Structure of peptide solutions: a light scattering and numerical study.

We investigated the interactions between protein molecules in solution, in particular for low salt concentrations and thus strong electrostatic interactions where a treatment based on the second virial coefficient is not sufficient. Static and dynamic light scattering experiments on solutions containing the peptide human calcitonin (hCT) were combined with calculations based on the Ornstein-Zernike equation with the hypernetted chain (HNC) closure and computer simulations within the primitive electrolyte model. The simulation illustrates the distribution of proteins in solution and the formation of (transient) protein aggregates. It furthermore allows us to predict the physical stability of hCT solutions in dependence of ionic strength, pH and hCT concentration.

Effect of dienogest-containing oral contraceptives on lipid metabolism.

In a double-blind, controlled, randomized, four-arm, bicentric clinical study, the effect of four oral contraceptives (OCs) on lipid metabolism was investigated. Four groups composed of 25 volunteers each (mean age 26.1 +/- 4.5 years; body mass index 21.9 +/- 2.8 kg/m(2)) were treated for six cycles with monophasic combinations containing 21 tablets with either 30 microg ethinyl estradiol (EE) + 2 mg dienogest (DNG) (30 EE/DNG), 20 microg EE + 2 mg DNG (20 EE/DNG), 10 microg EE + 2 mg estradiol valerate (EV) + 2 mg DNG (EE/EV/DNG), or 20 microg EE + 100 microg levonorgestrel (LNG; EE/LNG). The study was completed by 91 women. Blood samples were taken by venipuncture after at least 12 h fasting on Days 21-26 of the control cycle and Days 18-21 of the first, third, and sixth treatment cycle. There were clear differences between the effects of EE/LNG and the formulations containing estrogens and DNG. Although EE/LNG did not change the triglycerides levels, a significant increase was observed during treatment with the DNG-containing preparations. Although EE/LNG significantly reduced HDL-CH and HDL(2)-CH, there was a nonsignificant increase with the DNG-containing OCs. No change was observed in the levels of HDL(3)-CH. A significant rise in apolipoprotein A1 occurred during intake with the three DNG-containing formulations, but not with EE/LNG. In contrast to the women treated with combinations of estrogens and DNG, apolipoprotein B rose significantly in the women in the EE/LNG group. Lipoprotein (a) was significantly reduced by 30 EE/DNG and EE/LNG and remained unaltered with 20 EE/DNG and EE/EV/DNG. Altogether, the changes in lipid metabolism caused by the DNG-containing formulations appeared to be more favorable than those observed with EE/LNG. In OCs with DNG, the EE dose does not seem to play a major role with respect to the effect on lipids.

Low-temperature micronization of a peptide drug in fluid propellant: case study cetrorelix.

Aim of this study was to elaborate an efficient method for the micronization of the decapeptide cetrorelix (a GnRH-antagonist), in order to obtain a microsuspension as basis for other pharmaceutical preparations, such as e.g. inhalation aerosols. A modified pearl-mill coupled with a cryostat was used for the micronization of cetrorelix in fluid propellant and operated under different conditions. The obtained cetrorelix suspensions were analyzed for particle size distribution, purity of cetrorelix, and for metal contamination through abrasion from parts of the mill. The method allowed an effective micronization of cetrorelix. The mean particle size of the initial cetrorelix lyophilizate bulk ware was reduced from 52.5 microm (Volume Mean Diameter, VMD) down to 14.9, 6.1 and 3.1 microm, respectively, respectively. The HPLC analysis of all cetrorelix suspensions after micronization did not show signs of decomposition as compared to the initial product. The elementary analysis of the suspensions performed by inductively coupled plasma mass spectrometry revealed a negligible amount of contaminants in the suspension (Zr = max. 0.6 ppm; Fe, Cr, Ni, Ba, below limit of quantification, i.e. < 0.14 ppm). The only appreciable contaminant, Aluminum (Al = 1.1 ppm), was derived from the mechanical capping of aluminum canisters prior to analysis. The Zr determination in the suspension of 0.6 ppm, is still considered to be negligible as compared to the legally tolerated limit of air contamination. By low-temperature micronization in fluid propellant, fine drug suspensions of cetrorelix for pMDIs can be directly manufactured in one-step procedure without destruction of the peptide structure and without appreciable product contamination.

Synthesis of 1-aldofosfamide-perhydrothiazines.

Aldofosfamide-perhydrothiazine derivatives are a new class of prodrugs which spontaneously, with half-life times of 2 to > 12 h hydrolyse to the corresponding aldophosphamide in aquous solution. Synthesis of 1-aldofosfamide-perhydrothiazine (N,N'-(2-chloroethyl)-phosphorodiamide-2-(2'-[4'-carboxy-1',3'- perhydrothiazinyl])-ethylester) and a derivative, in which one 2-chlorethyl group of the alkylating function is substituted by a mesyl-ethyl-group (N-(2-Chloroethyl)-N'-(methanesulphonylethyl)- phosphorodiamide-2-(2'-[4'-carboxy-1',3'-perhydro-thiazinyl] )-ethylester), is described.

Synthesis and characterization of 2-mercaptoethanesulfonic acid albumin.

Autoimmune patients treated with ifosfamide (CAS 3778-73-2) and mesna (2-mercaptoethanesulfonic acid, CAS 3375-50-6) in some cases suffered from severe allergic reactions that were proposed to be due to mesna linked to serum albumin by a disulfide bond. To prove the existence of the hypothetic mesna albumin adduct in vivo it was synthesized: The free thiol group of albumin (molecular mass determined by MALDI spectroscopy: 67009 Da) was converted to S-phenylsulfonyl albumin and reacted with mesna to albumin mesna (molecular mass: 67159 Da). In an alternative synthesis albumin was incubated with mesna at pH 8, 40 degrees C (molecular mass of the adduct: 67166 Da).

Treatment of thrombotic saphenous vein bypass grafts using local urokinase infusion therapy with the Dispatch catheter.

Percutaneous treatment of thrombotic stenoses or total occlusions in aged saphenous vein bypass grafts is associated with a significant incidence of complications primarily related to distal embolization. The purpose of this study was to assess the efficacy of local urokinase delivery with the Dispatch catheter prior to balloon angioplasty and/or intragraft stent placement as a new technique of vein graft revascularization. Local urokinase delivery with the Dispatch catheter was performed in 15 saphenous vein grafts (mean age = 11.7 +/- 2.5 yr) in 13 patients with unstable or postinfarction angina. The target lesion was a total occlusion in 5 of the procedures and a severe vein graft stenosis in the remaining 10. In all cases, urokinase was administered directly to the site of the stenosis/occlusion via the Dispatch catheter at 0.5 cc/min and at a concentration of 30,000 units/cc. The mean urokinase infusion time for the 15 procedures was 33 +/- 10 min (range = 10-60 min) and the mean urokinase dose was 495,000 +/- 158,000 units (range = 150,000-900,000 units). Following Dispatch therapy, mean minimal lumen diameter increased from 0.34 +/- 0.32 to 1.81 +/- 0.78 mm (P < 0.01), mean TIMI flow increased from 1.9 +/- 1.4 to 2.8 +/- 0.8 (P < 0.06), and mean thrombus score was reduced from 2.3 +/- 0.6 to 0.3 +/- 0.8 (P < 0.01). Mild no reflow was noted in two cases, although no patient demonstrated angiographic evidence of gross distal embolization. One of the patients with no reflow also demonstrated a small increase in cardiac enzymes. Subsequent balloon angioplasty/stent placement was successful in 14 of the 15 procedures (93% success rate). This preliminary report suggests that pretreatment of thrombotic saphenous vein graft stenoses with local urokinase delivery via the Dispatch catheter may decrease intragraft thrombus and possibly decrease the incidence of vascular complications associated with percutaneous intervention. This technique may allow for recanalization of totally occluded vein grafts with large clot burdens by using significantly less urokinase and shorter drug administration times than conventional infusion protocols.

Congenital coronary arteriovenous fistula: spontaneous rupture and cardiac tamponade.

Congenital coronary arteriovenous fistula is an unusual, but not rare, coronary anomaly. Management of asymptomatic fistulas is controversial because of great variability in natural history. We describe an 82-year-old female patient with spontaneous rupture of a previously undetected left main coronary artery-to-pulmonary artery coronary arteriovenous fistula, with resulting hemopericardium and cardiac tamponade. Emergent surgical exploration and repair provided successful treatment.

Recanalization of totally occluded saphenous vein grafts using local urokinase delivery with the Dispatch catheter.

Current techniques for the percutaneous revascularization of totally occluded vein grafts are limited by a low initial success rate, a significant incidence of distal embolization, and a high rate of early graft reclosure. This case report describes two patients in whom graft recanalization was attempted with the combined use of balloon angioplasty/intra-graft stent placement and local urokinase delivery using a new angiotherapy catheter. Successful recanalization was achieved in both patients without major complications, in spite of a large thrombus burden as demonstrated by angiography.

Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin.

The human calcitonin (hCT) peptide hormone has a marked tendency to aggregate in aqueous solutions and to form long, thin fibrillar aggregates resulting in viscous and turbid dispersions. In this study, the in vitro aggregation products of hCT were systematically investigated using conventional transmission electron microscopy (CTEM) and in-lens field emission scanning electron microscopy. The mass per length of unstained/air-dried specimens was determined by scanning transmission electron microscopy. Irrespective of the sample preparation method and electron microscopic (EM) imaging mode employed, similar supramolecular assemblies were observed. Based on these EM data, it is proposed that hCT aggregation begins with the formation of approximately 4-nm-thick protofibrils. These protofibrils further interact via lateral association and coiling to form higher-order fibrillar assemblies, i.e., protofibril-ribbons, fibrils, fibril-ribbons, tubes, and multistranded cables. The concentration and history of the aggregated hCT solutions strongly influenced the relative frequency of the various hCT assemblies depicted. The supramolecular assemblies of hCT revealed distinct helical symmetries at their different levels of aggregation. A hypothetical mechanism assumed for aggregating solutions to form polymorphic fibrillar hCT assemblies is presented in a schematic model, and the supramolecular arrangement of hCT within the various polymorphic fibrillar aggregates is delineated.

Interfacial adsorption and aggregation associated changes in secondary structure of human calcitonin monitored by ATR-FTIR spectroscopy.

The peptide hormone human calcitonin (hCT) has a marked tendency to aggregate in aqueous solutions, resulting in viscous and turbid dispersions consisting of long fibrils approximately 80 A in diameter. Both transmission (T-FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) experiments were applied on hCT adsorption and aggregation kinetics. By means of the surface sensitive ATR-FTIR spectroscopy at a hydrophobic/hydrophilic interface, early adsorption and aggregation steps of hCT could be followed in situ under real time conditions. Since the aggregation of hCT is associated with conformational changes, the secondary structure sensitive amide I'-band (D2O) could be used as a diagnostic marker. ATR-FTIR spectra recorded during the aggregation kinetics of hCT showed an increase of the amide I'-band intensity by a factor of 3.4, interpreted as pronounced adsorption of hCT molecules from bulk solution to the germanium plate. Furthermore, variations in the line shape of the amide I'-band were interpreted. At the beginning, hCT adopted a random coil conformation followed by distinct formations of alpha-helical and intermolecular parallel beta-sheet structures. Finally, the random coil content declined to 63%, whereas alpha and beta contents rose to 8% and 29%, respectively. From our kinetics results the alpha-structures were formed faster than the beta-structures. This was interpreted as an initial induction of amphiphilic helices during the adsorption process of hCT monomers. ATR-FTIR spectroscopy provides a sensitive analytical tool suggested to monitor interfacial adsorption and aggregation phenomena also of other peptides and proteins.

[Evaluation of transitory evoked otoacoustic emissions in children with impaired tubal ventilation]. / Zur Evaluation transitorisch evozierter otoakustischer Emissionen bei Kindern mit Tubenbelüftungsstörungen.

Transient evoked otoacoustic emission (TEOAE) has proven to be a useful clinical screening test for auditory dysfunction. Problems concerning the evaluation are mainly due to irregularities of the physiological middle ear functions. In this study different degrees of middle ear alterations of otherwise normal-hearing children were quantified by means of tympanometry, otoscopy and pure-tone audiometry and then related to the results of TEOAE registration which were classified by the coefficient of the crosscorrelation and the frequency response spectrum. Data were obtained from 266 ears of 148 children between the age of 3 months and 11 years. The results indicate that even slight middle ear irregularities, e.g. negative pressure between -50 and -100 mm H2O, can reduce the rate of successfully recorded TEOAE from 95% (70/74) in the normal group to 78% (34/47). When children displayed a tympanogramm with no observable peak, a successful registration of TEOAE was possible in only 12% (n = 34) of the cases. Such results are related to serous or mucoid effusions which probably prevent a transmission of the emission from the cochlea to the meatus. In analysing the relation between conductive losses in pure-tone audiometry and TEOAE it was demonstrated that no TEOAE could be recorded in ears with a conductive loss above 20 dB HL. Where the conductive loss is smaller than 20 dB HL it is not possible to predict whether a reliable emission can be recorded or not. This study emphasizes the need to reexamine children who do not show positive emissions connected with middle ear imbalances.(ABSTRACT TRUNCATED AT 250 WORDS)

[Definition of psychogenic voice disorders]. / Zur Definition psychogener Stimmstörungen.

The clinical designation of the psychogenic voice disorder is discussed. "Psychogenic" is aetiologically by no means an apposite, or adjective, to organic diseases, for the occurrence of factors that can be defined as psychopathological (either primary or secondary) is always practically and clinically important--especially if these factors are of general psychosocial relevance, or of a latent depressive and neurotic nature. Hence, biographical anamnesis can be obligatory, supplying information that is essential for a therapeutic approach.

Preliminary study on subcortical slow potentials related to the readiness potential in monkey.

Two female rhesus monkeys with cortical and subcortical DC-electrodes were studied during self-paced operant responding to determine the feasibility of mapping the intracerebral distribution of readiness potentials (RP). These potentials reflect preparatory aspects of motor activation and may be useful indicators of subcortical areas involved in preparatory set. The monkeys had learned to execute the task appropriately after 17 and 28 sessions respectively; one monkey's performance was very stable, and the other's erratic. However, relatively comparable RPs were recorded from the monkeys when recordings from sessions involving good performance were considered. RPs of relatively large amplitude appeared in electrodes positioned near the substantia nigra and pretectal/collicular region; smaller responses were observed in cortex and midbrain reticulum and, in one monkey, the caudate nucleus. These findings indicate the feasibility of such studies and suggest that, like the contingent negative variation, RPs occur in many subcortical regions.

In vitro protein production by the human endometrium.

Protein secretion by the human endometrium was studied in vitro in medium after incubation of tissue minces (n = 10) or glands isolated by collagenase digestion (n = 4) from tissues obtained at the time of curettage from normal women. Samples were incubated in a serum-free medium for 24 h at 38 degrees C in the presence of radiolabeled methionine. Dialyzed medium from each sample was subjected to two-dimensional gel separation, and detected by protein staining. Although 5 of the 27 proteins were considered to be present in the labeling experiments by only one of the three observers, there was agreement about the presence of the 22 others. In addition, the observers categorized the proteins into three groups for purposes of analysis: a) those associated with the follicular phase of the cycle; b) those associated with the luteal phase; and c) those not cycle-related. One protein triplet, labeled #27, showed a significant relation to the luteal phase (p less than 0.01). A complete lack of similarity between the pattern of labeled proteins obtained from the medium and labeled proteins obtained from lysates of cells incubated in the same experiments makes it unlikely that cellular lysis, as opposed to secretion, contributed to the pattern of proteins studied in these experiments.

Analysis of proteins secreted by the human endometrium in vivo and in vitro.

Human uterine luminal fluids contain over two dozen proteins distinct from those of serum as detected by two dimensional gel electrophoresis and silver-type protein staining. Eighty-one percent of these uterine fluid proteins can be detected in vitro by radiolabeled methionine incorporation studies and the vast majority of these products are epithelial in origin. The major recognizable menstrual cycle phase-dependent change in the protein pattern in these gels was the appearance of a protein group (number 27) of approximately 25,000 mw and pI of 5.8 - 6.3. This group of proteins was found in nearly all mid- and all late secretory phase fluids or culture media and in none obtained earlier in the cycle. As yet, however, it has not been possible to induce these proteins in proliferative specimens in vitro by the addition of estrogens and/or progestins, though studies along these lines are continuing. Although we cannot be certain, it appears as though protein group number 27 is distinct from, but similar in several respects to, other proteins of human endometrium reported in the literature.

Two-dimensional gel electrophoresis of endometrial protein in human uterine fluids: qualitative and quantitative analysis.

By combining two-dimensional gel electrophoresis, protein staining and a sensitive computer-assisted gel scanning system, it was possible to examine human uterine fluid (n = 56) qualitatively and quantitatively for the presence of endometrial proteins. The protein concentration of uterine fluids ranged from 0.1 to 12.0 mg/ml with early secretory phase samples (n = 15) having significantly less protein (0.72 +/- 0.2 SEM mg/ml p less than or equal to 0.05) than the proliferative phase (n = 57) samples (1.58 +/- .29 SEM mg/ml). Whole blood contamination of uterine fluid, as measured by hemoglobin content, averaged 6.2 +/- 0.88% throughout the menstrual cycle. Human uterine fluids collected throughout the menstrual cycle were found to contain serum and up to 24 other proteins in addition to those previously described (MacLaughlin and Richardson, 1983). These proteins represent approximately 1% of the total protein in the gels and exhibit isoelectric points from 4.5 to 7.0 and molecular weights in the 26,000 to 60,000 range. These proteins are absent from human serum, which exhibits an identical pattern whether obtained in the proliferative or secretory phase of the menstrual cycle. These secreted endometrial proteins now become the standard against which to compare proteins identified in vitro using organ, gland and cell culture techniques and to characterize proteins that are regulated by steroid hormones in vivo.