Altered ligand dissociation rates in thyrotropin-releasing hormone receptors mutated in glutamine 105 of transmembrane helix III

Glutamine 105 in the third transmembrane helix of the thyrotropin-releasing hormone receptor (TRH-R) occupies a position equivalent to a conserved negatively charged residue in receptors for biogenic amines where it acts as counterion interacting with the cationic amine moiety of the ligand. Maximum levels of response to TRH in oocytes expressing wild-type TRH-Rs were indistinguishable from those of oocytes expressing receptors mutated to Glu, Asn, or Asp in position 105. However, the EC50 values for activation of oocyte responses increased more than 500 times in oocytes expressing mutant Glu105 receptors, in which the amido group of Gln105 has been removed by site-directed mutagenesis. Charge effects do not seem to be involved in the huge effect of mutating Gln105 to Glu, since mutation of Gln105 to Asp induces only a 15-fold increase in EC50. Furthermore, no change in EC50 is observed after mutation of Asn110 to Asp. The affinity shift (identified by changes in EC50 values for systems of comparable efficacy) in Glu105 mutant receptors was partially recovered in oocytes expressing Asn105 mutant receptors. These results and those obtained after substitution of Lys, Leu, Tyr, and Ser for Gln105 suggest that the presence and the correct position of the Gln hydrogen bond-donor amido group are important for normal functionality of the receptor. In wild type or Asp105 mutant receptors showing the same maximal responses, decreases in affinity with TRH and methyl-histidyl-TRH correlated with increased dissociation rates of hormone from the receptor. Rapid dilution experiments following subsecond stimulation indicate that the TRH-R is converted rapidly from a form showing fast dissociation kinetics to a form from which the hormone dissociates slowly. Mutation of residue 105 impairs the receptor shift between these two forms. This effect was demonstrated in a direct way by comparing [3H]methyl-histidyl-TRH dissociation rates in COS-7 cells transfected with either wild type or Asp105 mutant TRH-Rs. Thus, residues located in transmembrane helix III positions equivalent to those of the counterions for biogenic amines, regulate hormone-receptor interactions in the TRH receptor (and perhaps other receptors). Furthermore, the nature of the amino acid in these positions may also play a role, directly or indirectly, in conformational changes leading to receptor activation, and hence to signal transduction.     

Should patients with sleep apnoea/hypopnoea syndrome be diagnosed and managed on the basis of home sleep studies?

The purpose of this study was to analyse the validity and the economic efficiency of a portable monitor of respiratory parameters (PMRP), used in a home setting for the diagnosis of sleep apnoea/hypopnoea syndrome (SAHS). Eighty nine patients with suspected SAHS were studied in two settings: in the sleep laboratory using full-polysomnography (full-PSG); and at the patient's home using a PMRP. In the home setting, 50 patients were assisted by a technician and 39 set up the equipment themselves. SAHS (apnoea/hypopnoea index (AHI) >10 events x h(-1) by means of full-PSG) was diagnosed in 75 of the 89 patients. An acceptable agreement was obtained between the AHI measured by full-PSG and PMRP, according to the Bland and Altman method of concordance (mean bias 2.56; 95% confidence interval 3.25). Sensitivity and specificity of PMRP were adequate for diagnostic purposes; however, their values rely on the prior PMRP-AHI cut-off point selected with reference to full-PSG-AHI >10. The clinical therapeutic decision taken after PMRP agreed with that taken with full-PSG in 79 patients (89%). Although 10% of the studies with an individual set-up needed repetition, both of the domiciliary modalities (with and without a technician's intervention) were, economically, about three times more efficient than full-PSG. In conclusion, we believe that patients with a suspected sleep apnoea/hypopnoea syndrome should initially be studied in a home setting with a portable monitor of respiratory parameters, since it is a reliable method with an acceptable cost-effective profile.     

Cytogenetic abnormalities and clonal evolution in an adult hepatoblastoma

Hepatoblastomas usually occur in children < 3 years of age, and only occasional adult cases have been described. To date, 20 cytogenetically abnormal childhood hepatoblastomas have been reported. Karyotypic investigations have shown that most hepatoblastomas are diploid or hyperdiploid, often displaying trisomies for chromosomes 2 and 20. We have cytogenetically investigated an adult hepatoblastoma for which no previous karyotypic data exist. A hypertriploid stemline with multiple numerical and structural chromosomal aberrations, including +2 and +20, was found. In addition, the tumor displayed extensive clonal evolution with 11 subclones. Although the tumor thus displayed some chromosomal abnormalities commonly observed in childhood tumors, providing further support for the importance of these abnormalities in the development of hepatoblastoma, the level of genomic complexity seen in the present case has never been described in childhood hepatoblastomas and may suggest a different etiology or pathogenesis.     

Pancreas anulare as a rare differential diagnosis of duodenal stenosis in adulthood]

Large spontaneous intrahepatic portosystemic venous shunts are occasionally found and their diagnosis by Doppler sonography is rarely reported. The authors describe a case of spontaneous intrahepatic porto-systemic venous shunt in liver cirrhosis diagnosed by color Doppler and characterized by an unusual pulsed Doppler spectrum: a continuous flat portal-like pattern of flow in the portal branch, and in both the shunt and the hepatic vein.     

Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients

The liposomal formulation of amphotericin B (AmBisome) greatly reduces the acute and chronic side effects of the parent drug. The present study describes the pharmacokinetic characteristics of AmBisome applied to 10 patients at a dose of 2.8 to 3.0 mg/kg of body weight and compares them to the pharmacokinetics observed in 6 patients treated with amphotericin B deoxycholate at the standard dose of 1.0 mg/kg. Interpatient variabilities of amphotericin B peak concentrations (Cmax) and areas under concentration-time curves (AUC) were 8- to 10-fold greater for patients treated with AmBisome than for patients treated with amphotericin B deoxycholate. At the threefold greater dose of AmBisome, median Cmaxs were 8.4-fold higher (14.4 versus 1.7 microg/ml) and median AUCs exceeded those observed with amphotericin B deoxycholate by 9-fold. This was in part explained by a 5.7-fold lower volume of distribution (0.42 liters/kg) in AmBisome-treated patients. The elimination of amphotericin B from serum was biphasic for both formulations. However, the apparent half-life of elimination was twofold shorter for AmBisome (P = 0.03). Neither hemodialysis nor hemofiltration had a significant impact on AmBisome pharmacokinetics as analyzed in one patient. In conclusion, the liposomal formulation of amphotericin B significantly (P = 0.001) reduces the volume of drug distribution, thereby allowing for greater drug concentrations in serum. The low toxicity of AmBisome therefore cannot readily be explained by its serum pharmacokinetics.     

A study of the ethical duty of physicians to disclose errors

A quantitative immunocytochemical procedure was used for evaluation of the blood-brain barrier (BBB) to endogenous albumin in plaque-forming (PF) and non-plaque-forming (NPF) groups of scrapie-infected mice at the clinical stage of disease. Ultrathin sections of brain samples (cerebral cortex, hippocampus and cerebellum) embedded in resin (Lowicryl K4M) were exposed to anti-mouse albumin antiserum followed by protein A-gold. Using morphometry, the density of immunosignals (gold particles per microns2) was recorded over four compartments: vascular lumen, endothelium, subendothelial space, and brain parenchyma (neuropil). Morphometric and statistical analyses did not reveal significant differences in the barrier function of the microvasculature of the cerebral cortex and hippocampus in either group of mice, although a slight increase in the number of leaking vessels in the PF group was noted. In contrast, in the cerebellum, the permeability of the microvessels to albumin was significantly higher in the PF than in the NPF mouse group, and this was paralleled by the infiltration of the walls of numerous vascular profiles with amyloid deposits (amyloid angiopathy). These data also indicate the existence of distinct regional differences in BBB function in the brain of scrapie-infected mice. The vascular amyloid deposits and the amyloid plaques present in the cerebral cortex of PF mice were labeled with numerous immunosignals suggesting the affinity of extravasated albumin to these deposits. In conclusion, no convincing evidence was obtained indicating that impairment of the BBB, manifested by increased permeability of vascular segments, is directly related to the deposition of amyloid in the vascular wall and in plaques. Segmental impairment of the barrier function seems to be rather the result of disturbed structural integrity of the components of the vascular wall.     

Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).     

Renal sodium excretion after oral or intravenous sodium loading in sodium-deprived normotensive and spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats that had been on a low sodium diet for 3 days were given 1.5 mmol sodium chloride kg-1 body weight either orally or intravenously. The rats receiving an oral sodium load showed a greater natriuresis than those receiving the same saline load intravenously. No increase of renal sodium excretion was observed when the rats received a hypertonic mannitol solution orally. The cumulative sodium excretion during the 8 h following oral loading was two to three times larger in SHR than in WKY, whereas no difference between strains could be demonstrated after giving saline intravenously. Furthermore, after switching from normal to low sodium diet the rate of decrease of renal sodium excretion was greater in SHR than in WKY rats. It is proposed that there exists a gastrointestinal sensory mechanism for sodium controlling the renal sodium excretion. Furthermore, it is suggested that the function of this mechanism differs between SHR and WKY.     

Pharmacokinetics of newer cephalosporins after subconjunctival and intravitreal injection in rabbits

Pharmacologic considerations suggest that third-generation cephalosporins might penetrate the vitreous humor better after periocular injection and might be eliminated less readily after intravitreous injection than older agents. We studied the sodium salts of ceftizoxime, ceftriaxone, and ceftazidime, and of an investigational cephalosporin, cefepime, in rabbits. After a single subconjunctival injection in animals with normal eyes, vitreous levels ranged from 3 to 13 mg/L. After five subconjunctival injections in rabbits with infected eyes, vitreous concentrations ranged from 12 to 34 mg/L. These concentrations are not appreciably greater than those found with older beta-lactams. The vitreous half-life of the four drugs after intravitreous injection varied from 5.7 to 20 hours in rabbits with uninflamed eyes and from 9.4 to 21.5 hours in rabbits with infected eyes. Except for ceftizoxime, the half-lives were substantially longer than those for older beta-lactams and suggest predominantly anterior route elimination. Vitreous penetration of these new agents after subconjunctival injection does not appear to be sufficient to overcome the need for intravitreous injections in the treatment of endophthalmitis. However, the longer vitreous half-lives of some of the newer agents may be useful if the drugs are to be given intravitreally.