Development of an in vitro model to study tooth cystogenesis.

AIM: To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. METHODOLOGY: To form spheroid structures, various numbers (1 × 104 , 5 × 104 or 1 × 105 ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis. RESULTS: Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. CONCLUSIONS: This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.

RNase 7 downregulates TH2 cytokine production by activated human T cells.

BACKGROUND: The antimicrobial peptide (AMP) RNase 7 is constitutively expressed in the epidermis of healthy human skin and has been found to be upregulated in chronic inflammatory skin diseases such as atopic dermatitis and psoriasis. Activated T cells in lesional skin of patients with atopic dermatitis (AD) and psoriasis (PSO) might be directly exposed to RNase 7. In addition to their antimicrobial activity, immunoregulatory functions have been published for several AMPs. In this study, we investigated immunoregulatory effects of the antimicrobial peptide RNase 7 on activated T cells. METHODS: Isolated human CD3+T cells were stimulated with RNase 7 and screened for possible effects by mRNA microarray analysis. The results of the mRNA microarray were confirmed in isolated CD4+T cells and in polarized TH2 cells using skin-derived native RNase 7 and a recombinant ribonuclease-inactive RNase 7 mutant. Activation of GATA3 was analysed by electrophoretic mobility shift assay. RESULTS: Treatment of activated human CD4+T cells and TH2 cells with RNase 7 selectively reduced the expression of TH2 cytokines (IL-13, IL-4 and IL-5). Experiments with a ribonuclease-inactive recombinant RNase 7 mutant showed that RNase 7 ribonuclease activity is dispensable for the observed regulatory effect. We further demonstrate that CD4+T cells from AD patients revealed a significantly less pronounced downregulation of IL-13 in response to RNase 7 compared to healthy control. Finally, we show that GATA3 activation was diminished upon cultivation of T cells with RNase 7. CONCLUSION: Our data indicate that RNase 7 has immunomodulatory functions on TH2 cells and decreases the production of TH2 cytokines in the skin.

Clinical reasoning of junior doctors in emergency medicine: a grounded theory study.

INTRODUCTION: Emergency medicine (EM) has a high case turnover and acuity making it a demanding clinical reasoning domain especially for junior doctors who lack experience. We aimed to better understand their clinical reasoning using dual cognition as a guiding theory. METHODS: EM junior doctors were recruited from six hospitals in the south of England to participate in semi-structured interviews (n=20) and focus groups (n=17) based on recall of two recent cases. Transcripts were analysed using a grounded theory approach to identify themes and to develop a model of junior doctors' clinical reasoning in EM. RESULTS: Within cases, clinical reasoning occurred in three phases. In phase 1 (case framing), initial case cues and first impressions were predominantly intuitive, but checked by analytical thought and determined the urgency of clinical assessment. In phase 2 (evolving reasoning), non-analytical single cue and pattern recognitions were common which were subsequently validated by specific analytical strategies such as use of red flags. In phase 3 (ongoing uncertainty) analytical self-monitoring and reassurance strategies were used to precipitate a decision regarding discharge. CONCLUSION: We found a constant dialectic between intuitive and analytical cognition throughout the reasoning process. Our model of clinical reasoning by EM junior doctors illustrates the specific contextual manifestations of the dual cognition theory. Distinct diagnostic strategies are identified and together these give EM learners and educators a framework and vocabulary for discussion and learning about clinical reasoning.

Striatal adenosine A2A receptor involvement in normal and large nest building deer mice: Perspectives on compulsivity and anxiety.

Obsessive-compulsive disorder (OCD) is characterized by recurring obsessive thoughts and repetitive behaviors that are often associated with anxiety and perturbations in cortico-striatal signaling. Given the suboptimal response of OCD to current serotonergic interventions, there is a need to better understand the psychobiological mechanisms that may underlie the disorder. In this regard, investigations into adenosinergic processes might be fruitful. Indeed, adenosine modulates both anxiety- and motor behavioral output. Thus, we aimed to explore the potential associations between compulsive-like large nest building (LNB) behavior in deer mice, anxiety and adenosinergic processes. From an initial pool of 120 adult deer mice, 34 normal nest building (NNB)- and 32 LNB-expressing mice of both sexes were selected and exposed to either a normal water (wCTRL) or vehicle control (vCTRL), lorazepam (LOR) or istradefylline (ISTRA) for 7- (LOR) or 28 days after which nesting assessment was repeated and animals screened for anxiety-like behavior in an anxiogenic open field. Mice were then euthanized, the striatal tissue removed on ice and the adenosine A2A receptor expression quantified. Our findings indicate that NNB and LNB behavior are not distinctly associated with measures of generalized anxiety and that ISTRA-induced changes in nesting expression are dissociated from changes in anxiety scores. Further, data from this investigation show that nesting in deer mice is directly related to striatal adenosine signaling, and that LNB is founded upon a lower degree of adenosinergic A2A stimulation.

Effects of melamine in young broiler chicks.

A study was conducted to determine the toxicity of melamine in young broilers fed graded levels of melamine. An additional objective was to determine melamine residual levels in selected tissues. One hundred and seventy-five 1-d-old male Ross broiler chicks were sorted to a randomized block design in stainless steel battery pens. Chicks were assigned to 7 dietary treatments containing 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% melamine. Each dietary treatment was fed to 5 replicate pens of 5 chicks for 21 d. Mortality increased quadratically (P<0.001) with increasing dietary concentrations of melamine. However, compared with controls, mortality was only higher (P<0.001) in birds fed≥2.5% melamine. Feed intake decreased linearly (P<0.001), whereas BW gain decreased quadratically (P<0.02) with increasing dietary concentrations of melamine. Compared with controls, both feed intake and BW gain were lower (P<0.001) only in birds fed≥1.0% melamine. Relative kidney weights increased linearly (P<0.001), whereas relative liver weights increased quadratically (P<0.05) with increasing dietary concentrations of melamine. Melamine residues in breast muscle and liver tissue increased linearly (P<0.001) with increasing dietary concentrations of melamine, whereas melamine residues in kidney and bile increased quadratically (P<0.02) with increasing dietary concentrations of melamine. Compared with controls, melamine concentrations in liver and kidney were higher (P<0.001) in birds fed all levels of melamine, whereas melamine concentrations in breast muscle and bile were only higher (P<0.001) in birds fed≥1.0% melamine. Serum albumin, total protein, globulin, and calcium increased quadratically (P<0.02) in birds as dietary melamine increased, whereas serum aspartate transaminase and gamma gluatamyltransferase increased linearly (P<0.01) with increasing levels of melamine in the diet. Renal histopathology revealed nonpolarizable melamine crystals in the collecting tubules and ducts of birds fed≥1.5% melamine. In summary, dietary melamine was toxic to broilers at concentrations≥1.0%.

Proliferation of microalgae and enterococci in the Lake Okeechobee, St. Lucie, and Loxahatchee watersheds.

This study is an analysis of relationships between microalgae (measured as chlorophyll a) and the fecal indicator bacteria enterococci. Microalgae blooms and enterococci exceedances have been occurring in Florida's recreational waterways for years. More recently, this has become a management concern as microalgae blooms have been attributed to potentially toxic cyanobacteria, and enterococci exceedances link to human infection/illness. Since both the microalgal blooms and bacterial exceedances occur in regions that receive managed freshwater releases from Lake Okeechobee, we hypothesized that both the blooms and exceedances are related to excess nutrients from the lake. Two experimental sites, on Lake Okeechobee and the St. Lucie River (downstream of the lake), plus a control site on the Loxahatchee River (which does not receive lake flow) were evaluated. The hypothesis was evaluated through three study components: 1) analysis of available long-term data from local environmental databases, 2) a year-long monthly sampling and analysis of chlorophyll a, enterococci, nutrients, and physical-chemical data, and 3) microcosm experiments with altered water/sediment conditions. Results support the hypothesis that excess nutrients play a role in both chlorophyll a and enterococci levels. For the St. Lucie River, analyses indicate that chlorophyll a correlated significantly with total Kjeldahl nitrogen (TKN) (R2 = 0.30, p = 0.008) and the strongest model for enterococci included nitrate-nitrite, TKN, total phosphorus, orthophosphorus, and turbidity in our long-term analysis (n = 39, R2 = 0.83, p ≤ 0.001). The microcosm results indicated that chlorophyll a and enterococci only persisted for 36 h in water from all sources, and that sediments from Lake Okeechobee may have allowed for sustained levels of chlorophyll a and enterococci levels. Overall similarities were observed in chlorophyll a and enterococci relationships with nutrient concentrations regardless of a Lake Okeechobee connection, as underscored by a study of flow out of the lake and downstream areas. This suggests that both nutrient-rich lake water and untreated surface water runoff contribute to microalgae blooms and enterococci exceedances in southeast Florida.

Solvent dependence of the luminescence of N-arylaminonaphthalenesulfonates.

N-Methyl,N-phenyl-2-aminonaphthalene-6-sulfonate, a fluorescence probe, adsorbs to cycloheptaamylose with a stoichiometry of 1 : 1. The fluorescence of the complex is similar to that observed when the dye is dissolved in organic solvents. Similar fluorescence is observed with the dye in ice. The results are interpreted in terms of "solvent" relaxation during the excited state lifetime of the dye.

Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis.

As an alternative to traditional, morphology-based methods, molecular techniques can provide detection of multiple species within the HAB community and, more widely, the phytoplankton community in a rapid, accurate and simultaneous qualitative analysis. These methods require detailed knowledge of the molecular diversity within taxa in order to design efficient specific primers and specific probes able to avoid cross-reaction with non-target sequences. Isolates from Florida coastal communities were sequence-analyzed and compared with the GenBank database. Almost 44% of the genotypes obtained did not match any sequence in GenBank, showing the existence of a large and still unexplored biodiversity among taxa. Based on these results and on the GenBank database, we designed 14 species-specific probes and 4 sets of specific primers. Multiple simultaneous detection was achieved with a bead array method based on the use of a flow cytometer and color-coded microspheres, which are conjugated to the developed probes. Following a parallel double PCR amplification, which employed universal primers in a singleplex reaction and a set of species-specific primers in multiplex, detection was performed in a cost effective and highly specific analysis. This multi-format assay, which required less than 4 h to complete from sample collection, can be expanded according to need. Up to 100 different species can be identified simultaneously in a single sample, which allows for additional use of this method in community analyses extended to all phytoplankton species. Our initial field trials, which were based on the 14 species-specific probes, showed the co-existence and dominance of two or more species of Karenia during toxic blooms in Florida waters.

Tracing the Early Development of Harmful Algal Blooms on the West Florida Shelf with the Aid of Lagrangian Coherent Structures.

Several theories have been proposed to explain the development of harmful algal blooms (HABs) produced by the toxic dinoflagellate Karenia brevis on the West Florida Shelf. However, because the early stages of HAB development are usually not detected, these theories have been so far very difficult to verify. In this paper we employ simulated Lagrangian coherent structures (LCSs) to trace potential early locations of the development of a HAB in late 2004 before it was transported to a region where it could be detected by satellite imagery. The LCSs, which are extracted from surface ocean currents produced by a data-assimilative HYCOM (HYbrid-Coordinate Ocean Model) simulation, constitute material fluid barriers that demarcate potential pathways for HAB evolution. Using a simplified population dynamics model we infer the factors that could possibly lead to the development of the HAB in question. The population dynamics model determines nitrogen in two components, nutrients and phytoplankton, which are assumed to be passively advected by surface ocean currents produced by the above HYCOM simulation. Two nutrient sources are inferred for the HAB whose evolution is found to be strongly tied to the simulated LCSs. These nutrient sources are found to be located nearshore and possibly due to land runoff.

Predicting abundance of desert riparian birds: validation and calibration of the Effective Area Model.

Reliable prediction of the effects of landscape change on species abundance is critical to land managers who must make frequent, rapid decisions with long-term consequences. However, due to inherent temporal and spatial variability in ecological systems, previous attempts to predict species abundance in novel locations and/or time frames have been largely unsuccessful. The Effective Area Model (EAM) uses change in habitat composition and geometry coupled with response of animals to habitat edges to predict change in species abundance at a landscape scale. Our research goals were to validate EAM abundance predictions in new locations and to develop a calibration framework that enables absolute abundance predictions in novel regions or time frames. For model validation, we compared the EAM to a null model excluding edge effects in terms of accurate prediction of species abundance. The EAM outperformed the null model for 83.3% of species (N=12) for which it was possible to discern a difference when considering 50 validation sites. Likewise, the EAM outperformed the null model when considering subsets of validation sites categorized on the basis of four variables (isolation, presence of water, region, and focal habitat). Additionally, we explored a framework for producing calibrated models to decrease prediction error given inherent temporal and spatial variability in abundance. We calibrated the EAM to new locations using linear regression between observed and predicted abundance with and without additional habitat covariates. We found that model adjustments for unexplained variability in time and space, as well as variability that can be explained by incorporating additional covariates, improved EAM predictions. Calibrated EAM abundance estimates with additional site-level variables explained a significant amount of variability (P < 0.05) in observed abundance for 17 of 20 species, with R2 values >25% for 12 species, >48% for six species, and >60% for four species when considering all predictive models. The calibration framework described in this paper can be used to predict absolute abundance in sites different from those in which data were collected if the target population of sites to which one would like to statistically infer is sampled in a probabilistic way.

Persistent transport barrier on the West Florida Shelf.

Analysis of drifter trajectories in the Gulf of Mexico has revealed the existence of a region on the southern portion of the West Florida Shelf (WFS) that is not visited by drifters that are released outside of the region. This so-called "forbidden zone" (FZ) suggests the existence of a persistent cross-shelf transport barrier on the southern portion of the WFS. In this letter a year-long record of surface currents produced by a Hybrid-Coordinate Ocean Model simulation of the WFS is used to compute Lagrangian coherent structures (LCSs), which reveal the presence of a persistent cross-shelf transport barrier in approximately the same location as the boundary of the FZ. The location of the cross-shelf transport barrier undergoes a seasonal oscillation, being closer to the coast in the summer than in the winter. A month-long record of surface currents inferred from high-frequency (HF) radar measurements in a roughly 60 km × 80 km region on the WFS off Tampa Bay is also used to compute LCSs, and these also reveal the presence of transient transport barriers. While the HF-radar-derived transport barriers cannot be unambiguously linked to the boundary of the FZ, this analysis does demonstrate the feasibility of monitoring transport barriers on the WFS using a HF-radar-based measurement system. The implications of a persistent cross-shelf transport barrier on the WFS for the development of harmful algal blooms on the shoreward side of the barrier are considered.

[Acute abdominal pain in children caused by pneumonia]. / Acute buikpijn door longontsteking bij kinderen.

Three children presented with symptoms of an acute abdomen. In all three a diagnosis of pneumonia was subsequently established, and the patients made a full recovery following antibiotic therapy. When a paediatric patient presents with symptoms of an acute abdomen, the possibility of pneumonia should be considered. It can be difficult to differentiate between appendicitis and pneumonia because of the subtle clinical signs. Early recognition is, however, important in order to start the correct therapy and to avoid an unnecessary laparotomy.

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.

Fluorescence measurements of environmental relaxation at the lipid-water interface region of bilayer membranes.

Nanosecond time-resolved emission spectroscopy is used to characterize the complex fluorescence behavior of the probe 2-p-toluidinonaphthalene 6-sulfonate (2,6 p-TNS) when adsorbed to several bilayer membrane system. These include egg phosphatidylcholine vesicles with and without added cholesterol as well as erythrocyte ghost membranes. In each case a nanosecond time-dependent shift of the fluorescence emission to lower energy follows pulsed photoexcitation. The properties of the time-resolved surfaces obtained are consistent with a non-exponential decay law which describes a continuous interaction process of 2,6 p-TNS with its local environment in the membrane. This environment consists in part of polar residues (water plus polar head region) undergoing nanosecond motions. The pure phosphatidylcholine bilayer system was studied at four temperatures and electronic and spectral relaxation contributions to the total fluorescence decay were separated. Temperature coefficients for empirical rate parameters derived for the separated processes were obtained. It appears that a treatment of the fluorescence behavior of amphiphilic probes such as 2,6 p-TNS adsorbed to bilayer membranes at temperatures near ambient in which a single lifetime and radiative decay channel have been assumed is inappropriate.

Nanosecond time-resolved emission anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid.

The fluorescence emission anisotropy of 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid has been studied using nanosecond time-resolved emission techniques. These studies demonstrate that the previously reported decrease in the steady state emission anisotropy, [r], with gestational age is due to a change in the rate of rotational motion of the probe. The emission anisotropy decays to a limiting value (r infinity) greater than zero, suggesting a hindered rotation of the probe, and this is independent of gestational age. The decay function for the emission anisotropy of amniotic fluids from 17, 29, 40 and 41 weeks in gestational age can be best expressed as a single exponential plus a constant term, with rotational correlation times varying from 17 ns to 2.2 ns, respectively. The zero time emission anisotropy remains approx. 0.30 for both early and late gestational times.

Unintended consequences of management actions in salt pond restoration: cascading effects in trophic interactions.

Salt evaporation ponds have played an important role as habitat for migratory waterbirds across the world, however, efforts to restore and manage these habitats to maximize their conservation value has proven to be challenging. For example, salinity reduction has been a goal for restoring and managing former salt evaporation ponds to support waterbirds in the South Bay Salt Pond Restoration Project in San Francisco Bay, California, USA. Here, we describe a case study of unexpected consequences of a low-dissolved oxygen (DO) event on trophic interactions in a salt pond system following management actions to reduce salinity concentrations. We document the ramifications of an anoxic event in water quality including salinity, DO, and temperature, and in the response of the biota including prey fish biomass, numerical response by California Gulls (Larus californicus), and chick survival of Forster's Tern (Sterna forsteri). Management actions intended to protect receiving waters resulted in decreased DO concentrations that collapsed to zero for ≥ 4 consecutive days, resulting in an extensive fish kill. DO depletion likely resulted from an algal bloom that arose following transition of the pond system from high to low salinity as respiration and decomposition outpaced photosynthetic production. We measured a ≥ 6-fold increase in biomass of fish dropped on the levee by foraging avian predators compared with weeks prior to and following the low-DO event. California Gulls rapidly responded to the availability of aerobically-stressed and vulnerable fish and increased in abundance by two orders of magnitude. Mark-recapture analysis of 254 Forster's Tern chicks indicated that their survival declined substantially following the increase in gull abundance. Thus, management actions to reduce salinity concentrations resulted in cascading effects in trophic interactions that serves as a cautionary tale illustrating the importance of understanding the interaction of water quality and trophic structure when managing restoration of salt ponds.

Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.

A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.

Hypoglycemic prodrugs of 4-(2,2-dimethyl-1-oxopropyl)benzoic acid.

SAH 51-641 (1) is a potent hypoglycemic agent, which acts by inhibiting hepatic gluconeogenesis. It is a prodrug of 4-(2, 2-dimethyl-1-oxopropyl)benzoic acid (2) and 4-(2, 2-dimethyl-1-hydroxypropyl)benzoic acid (3), which sequester coenzyme A (CoA) in the mitochondria, and inhibits medium-chain acyltransferase. 1-3 and 4-tert-butylbenzoic acid all cause testicular degeneration in rats at pharmacologically active doses. 14b (FOX 988) is a prodrug of 3, which is metabolized in the liver at a rate sufficient enough to have hypoglycemic potency (an ED50 of 65 micromol/kg, 28 mg/kg/day, for glucose lowering), yet by avoiding significant escape of the metabolite 3 to the systemic circulation, it avoids the testicular toxicity at doses up to 1500 micromol/kg/day. 14b was selected for clinical studies.

Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.