Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Regional lipid peroxidation and protein oxidation in rat brain after hyperbaric oxygen exposure

Reactive oxygen species may participate in development of neurological toxicity resulting from hyperbaric oxygen exposure. To explore the possibility that increased reactive O2 metabolite generation may result in oxidative modification of lipids and proteins, rats were exposed to five atmospheres (gauge pressure) of O2 until development of an electroencephalographic seizure. Lipid peroxidation (as thiobarbituric acid-reactive substances) and protein oxidation (as 2,4-dinitrophenyl-hydrazones) were measured in five brain regions. Oxidized and reduced glutathione were also determined because of their role in regulating lipid peroxidation. Lipid peroxidation was confined to the frontal cortex and hippocampus, while protein oxidation (in both cytoplasmic and membranous fractions) and increased oxidized glutathione was evident throughout the brain. These results support a role for formation of reactive O2 metabolites from hyperbaric O2 exposure and suggest that protein oxidation, especially in soluble proteins, may be one of the most sensitive measures.     

Chronic oral etoposide and tamoxifen in the treatment of far-advanced hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is a chemoresistant tumor that frequently expresses a high level of p 170 glycoprotein of the multidrug-resistance (MDR) gene. Preliminary data suggested that VP-16 showed modest activity in HCC. Recently, schedule-dependent cytotoxicity of VP-16 has been demonstrated. In this study, we tested the therapeutic efficacy of chronic oral VP-16 plus tamoxifen, a potential MDR-reversing agent, in patients with far-advanced HCC. METHODS: A prospective single-arm study was conducted in the National Taiwan University Hospital. To be eligible, patients must have had unresectable and non-embolizable HCC, objectively measurable tumors, adequate hemogram with absolute granulocyte count greater than or equal to 2,000/mm3, and platelet count greater than or equal to 1x10 (5)/mm3, total serum bilirubin less than or equal to 3.0 mg/dl, age less than or equal to 75 years, and a Karnofsky performance status of greater then or equal to 50%. The treatment included VP-16 (Bristol-Myers-Squibb, Princeton, NJ), 50 mg/m2/day, orally, Days 1 to 21, and tamoxifen (Pharmachemie B.V. Haarlem, Netherlands), 40 mg/day, orally, Days 1 to 21; repeated every 5 weeks. RESULTS: Between December 1990 and December 1993, a total of 33 patients were enrolled in the study. There were 28 men and 5 women, with a median age of 51 years. They received an average of 3.2 (range: 1-10) courses of chemotherapy. ECOG (Eastern Cooperative Oncology Group) Grade 3 and Grade 4 leucopenia developed in 6 patients (18.2%) and 4 (12.1%) patients, respectively. Grade 3 and 4 thrombocytopenia developed in 2 patients (6.1%). Treatment-related death occurred in one patient due to sepsis. Mild gastrointestinal toxicities were common with Grade 1 and 2 nausea. Grade 1 and 2 vomiting, Grade 1 and 2 diarrhea, and Grade 1 and 2 stomatitis, developed in 13 (39.4%), 7 (21.2%), 12 (36.4%), and 16 (48.5%) patients, respectively. Grade 3 and 4 gastrointestinal toxicities were rare. Deep vein thrombosis occurred in one patient (3.0%). Eight patients (24.2%, 95% confidence interval 11%-42%) had achieved a partial remission, with a median time-to-progression of 6 months (2-11). Median survivals of the responders and non-responders were 8.0 and 3.0 months, respectively (P < 0.05). The median Karnofsky performance status of the responders improved from 70% to 80%. CONCLUSIONS: Chronic oral VP-16 and tamoxifen has modest activity and acceptable toxicity in far-advanced HCC, and is a useful palliative treatment in about a quarter of such patients.     

Role strain in couples with and without a child with a chronic illness: associations with marital satisfaction, intimacy, and daily mood

The tryptophan depletion test is a research strategy to investigate the functional consequences of decreasing the brain serotonin metabolism. Because serotonin is involved in sleep regulation and the regulation of affective states, we studied the acute polysomnographic effects of tryptophan depletion and expected to induce similar changes of sleep EEG as observed in depressed patients. A total of 12 healthy subjects (mean age 34 +/- 3 years) had eight polysomnograms, divided in two blocks of 4 consecutive nights. After one adaptation and 1 baseline night, subjects received a low-protein diet on day 3 and 4 until midday. On day 4 at 18.00 h, they drank an amino acid mixture either devoid of tryptophan or containing 2.3 g of tryptophan (placebo control) in randomized and double-blind order, resulting in an 85% decrease (tryptophan depletion) and a 144% increase (placebo control) of serum tryptophan at 22.00 h. After tryptophan depletion but not placebo, significant effects on sleep EEG were observed in terms of decreased non-rapid eye movement (non-REM) stage 2, increase of wake %, and of rapid eye movement (REM) density compared with baseline. REM latency was not altered, however the first and second REM period interval were significantly shorter after tryptophan depletion. This study underlines the impact of the serotonergic system on sleep maintenance and on REM sleep.     

Conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interactions with hydrophobic regions of unfolded proteins. The activities and the conformations of the N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recently reported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which are located in the nucleotide-binding domain. To further characterize conformational changes associated with interdomain coupling and to determine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p is more oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEM modification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which is located in a C-terminal alpha-helical region adjacent to the polypeptide-binding cleft, was quenched in the presence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsic fluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alpha-helical lid that is open in the presence of ATP, but closed in the presence of ADP.     

Effects of Plasmodium berghei infection on arteether metabolism and disposition

Arteether (AE) is primarily deethylated to dihydroqinghaosu (DQHS) in rats and humans. Conversion of AE to DQHS was impaired in microsomes from rats infected with Plasmodium berghei. The Km for AE was 175.1 +/- 49.1 and 124.4 +/- 115.1 mumol/l, and Vmax was 2.24 +/- 0.45 and 1.22 +/- 0.67 nmol AE formed/mg protein/min in control and infected microsomes (p < 0.05), respectively. Calculated intrinsic clearance (CLint = initial Vmax/Km) for AE was only 4% lower in infected microsomes. Apparent pharmacokinetic parameter estimates for AE using the isolated perfused rat liver demonstrated no differences (p > 0.05) in volume of distribution, clearance, and half-life between normal and infected animals. Malaria infection resulted in decreased biliary excretion of free AE and DQHS. The majority of AE is eliminated via biliary excretion of conjugated DQHS, which is approximately 500-fold higher than free DQHS and 75-fold higher than free AE on a molar basis.     

Inhibition of TNFalpha attenuates infarct volume and ICAM-1 expression in ischemic mouse brain

The family physician occupies a front-line position in the detection and treatment of emotional problems and psychiatric illnesses. The practice pattern of the family physician necessitates an efficient, effective model of psychotherapy The BATHE technique is a brief psychotherapeutic method that addresses the patient's background issues, affect and most troubling problem. The emphasis of the interview then shifts to how the patient is handling the problem and a demonstration of empathy by the physician. Some of the challenges in psychotherapy are presented, and cases in which the BATHE technique was used are described.     

Deficient insulin-like growth factor I in chronic heart failure predicts altered body composition, anabolic deficiency, cytokine and neurohormonal activation

BACKGROUND: Recent studies of growth hormone supplementation in chronic heart failure have been associated with variable results. Acquired abnormalities of biochemical parameters of the growth hormone insulin-like growth factor I axis have been associated with severe chronic heart failure. There are suggestions of an acquired growth hormone resistance with deficient insulin-like growth factor I in some patients. OBJECTIVES: Therefore, we set out to investigate the clinical and functional status and the degree of cytokine and neurohormonal alteration of chronic heart failure patients with deficient insulin-like growth factor I responses. METHODS: Patients with chronic heart failure were divided into two groups according to their insulin-like growth factor I levels (classified according to the manufacturer's assay range in normal controls): low insulin-like growth factor I <104 (n = 20; 89 +/- 9.6 ng/ml), and normal/high >104 ng/ml (n = 32; 169 +/- 52 ng/ml). Between groups there was no difference in age (low versus high: 65.3 +/- 12.1 versus 61.6 +/- 9.1 years, p = 0.21), body mass index, aerobic capacity (peak oxygen consumption: low versus high: 15.5 +/- 5.2 versus 17.3 +/- 6.3 mL/kg/min, p = 0.23), left ventricular ejection fraction, New York Heart Association classification. RESULTS: During quadriceps strength testing, patients with low insulin-like growth factor I had reduced absolute strength (-24%), and strength per unit area muscle (- 14%) than patients with normal/high insulin-like growth factor I. Leg muscle cross-sectional area was lower in the low insulin-like growth factor I group (-12% and -13% for right and left legs, respectively). These alterations were accompanied by increased levels of growth hormone (+145%), tumor necrosis factor-alpha (+46%), cortisol/ dehydroepiandrosterone ratio (+60%), noradrenaline (+49%) and adrenaline (+136%) (all at least p < 0.05). CONCLUSIONS: Patients with low insulin-like growth factor I levels show signs of altered body composition, cytokine and neuroendocrine activation, to a greater extent than patients with normal/high levels.