferred from the similarity of these constants.     

The biosynthesis of phosphatidylinositol in human platelets

Homogenates of human platelets can mediate the synthesis of phosphatidylinositol from myoinositol and cytidine diphosphate diglyceride. The cytidine diphosphate diglyceride: myoinositol, phosphatidyl transferase activity is particulate-bound, and the highest specific activity is found in the membrane fraction. The production of phosphatidylinositol is decreased by sulfhydryl-binding agents, and the addition of thiols to the platelet homogenates increases the enzymatic activity. The reaction exhibits a pH optimum of 8.5-9.0. Divalent cations stimulate the reaction, and manganous chloride was the most effective of those investigated. The K_m of the enzyme for myoinositol is 0.27 mM, and the K_m for cytidine diphosphate diglyceride is 0.53 mM. The enzymatic activity of platelets isolated from patients with several diseases known to interfere with platelet clotpromoting function is similar to the enzymatic activity of platelets from normal donors.     

Thermally controlled biotransformation of glycyrrhizic acid via an asymmetric temperature-responsive polyurethane membrane

Separating a target product from a relatively complex bioreaction system is often difficult. In this work, a “smart” bioreaction system was developed by using the special characteristic of temperature-responsive polyurethane (TRPU). By combining solvent evaporation with a wet phase inversion technique, an asymmetric membrane consisting of an integral and dense skin layer supported by a porous sublayer was prepared from a thermally responsive polyurethane that experiences a sudden free volume increase upon heating through a phase transition temperature of 56 °C. Subsequently, the asymmetric TRPU membrane served as the carrier of an immobilized enzyme, wherein β-glucuronidase was multipoint-conjugated by using biotin and streptavidin on the porous sublayer. Then, the material-asymmetric TRPU membrane served jointly as the antennae as well as the actuator, which reversibly responds to temperature to switch (on–off) the access of the reactant glycyrrhizic acid (GL). Under the optimal temperature (40 °C) and pH (7.0) conditions, the immobilized β-glucuronidase contributed to almost 33% yield of glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) of the isolated counterpart for the same concentration of substrate (250 mg L⁻¹) reaction for 24 h, while costing 1% of that of the isolated β-glucuronidase. Kinetic results showed that V_max and K_m values were 8.89 × 10³ mg L⁻¹ and 2.30 × 10³ mg L⁻¹ h⁻¹, respectively. The specific functional polymer-immobilized β-glucuronidase design serves as a bioreactor of GL into GAMG, as well as a separator deliberately irritated and controlled by temperature. This “smart” support material presents a potential facilitator for the separation of complex biotransformation reactions.

A “smart” bioreaction system was developed by using the special characteristic of a temperature-responsive polyurethane (TRPU). This “smart” support material presents a potential benefit of separation for complex biotransformation reactions.     

Beta-Lactamases Produced by a Pseudomonas aeruginosa Strain Highly Resistant to Carbenicillin

A Pseudomonas aeruginosa strain isolated at Besançon Hospital, France, proved to be highly resistant to carbenicillin and showed a high hydrolytic activity toward this antibiotic. We clearly demonstrated that two β-lactamases were synthetized: one of them, constitutive, has its enzymatic activity directed mainly toward penicillins, and carbenicillin appears to be its best substrate (higher V_max); thus, this β-lactamase is a “carbenicillinase” that differs from the well-known “TEM-like” enzymes. The isoelectric point of this carbenicillinase is 5.30 ± 0.03. The other one is an inducible cephalosporinase, very similar to the cephalosporinases usually found in these organisms. Its isoelectric point is 8.66 ± 0.04. These two enzymes have been separated by affinity chromatography and isoelectric focusing. The kinetic constants were measured by computerized microacidimetry.     

Multitissue insulin resistance despite near-normoglycemic remission in Africans with ketosis-prone diabetes

OBJECTIVE—To characterize insulin action in Africans with ketosis-prone diabetes (KPD) during remission.RESEARCH DESIGN AND METHODS—At Saint-Louis Hospital, Paris, France, 15 African patients with KPD with an average 10.5-month insulin-free near-normoglycemic remission period (mean A1C 6.2%) were compared with 17 control subjects matched for age, sex, BMI, and geographical origin. Insulin stimulation of glucose disposal, and insulin suppression of endogenous glucose production (EGP) and nonesterified fatty acids (NEFAs), was studied using a 200-min two-step (10 mU · m⁻² body surface · min⁻¹ and 80 mU · m⁻² · min ⁻¹ insulin infusion rates) euglycemic clamp with [6,6-²H₂]glucose as the tracer. Early-phase insulin secretion was determined during an oral glucose tolerance test.RESULTS—The total glucose disposal was reduced in patients compared with control subjects (7.5 ± 0.8 [mean ± SE] vs. 10.5 ± 0.9 mg · kg⁻¹ · min⁻¹; P = 0.018). EGP rate was higher in patients than control subjects at baseline (4.0 ± 0.3 vs. 3.0 ± 0.1 mg · kg⁻¹ · min⁻¹; P = 0.001) and after 200-min insulin infusion (10 mU · m⁻² · min⁻¹: 1.6 ± 0.2 vs. 0.6 ± 0.1, P = 0.004; 80 mU · m⁻² · min⁻¹: 0.3 ± 0.1 vs. 0 mg · kg⁻¹ · min⁻¹, P = 0.007). Basal plasma NEFA concentrations were also higher in patients (1,936.7 ± 161.4 vs. 1,230.0 ± 174.1 μmol/l; P = 0.002) and remained higher after 100-min 10 mU · m⁻² · min⁻¹ insulin infusion (706.6 ± 96.5 vs. 381.6 ± 55.9 μmol/l; P = 0.015).CONCLUSIONS—The triad hepatic, adipose tissue, and skeletal muscle insulin resistance is observed in patients with KPD during near-normoglycemic remission, suggesting that KPD is a form of type 2 diabetes.Impairment of insulin sensitivity is considered the background defect that interplays with the add-on progressive β-cell dysfunction to underlie the development of type 2 diabetes (1,2). An atypical form of diabetes, ketosis-prone diabetes (KPD), has been described over the past 2 decades and may represent a significant proportion of diabetes cases in people of sub-Saharan African origin (3,4). Patients with KPD present at onset with acute hyperglycemia, usually >30 mmol/l, and ketosis or ketoacidosis as type 1 diabetes but do not have autoimmune markers against the islet β-cell (3,5–7). The correction of those insulin-requiring acute-phase disorders is followed in >50% of cases by an insulin-free near-normoglycemic remission weeks to months later (8–10), thus resembling the course of type 2 diabetes. The pathogenesis and, consequently, the classification of KPD are still debated. It was classified under idiopathic type 1 diabetes or type 1B diabetes (11). However, growing evidence based on clinical and metabolic studies suggests its high phenotypical likeness to type 2 diabetes, and “ketosis-prone type 2 diabetes” has been proposed as a provisional name and is being used elsewhere (4,8,12). Metabolic studies have evidenced insulin secretion deficiency as the major determinant of the ketotic onset (8–10). This deficit is marked by a loss of acute-phase insulin secretion in response to intravenous glucose (10) or a decrease in C-peptide response to glucagon (9,10). The subsequent remission process is due to a restoration, at least partial, of the β-cell insulin secretory capacity after achievement of good metabolic control (8,10). Insulin action was assessed in three reports, but only toward glucose metabolism, and was found to be normal or decreased while patients were in good metabolic control (6,8,10). Moreover, most studies on KPD have been reported in African-Americans who are more overweight than native Africans and may be metabolically different from them, as suggested earlier (13).In this study, we aimed at characterizing all aspects of insulin action in Africans with KPD when in the near-normoglycemic state without insulin treatment compared with control subjects of the same geographic origin.     

Biochemical and kinetic characterization of laccase and manganese peroxidase from novel Klebsiella pneumoniae strains and their application in bioethanol production

Laccase (lac) and manganese peroxidase (MnP) enzymes from the novel Klebsiella pneumoniae isolates, grown on lignin basic media (LBM) were purified by 80% ammonium sulphate fractionation, dialysis and DEAE-sepharose column chromatography. The optimum temperatures for laccase production were 60 °C, 50 °C and 50 °C and for MnP production were 50 °C, 70 °C and 60 °C from NITW715076_2, NITW715076_1 and NITW715076 isolates, respectively. The optimal pH for production was found to be 5 for production of both the enzymes from all the isolates. 2.8–3.5 fold enzyme purification was achieved retaining around 60–70% of the initial activity. SDS-PAGE revealed the molecular mass of laccase and MnP to be 66 kDa and 48 kDa, respectively. The substrate ABTS and MnSO₄ exhibited more specificity towards NITW715075_2 derived laccase and MnP (lac: K_m = 0.38 mM, V_max = 71.42 U ml⁻¹; MnP: K_m = 0.17 mM, V_max = 106.38 U ml⁻¹) compared to NITW715076_1 (lac: K_m = 3.97 mM, V_max = 148.8 U ml⁻¹; MnP: K_m = 0.90 mM, V_max = 114.67 U ml⁻¹) and NITW715076 (lac: K_m = 0.46 mM, V_max = 23.42 U ml⁻¹; MnP: K_m = 0.19 mM, V_max = 108.10 U ml⁻¹) derived. l-Cysteine and sodium azide imposed a strong inhibitory effect on the activities of both the enzymes. EDTA inhibited laccase and MnP activity at higher concentration. SDS strongly inhibited activity while for MnP it showed less inhibitory effect. The enzymes were employed for ethanol production from rice and wheat bran biomass which showed 39.29% improved production compared to control. After evaluating the applicability of these enzymes it can be suggested that the ligninolytic enzyme of Klebsiella pneumoniae isolates could be effectively employed in enhanced ethanol production and could be explored for other putative applications.

Upto 3 fold purified laccase and MnP from novel Klebsiella isolates, mediated ethanol production from rice and wheat bran substrates lead to almost 40% improvement in production profile.     

Arylbenzazepines Are Potent Modulators for the Delayed Rectifier K+ Channel: A Potential Mechanism for Their Neuroprotective Effects

(±) SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D₁-like dopamine receptor agonistic activity. The precise mechanism for the (±) SKF83959-mediated neuroprotection remains elusive. We report here that (±) SKF83959 is a potent blocker for delayed rectifier K⁺ channel. (±) SKF83959 inhibited the delayed rectifier K⁺ current (I_K) dose-dependently in rat hippocampal neurons. The IC₅₀ value for inhibition of I_K was 41.9±2.3 µM (Hill coefficient=1.81±0.13, n=6), whereas that for inhibition of I_A was 307.9±38.5 µM (Hill coefficient=1.37±0.08, n=6). Thus, (±) SKF83959 is 7.3-fold more potent in suppressing I_K than I_A. Moreover, the inhibition of I_K by (±) SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) SKF83959 with the K⁺ channel. The intracellular application of (±) SKF83959 had no effects of on I_K, indicating that the compound most likely acts at the outer mouth of the pore of K⁺ channel. We also tested the enantiomers of (±) SKF83959, R-(+) SKF83959 (MCL-201), and S-(−) SKF83959 (MCL-202), as well as SKF38393; all these compounds inhibited I_K. However, (±) SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I_K , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) SKF83959.     

Supramolecular interaction of sanguinarine dye with sulfobutylether-β-cyclodextrin: modulation of the photophysical properties and antibacterial activity

The noncovalent host–guest interaction of sanguinarine (SGR), a benzophenanthridine alkaloid, with a nontoxic, water soluble sulfobutylether-β-cyclodextrin (SBE₇βCD, commercially available as Captisol) macrocyclic host has been investigated using ground-state optical absorption, and steady-state and time-resolved fluorescence measurements. The pH-dependent changes in the absorbance of the dye at 327 nm showed a pK_a value of 7.5, which has been shifted to 8.1 in the presence of SBE₇βCD. The changes in the pK_a values, absorption and fluorescence spectra, and fluorescence lifetime values of these two forms of SG with SBE₇βCD indicate complex formation between them. The cationic form shows 3 times higher interaction towards SEB₇βCD (K = 1.2 × 10⁴ M⁻¹) as compared to the neutral form (K = 3.9 × 10³ M⁻¹) which leads to a moderate upward pK_a shift (pK_a values of SGR shifted by more than 0.6 units). The subsequent fluorescence “turn off” was demonstrated to be responsive to chemical stimuli, such as metal ions (Ca²⁺ ions). Upon addition of Ca²⁺ ions, nearly quantitative dissociation of the complex was established to regenerate the free dye and result in fluorescence “turn on”. Apart from improving the stability under ambient light conditions, the upward pK_a shift of SGR in the presence of SBE₇βCD results in increasing the antibacterial activity of the SBE₇βCD:SGR complex compared to that of the free dye towards four pathogenic micro-organisms at the physiological pH range. This work further compares SGR interaction with parent β-cyclodextrin.

The noncovalent host-guest interaction of sanguinarine (SGR) with a nontoxic, water soluble sulfobutylether-beta-cyclodextrin macrocyclic host modulates the photophysical properties, improves the photostability and antibacterial activity of SGR.     

An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (K_m = 30 ± 4 μM) at the C-2 position of the fucofuranose ring in the presence of ATP (K_m = 200 ± 20 μM) or GTP (K_m = 350 ± 60 μM) with a k_cat of 2.2 ± 0.1 min⁻¹. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k_cat and K_m values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5-dihydrohygromycin A and 5-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.     

Monophenol Monooxygenase and Lincomysin Biosynthesis in Streptomyces lincolnensis

Monophenol monooxygenase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase EC 1.14.18.1) was studied in melanin-positive and melanin-negative mutants of Streptomyces lincolnensis NCIB 9413, varying in the lincomycin synthesizing ability. The activities of laccase and tyrosine phenol lyase (EC 4.1.99.2) are absent in this organism. The monophenol monooxygenase catalyzes hydroxylation of monophenols (K_m and V_max for l-tyrosine, 2 × 10⁻⁴ M and 8.0 nmol of O₂/min per ml, respectively) at a slower rate than it dehydrogenates diphenols to o-quinones (K_m and V_max for l-3,4-dihydroxyphenylalanine, 7 × 10⁻⁵ M and 51.7 nmol of O₂/min per ml, respectively. It is inhibited by KCN, β-mercaptoethanol, ethylenediaminetetraacetate, dipyridyl, thiourea, p-aminobenzoic acids and by some tryptophan metabolites. Changes in the activity of monophenol monooxygenase caused by mutation or by inhibitors are reflected in the synthesis of the antibiotic. Its participation in the biogenesis of the propylhygric moiety of lincomycin is discussed.     

Increased daily walking improves lipid oxidation without changes in mitochondrial function in type 2 diabetes

OBJECTIVE—To determine whether increased daily physical activity improves mitochondrial function and/or lipid oxidation in type 2 diabetes.RESEARCH DESIGN AND METHODS—Volunteers with (n = 10) and without (n = 10) type 2 diabetes were matched for habitual physical activity, age, sex, and weight. Basal and maximal mitochondrial activity, physical activity, and resting substrate oxidation were measured at baseline and after 2 and 8 weeks of increased physical activity.RESULTS—Baseline physical activity (6,450 ± 851 vs. 7,638 ± 741 steps/day), basal ATP use (12 ± 1 vs. 12 ± 1 μmol · ml⁻¹ · min⁻¹), phosphocreatine recovery from exercise (31 ± 5 vs. 29 ± 3 s), and basal lipid oxidation (0.57 ± 0.07 vs. 0.65 ± 0.06 mg · kg body wt⁻¹ · min⁻¹) were similar in people with and without type 2 diabetes. There was a significant increase in physical activity after 8 weeks (12,322 ± 1,979 vs. 9,187 ± 1,159 steps/day, respectively). Following increased physical activity, there were no changes in basal ATP use or phosphocreatine recovery after exercise in either group. Basal lipid oxidation increased after 8 weeks of increased physical activity in people with type 2 diabetes (0.79 ± 0.08 mg · kg⁻¹ · min⁻¹) but not people without (0.68 ± 0.13 mg · kg body wt⁻¹ · min⁻¹).CONCLUSIONS—Resting and maximal ATP turnover are not impaired in people with well-controlled type 2 diabetes compared with control subjects matched for physical activity as well as age and weight. Increased unsupervised daily physical activity is sustainable and improves lipid oxidation independent of change in mitochondrial activity in people with type 2 diabetes.The potential role of the mitochondria in the development of insulin resistance and type 2 diabetes has recently attracted much interest. Muscle biopsies taken from people with type 2 diabetes demonstrate smaller mitochondria and lower activities of oxidative enzymes compared with those of lean individuals without diabetes (1). Insulin-resistant people with a family history of diabetes have reduced basal mitochondrial activity in skeletal muscle compared with insulin-sensitive individuals (2). These observations, in combination with others (3–6), raise the possibility that mitochondrial defects could underlie type 2 diabetes. Defects in oxidative function could possibly help explain the impaired fatty acid oxidation (7) and elevated intramyocellular lipid (IMCL) (8) characteristic of impaired insulin action and type 2 diabetes. The elevated intramuscular lipid may affect insulin signaling in skeletal muscle (5), exacerbating insulin resistance.However, other studies have not observed abnormalities in basal mitochondrial activity in skeletal muscle of people with type 2 diabetes (9). Recent biopsy work has also shown that differences in oxidative enzymes between people with and without type 2 diabetes disappear when corrected for mitochondrial density (10). These data raise the possibility that type 2 diabetes is associated with normal mitochondrial function but that the mitochondrial capacity is reduced. This is an important differentiation, as it holds implications for the therapeutic approach to type 2 diabetes.People with type 2 diabetes are more sedentary than those without diabetes (11). It is clear that reversing this sedentary lifestyle with physical activity and/or exercise can produce significant improvements in long-term glucose control (12). These benefits could be mediated, at least in part, by changes in mitochondrial function (13). In people with type 2 diabetes, moderate-intensity exercise combined with moderate weight loss produced a significant improvement in insulin sensitivity and mitochondrial density (14). However, such moderate intensity exercise programs are difficult to implement and usually require close supervision. In contrast, unsupervised walking has been shown to produce significant improvements in long-term glucose control and is a sustainable behavior over long periods of time (2 years) (15). Little is known about how low-intensity physical activity interventions such as walking influence muscle metabolism in people with type 2 diabetes.This study was designed to 1) determine whether there are differences in basal and stimulated mitochondrial activity in people with type 2 diabetes compared with physical activity–matched control subjects and 2) establish whether an increase in daily physical activity is associated with changes in mitochondrial ATP turnover and changes in lipid oxidation.     

Type 2 Diabetes Prevention in the Real World: Three-year results of the GOAL Lifestyle Implementation Trial

OBJECTIVE

We study the effectiveness of the GOAL Lifestyle Implementation Trial at the 36-month follow-up.

RESEARCH DESIGN AND METHODS

Participants (n = 352, type 2 diabetes risk score FINDRISC = 16.2 ± 3.3, BMI 32.6 ± 5.0 kg/m²) received six lifestyle counseling sessions over 8 months. Measurements were at baseline, 12 months (88.6%), and 36 months (77.0%).

RESULTS

Statistically significant risk reduction at 12 months was maintained at 36 months in weight (−1.0 ± 5.6 kg), BMI (−0.5 ± 2.1 kg/m²), and serum total cholesterol (−0.4 ± 1.1 mmol/l).

CONCLUSIONS

Maintenance of risk reduction in this “real world” trial proves the intervention''s potential for significant public health impact.The Goal Lifestyle Implementation Trial (1,2) replicated most of the findings from the Finnish Diabetes Prevention Study (DPS) (3,4) in primary health care settings, demonstrating that lifestyle counseling can be effective and feasible in routine care. We report findings on sustainability of the results at 3 years.     

Identification of the radicals formed in the reactions of some endogenous photosensitizers with oleic acid under the UVA irradiation

Electron spin resonance measurements were performed for the reactions of some endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid or β-nicotinamide adenine dinucleotide or β-nicotinamide adenine dinucleotide phosphate or pyridoxal-5''-phosphate or urocanic acid) with oleic acid under the ultraviolet light A irradiation using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin trap reagent. Of the endogenous photosensitizers, prominent electron spin resonance signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) were observed for the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid), suggesting that radical species form in the reaction mixtures. Singlet oxygen seems to participate in the formation of the radicals because the electron spin resonance peak heights increased for the reactions in D₂O to a great extent. A high performance liquid chromatography-electron spin resonance-mass spectrometry was employed to identify the radicals formed in the reactions of the endogenous photosensitizers (flavin mononucleotide or flavin adenine dinucleotide or folic acid) with oleic acid under the ultraviolet light A irradiation. The high performance liquid chromatography-electron spin resonance-mass spectrometry analyses showed that 7-carboxyheptyl and 1-(3-carboxypropyl)-4-hydroxybutyl radicals form in the reaction mixture of flavin mononucleotide (or flavin adenine dinucleotide or folic acid).     

Effect of Calcium on Superoxide Production by Phagocytic Vesicles from Rabbit Alveolar Macrophages

Phagocytic vesicles from rabbit lung macrophages produced superoxide in the presence of NADH or NADPH. At 37°C, these vesicles generated 51±7.8 nmol O₂⁻/min per mg protein in the presence of 0.5 mM NADPH. The apparent K_m for NADPH and NADH (66 and 266 μM, respectively), the pH optimum for the reaction (6.9), and the cyanide insensitivity were similar to properties of plasma membrane-rich fractions of stimulated polymorphonuclear leukocytes studied by others. The activity of the phagocytic vesicles was trypsin sensitive. The specific superoxide-generating activity of macrophage phagocytic vesicles isolated from cells incubated up to 90 min with phagocytic particles remained constant.     

Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K_m values of 4.70 ± 0.059 (mean ± standard error of the mean) and 9.75 ± 1.64 μM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC₅₀] = 68.6 ± 5.20 nM) than pyrimethamine (IC₅₀ = 55.0 ± 2.08 μM) and trimethoprim (IC₅₀ = 50 ± 12.5 μM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.     

Effects of Antiviral Drugs on Organic Anion Transport in Human Placental BeWo Cells

Placental drug transfer is important for achieving better pharmacotherapy in pregnant women and in fetuses. In the present study, we examined the effects of anti-hepatitis C virus (HCV) and anti-HIV drugs on organic anion transport in human placental BeWo cells. The cellular uptake of two fluorescence organic anions, 8-(2-[fluoresceinyl]aminoethylthio)adenosine-3′,5′-cyclic monophosphate (8-FcAMP) and fluorescein, was temperature and concentration dependent. The Michaelis constant (K_m) and the maximum uptake rate (V_max) for 8-FcAMP transport in BeWo cells were estimated to be 6.45 ± 0.75 μM and 25.55 ± 5.93 pmol/mg protein/10 min, respectively. The K_m and V_max values for fluorescein uptake were estimated to be 31.2 ± 11.8 μM and 510.9 ± 90.6 pmol/mg protein/10 min, respectively. Several known substrates of organic anion transporters in human placenta, including atorvastatin, glibenclamide, estrone-3-sulfate, and rifampin, inhibited cellular uptake of 8-FcAMP and fluorescein in BeWo cells. Transport of 8-FcAMP and fluorescein was inhibited by the antiviral drugs boceprevir, telaprevir, elvitegravir, and maraviroc. These findings suggest that some antiviral drugs are sufficiently potent to influence placental drug transfer and cause drug-drug interactions.