A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy

Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor-selective LU 112313 was the least selective compound in this model, indicating that non-NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor-selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.     

Reprioritization of liver protein synthesis resulting from recombinant human growth hormone supplementation in parenterally fed trauma patients: the effect of growth hormone on the acute-phase response

BACKGROUND: One of the major components of the metabolic response to severe trauma is the alteration in concentrations of a large number of plasma proteins referred to as acute-phase proteins (APP). The principle mediators of these liver-synthesized APP are mainly the cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We have measured the plasma levels of IL-6, TNF alpha, and 20 APP in 24 adult, severely injured, hypermetabolic and highly catabolic patients with multiple injuries within 48-60 hours after injury, when they were receiving maintenance fluids without calories or nitrogen, and subsequently during 7 days of total parenteral nutrition with (n = 12) or without (n = 12) recombinant human growth hormone supplementation (rhGH, 0.15 mg/kg/d). RESULTS: Baseline positive APP due to severe trauma include C-reactive protein (CRP), alpha-1 antichymotrypsin, alpha-1 acid glycoprotein, alpha-1 antitrypsin, fibronectin, and factor B. Negative APP include IgG, IgM, complement-3, prealbumin, transferrin, ceruloplasmin, and albumin. Except for CRP, alpha-1 antichymotrypsin, and albumin, all the APP levels increase during 7 days of nutritional support. Plasma levels of cytokines IL-6 and TNF-alpha, although initially markedly increased after injury, decrease with parenteral refeeding. There is a linear correlation between CRP and IL-6 levels and also between the transport proteins prealbumin and transferrin. Trauma-induced increases in CRP and IL-6 levels decreased with nutrition alone, but did not change with rhGH supplementation. An immunosuppressed state of injury is evident from the decreased immunoglobulin levels (IgG, IgM, IgA) in the trauma patients. Total parenteral nutrition alone increases the immunoglobulin levels to normal. However, with adjuvant rhGH, only IgA levels are normalized. CONCLUSIONS: Adjuvant rhGH therapy does not attenuate the reprioritization of acute liver protein synthesis and results in only limited restoration of host defenses. The clinical implications of these findings await further study.     

Use of full cortical allograft to repair a metatarsal fracture in a foal

A 4-month-old Quarter Horse colt was admitted for repair of an open, comminuted fracture of the proximal portions of the diaphyses of the left second, third, and fourth metatarsal bones. Initial repair included internal fixation and cancellous bone graft. However, the third metatarsal bone became infected and failed to heal. After removal of infected portions of the bone, a 5-cm, fullthickness cortical allograft was placed in the defect. Rigid internal fixation provided stability for the allograft and remaining fracture fragments so that the horse was able to bear weight on the second and fourth metatarsal bones. The allograft was ultimately resorbed; however, appositional bone growth permitted a massive, functional metatarsal bone to form that incorporated the second, third, and fourth metatarsal bones. The colt went on to compete successfully, long term, as a show horse.     

Penetrating injuries involving the intrathoracic great vessels

Approximately 50% of the forearm vasodilatation to intra-arterial infusions of acetylcholine is mediated by endothelium-derived nitric oxide. These conclusions have been derived from venous occlusion plethysmographic measurements of total forearm blood flow during co-infusions of acetylcholine and NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase. Since venous occlusion plethysmography measures total limb blood flow, the relative proportion of the measurement from skin cannot be determined precisely. To determine the effects of acetylcholine on skin specifically, we have used laser Doppler flowmetry to measure vascular responses to local iontophoresis of acetylcholine in the forearm of normal male volunteers. To elucidate the possible mechanisms of cutaneous vasodilatation to acetylcholine, vascular responses were measured before and after systemic inhibition of prostanoid production and nitric oxide synthesis by oral aspirin (600 mg daily for 3 days) and intravenous L-NMMA (3 mg/kg for 60 min), respectively. After aspirin administration, dose-dependent vascular responses to acetylcholine were reduced significantly by approximately 53% (p < 0.005, ANOVA). In contrast, intravenous L-NMMA appeared to have no significant effect on cutaneous vascular responses to acetylcholine. While the role of nitric oxide is uncertain, vasodilatation to acetylcholine in the forearm skin is mediated largely by a prostanoid-dependent mechanism. Assessment of cutaneous vascular responses to iontophoresis of acetylcholine may, therefore, be useful in diseases where abnormal endothelium-dependent prostanoid function has been implicated.     

Fetoplacental vascular tone during fetal circuit acidosis and acidosis with hypoxia in the ex vivo perfused human placental cotyledon

OBJECTIVES: Our purpose was to determine the effects of acidosis and acidosis-hypoxia on fetoplacental perfusion pressure and its response to angiotensin II. STUDY DESIGN: Perfused cotyledons from 14 placentas were studied with either an acidotic fetal circuit perfusate (n = 7) or an acidotic-hypoxic fetal circuit perfusate (n = 7). Each cotyledon's fetal vasculature was initially perfused under standard conditions and bolus injected with 1 x 10(-10) moles of angiotensin II. Fetoplacental perfusate was then replaced with either an acidotic medium (pH 6.90 to 7.00 and Po2 516 to 613 mm Hg) or an acidotic-hypoxic medium (pH 6.90 to 7.00 and Po2 20 to 25 mm Hg) followed by an angiotensin II injection. The vasculature was subsequently recovered with standard perfusate and again injected with angiotensin II. Perfusion pressures within each group were compared by one-way analysis of variance, and results were expressed as mean pressure +/- SEM. RESULTS: Resting fetoplacental perfusion pressure did not change when the fetal circuit perfusate was made acidotic (28 +/- 1 mm Hg vs 25 +/- 2 mm Hg) or acidotic-hypoxic (26 +/- 2 mm Hg vs 25 +/- 2 mm Hg). The maximal fetoplacental perfusion pressure achieved in response to angiotensin II did not differ with an acidotic perfusate (41 +/- 2 mm Hg vs 38 +/- 1 mm Hg) or with an acidotic-hypoxic perfusate (39 +/- 2 mm Hg vs 36 +/- 2 mm Hg). CONCLUSIONS: In the perfused placental cotyledon fetoplacental perfusion pressure and pressor response to angiotensin II are not affected by fetal circuit acidosis or acidosis-hypoxia. This suggests that neither fetal acidosis nor fetal acidosis combined with hypoxia has a direct effect on fetoplacental vascular tone.     

Baculovirus-mediated expression of Plasmodium falciparum erythrocyte binding antigen 175 polypeptides and their recognition by human antibodies

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein which mediates merozoite invasion of erythrocytes in a sialic acid-dependent manner. The purpose of this study was to produce recombinant EBA-175 polypeptide domains which have previously been identified as being involved in the interaction of EBA-175 with erythrocytes and to determine whether these polypeptides are recognized by malaria-specific antibodies. The eba-175 gene was cloned by PCR from genomic DNA isolated from the 3D7 strain of P. falciparum. The predicted protein sequence was highly conserved with that predicted from the published eba-175 gene sequences from the Camp and FCR-3 strains of P. falciparum and contained the F segment divergent region. Purified recombinant EBA-175 polypeptide fragments, expressed as glutathione S-transferase fusion proteins in insect cells by using the baculovirus system, were recognized by antibodies present in serum from a drug-cured, malaria-immune Aotus nancymai monkey. The fusion proteins were also recognized by antibodies present in sera from individuals residing in areas where malaria is endemic. In both cases the antibodies specifically recognized the EBA-175 polypeptide portion of the fusion proteins. Antibodies raised in rabbits immunized with the recombinant fusion proteins recognized parasite proteins present in schizont-infected erythrocytes. Our results suggest that these regions of the EBA-175 protein are targets for the immune response against malaria and support their further study as possible vaccine components.     

Molecular epidemiology of sporadic (endemic) serogroup C meningococcal disease

Understanding the basis of sporadic (endemic) meningococcal disease may be critical to prevention of meningococcal epidemic outbreaks and to understanding fluctuations in incidence. Active, prospective, population-based surveillance and molecular epidemiologic techniques were used to study sporadic serogroup C meningococcal disease in a population of 2.34 million persons (Atlanta area). During 1988-1994, in which no outbreaks or case clusters were reported, 71 patients developed sporadic serogroup C meningococcal disease (annual incidence, 0.51/100,000). Eighty-three percent of patients were >2 years old. By multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and serotyping, 84% (52/62) of the isolates available for study were identical or closely related members of the electrophoretic type 37 (ET 37) complex responsible for multiple serogroup C outbreaks in the United States in the 1990s. Sporadic disease caused by 9 clonal strains occurred over periods up to 4 years and accounted for 45% (28/62) of cases. Sporadic serogroup C meningococcal disease was most often due to a limited number of related strains that appear to slowly circulate in the population.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Production and systemic absorption of toxic byproducts of tissue combustion during laparoscopic surgery

Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one or more DHFR mutations in all the samples, but no relationship was found with the presence of parasites in the blood at day 7. The results suggest that alterations in the coding region for dihydropteroate synthetase (DHPS), the enzyme target for sulfadoxine, should be studied in order to predict resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely.     

The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation

To compare the relative sensitivity and specificity of bone turnover indexes for bone loss or gain in early postmenopausal women, we performed a multicenter trial in 236 menopausal women (mean age, 51 yr), who were randomized to hormone replacement therapy (HRT) or calcium supplementation (CS; 500 mg/day) for 1 yr. Two markers of bone formation, osteocalcin (OC) and bone alkaline phosphatase (BSAP), and two markers of bone resorption, urinary N-telopeptide (NTx) and urinary free deoxypyridinoline (fDpd), as well as spine and femoral neck bone mineral density (BMD) were measured at baseline and 3, 6, and 12 months after treatment. Women receiving HRT (n = 105) showed a significant increase in spine BMD (+2.5%; P < 0.0001) and hip BMD (+1.0%; P = 0.02) compared to women receiving CS, who showed a decline at both sites (-1.1%; P < 0.01). All four markers showed time-dependent decreases in women receiving HRT (P < 0.001) and no change in women receiving CS alone. When baseline indexes of turnover were stratified by quartile, there was a significantly greater increase in BMD among those with the highest NTx, OC, and BSAP levels compared to that in those with the lowest NTx, OC, and BSAP levels (P < 0.05). The highest quartile for percent change from baseline to 6 months in fDpd, BSAP, and NTx was also associated with the greatest change in spine BMD at 1 yr. Receiver operator characteristic curves for percent change from baseline to 6 months in an individual marker to 1 yr change in BMD during HRT revealed that the percent change in NTx provided the greatest discrimination between gain and loss of BMD. When subjects receiving HRT were compared by their positive or negative skeletal response at 1 yr and their baseline turnover marker, initial NTx values were significantly higher in those that gained bone than in those that lost bone (P = 0.0002). CS women in the highest quartile for NTx at baseline had significantly greater decreases in spine BMD than subjects with the lowest NTx values (P < 0.005), although this was not true for fDpd (P < 0.20). In conclusion, for early postmenopausal women there are differential responses of biochemical markers to HRT and CS. Baseline urinary NTx and serum OC were the most sensitive predictors of change in spine BMD after 1 yr of either HRT or CS. Similarly, the percent change in NTx and OC from baseline to 6 months best predicted bone gain or loss. We conclude that markers of bone formation and resorption can be used clinically to predict future BMD in early postmenopausal women.