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Summary The Brownian Dynamics (BD) method is applied to study the diffusive dynamics and interaction of two proteins, cytochrome c (CYTC) and cytochrome c peroxidase (CYP). We examine the role of protein electrostatic charge distribution in the facilitation of protein-protein docking prior to the electron transfer step, assessing the influence of individual charged amino acid residues. Accurate interaction potentials are computed by iterating the linearized Poisson-Boltzmann (PB) equation around the larger protein CYP. The low dielectric constant inside proteins, electrolyte screening effects and irregular protein surface topography are taken into account. We observe a large ensemble of electrostatically stable encounter complexes seemingly with acceptable geometric requirements for electron transfer rather than a single dominant complex. Stabilities of the large variety of docking complexes are rationalized in terms of generalized charged residue complementarities. However, it is found that the electrostatic interactions giving rise to complex stabilities are somewhat nonspecific in nature. A large series of additional simulations are performed in which individual charged residues on CYTC have been chemically modified. Resulting perturbations of the association rate are significant and qualitatively similar to results observed in comparable kinetics experiments. We therefore demonstrate the potential of the Brownian dynamics method to estimate the effects of site-directed mutagenesis on protein-protein and protein-ligand diffusional association rates.

Proopiomelanocortin (POMC), the precursor to adrenocorticotropic hormone and other related peptides, was originally identified in the corticotropic cell. Recent evidence shows that POMC products are also normally present in a variety of nonpituitary tissues. To investigate this phenomenon in humans we looked for the presence and characteristics of POMC transcripts in various adult tissues. Blot hybridization analysis of normal adrenal, thymus, and testis RNAs revealed a small RNA species approximately 400 nucleotides shorter than the 1200-nucleotide pituitary species. Primer extension and S1 nuclease mapping studies showed that this small RNA lacked exon 1 and exon 2 of the gene, and it corresponded to a set of at least six molecules starting 41 to 162 nucleotides downstream from the 5' end of exon 3. These RNAs appear to result from heterogeneous transcription initiation sites presumably under the control of "GC box" promoter sequences located in the 3' end of intron 2. They cannot encode a complete POMC molecule, and the only truncated POMC molecules that could be translated would lack a signal peptide necessary for membrane translocation and precursor processing. The use of highly sensitive S1 nuclease mapping techniques with uniformly labeled single-stranded DNA probes allowed the detection of a small but definite amount of the "normal," 1200-nucleotide, mRNA species. It is suggested that it is this POMC mRNA that is responsible for the local production of all the POMC peptides.

o,p'-DDD is an inhibitor of adrenal steroid synthesis currently used for therapy of Cushing's syndrome. Conflicting data have been published on the relationship between the plasma level of the drug and its clinical and biological effects. The levels of o,p'-DDD and o,p'-DDE in various tissues obtained from treated patients have been measured and are compared with data on in vitro steroidogenesis in adrenal tissues from the same patients. o,p'-DDD was found in all samples and o,p'-DDE in half of them, both levels being high when the tissue lipid concentration was high. There was considerable variability in lipid content from one tissue to another and within a tissue from one sample to another; only the drug to lipid ratio seems able to provide a reproducible index of drug entry into a tissue. No relationship was found between the tissue concentration of the drug and the total dose administered or inhibition of the steroid biosynthetic step studied.

Tissue concentrations of immunoreactive lipotrophin, β-endorphin, and met-enkephalin were determined in 10 phaeochromocytomas, 3 of which were responsible for the ectopic ACTH syndrome. Lipotrophin and β-endorphin immunoreactivities could be detected in all cases, whether or not Cushing's syndrome was present, and their tissue concentrations were significantly correlated (r = 0.95, P< 0.001). Chromatographic studies showed tha γ-lipotrophin and β-endorphin were the main peptides in the tumours. Met-enkephalin immunoreactivity was also found in all tumours examined, at much higher concentration and showing no correlation with either lipotrophin or β-endorphin immunoreactivity. Although β-endorphin and met-enkephalin are thought to originate from different precursor molecules, these data show that the two opioid peptides may be secreted by the same tumour. The evidence for excess secretion of opioid peptides and their pathological significance in phaeochromocytomas remain to be established.

Abstract. We evaluated the usefulness of measurement of urinary free corticoids (UFC) in the diagnosis of hypercorticism. One hundred and thirty patients with features suggestive of Cushing's syndrome were examined. Twenty-six patients were proved to have Cushing's syndrome. The remaining 104 patients were considered not to have this disease on the basis of other investigations, and were designated as 'suspected patients'. UFC and 17-hydroxycorticosteroids were measured in all these patients in the basal state and on the 5th day of a low-dose-dexamethasone suppression test (3 mg/day for 5 days). UFC values of patients with Cushing's syndrome were well above 125 μg/24 h in the basal state and higher than 10 μg/24 h during the suppression test. UFC values of 'suspected patients' were between 20 and 125 μg/24 h in the basal state and below 10 μg/24 h during the suppression test.It can be concluded that the measurement of UFC in the basal state and on the 5th day of a suppression test is not only a very sensitive but also a specific method for the diagnosis of Cushing's syndrome.

Abstract. In this study, we investigated the usefulness of the determination of evening urinary free corticoids/creatinine in samples collected from 20.00 to 24.00 h as a screening test in Cushing's syndrome. In controls (N = 61) the ratio values ranged from 1.1 to 9.4 μmol/mol, whereas in patients with Cushing's syndrome (N = 20), they ranged from 27.5 to 855.5 μmol/mol. However, in 28% of patients with major obesity (> 50% overweight) and no hypercortisolism, the ratio values were between 9.4 to 27.8 μmol/mol. A short (10 days) hypocaloric diet induced a decrease in the values in 75% of these patients; the normal range was reached in 50% of them. In addition, the evening urinary free corticoids/creatinine was slightly abnormal in 8 out of 10 patients with incidentally discovered 'silent' adrenal adenomas, whereas it was normal in all 6 with other adrenal masses. In conclusion, evening urinary free corticoids/creatinine is easy to obtain and it reaches a higher sensitivity (100%) and specificity (97%) than the 24 h urinary free corticoids. In the case of borderline values, the presence of overweight should be taken into account.

A procedure for simulating aerodynamic/dynamic interactions is described. The equations of motion of the flowing air and the lifting structure are integrated numerically by an iterative technique based on Hamming’s predictor-corrector method. The solution provides the flowfield and the motion of the lifting body simultaneously. The technique is modular and different models of the flow and the structure can be incorporated by simply changing subroutines. For the current examples of wingrock of highly swept delta wings and flutter of very flexible, very high-aspect-ratio wings, the flow-fields are considered incompressible and modeled by vortex-lattice and continuous vorticity-panel methods. These models are inherently nonlinear and provide a force-free wake as part of the solution. It is shown that the instabilities can be easily suppressed by means of actively controlled flaps.

It is generally accepted that human beta-melanocyte-stimulating hormone (h beta MSH) does not normally exist in humans but was merely an artifactually generated 22-amino acid peptide corresponding to a lipotropin (LPH) fragment (residues 35-56). We examined whether the shorter 18-amino acid peptide h beta MSH-(5-22) could be detected in some human tissues. Normal human pituitaries and hypothalami as well as corticotropin-secreting pituitary and nonpituitary tumors were extracted and chromatographed on Sephadex G-50, and the fractions were measured with two radioimmunoassays using either a COOH-terminal human gamma LPH (h gamma LPH) antiserum that recognized equally h gamma LPH, h beta MSH, and h beta MSH-(5-22) or a mid-portion h gamma LPH antiserum that recognized h gamma LPH and h beta MSH but not h beta MSH-(5-22). Normal pituitaries and pituitary tumors contained a single immunoreactive material coeluting with h gamma LPH. The hypothalami and the nonpituitary tumors all contained h gamma LPH and a smaller molecular weight material that was only detected in the COOH-terminal h gamma LPH radioimmunoassay; its elution volume (Ve/V, 0.75) was identical to that of h beta MSH-(5-22) but different from that of h beta MSH (Ve/V, 0.60); on reversed-phase HPLC, it coeluted with synthetic h beta MSH-(5-22) with a retention time different from that of h beta MSH. It is concluded that h beta MSH-(5-22) that corresponds to the 18-amino acid peptide h beta LPH-(39-56), flanked by two pairs of basic amino acids within the h beta LPH molecule, is a normal maturation product of proopiomelanocortin in human nonpituitary tissues.

Phaeochromocytoma is an occasional cause of the ectopic ACTH syndrome. The mechanisms of proopiomelanocortin (POMC) gene expression were analysed in 11 human tumours not associated with Cushing's syndrome, by detecting and characterizing the POMC mRNA. A DNA probe corresponding to most of the protein-coding region of the third exon was used in Northern blot studies of total and poly(A)+RNA. All tumours contained a short (800 bases) mRNA species different from the 1200 base mRNA species of the human pituitary. This short mRNA was also present in the normal adrenal, where S1mapping showed that it resulted from transcription initiation within the third exon. However, in two tumours, equivalent amounts of the 1200 base mRNA were also present, and in one of them a third POMC mRNA of approximately 1450 bases was detected. These data show that POMC gene expression occurs in all phaeochromocytomas. It is suggested that excess production of the 1200 bases (or the larger, 1450 base) mRNA in some tumours may be responsible for the rare occurrence of the ectopic ACTH syndrome.

Surgical treatment for primary hyperparathyroidism (HPT) is effective in 90% of cases. Recurrent or persistent HPT occurs in 10% of cases. Parathyroid imaging is indicated to confirm and locate an abnormal gland before reoperation. The aim of this study was to evaluate whether the combination of 99Tcm sestamibi scintigraphy, MRI and venous blood sampling (VBS) improved the overall sensitivity for abnormal parathyroid gland detection. 18 patients with recurrent or persistent HPT underwent sestamibi scintigraphy (n = 18), MRI (T1 weighted and STIR sequences) (n = 18) and venous blood sampling (n = 12) at different sites (internal jugular veins, innominate veins, and superior vena cava). All patients underwent surgical exploration. MRI yielded positive results in 15 cases (sensitivity 88%), sestamibi scintigraphy in 14 cases (83%) and VBS in 10 cases out of 12 (83%). Combined results of MRI, sestamibi and VBS yielded positive results in 16 cases (94%). The combination of MRI, sestamibi scintigraphy and VBS improved accuracy in detecting abnormal parathyroid glands before reoperation.

The effects of the intramuscular injection of lysine-8-vasopressin (LVP) were studied through direct measurement of plasma corticotrophic activity.In 9 controls cortisol rises constantly and markedly. The rise of ACTH is limited but it was pointed out that this minimal rise is sufficient to induce a submaximal stimulation of the adrenal cortex. 63 subjects suffering from adreno-pituitary disorders were submitted to the LVP test.In idiopathic hypopituitarism with ACTH deficiency established through metopirone test and water load, the rise of plasma corticotrophic activity is normal. In hypocortisolism (Addison's disease, congenital adrenal hyperplasia) the ACTH basal level is high but the increase after LVP is proportionately the same as in the normal controls.38 Cushing's syndrome were studied. In case of adrenal tumour, LVP does not induce any change in the parameters. In Cushing's disease with bilateral adrenal hyperplasia without pituitary tumour, the rise of plasma ACTH is identical as in the normal controls. After bilateral adrenalectomy, the response to LVP is comparable to suitable controls (Addison's disease). But in case of the development of a pituitary tumour, before and especially after adrenalectomy, LVP induces a large increase of plasma corticotrophic activity. Tumoral tissue can also be detected even before the onset of clinical or radiological symptoms.The mechanism of action of LVP is discussed.

Plasma insulin levels have been measured by radio-immunoassay in 15 patients with untreated Cushing's syndrome, using oral glucose tolerance tests (O.G.T.T.), arginine infusions (A.I.) or both of these stimuli. During O.G.T.T., glucose tolerance was impaired in 12 subjects, at the upper limit of the normal range in two, and normal in only one case. Statistically significant hyperinsulinaemia occured in 5 patients. Nine subjects had plasma insulin levels in the control's range, though their glycaemic curve was of the diabetic type. During arginine infusion, insulin peak was not reduced in these 9 patients. A negative response to each test was observed in one patient. These heterogeneous results are discussed in the light of experimental data and known physiological effects of glucocorticoids. L'insulinémie a été étudiée avant tout traitement chez 15 malades atteints d'un syndrome de Cushing lors de la stimulation par le glucose oral, l'arginine intraveineuse ou les deux. Les courbes glycémiques étaient de type diabétique dans 12 cas, à la limite supérieure de la normale chez deux patients et normale dans un seul cas. Cinq de ces sujets seulement ont un hyperinsulinisme statistiquement significatif lors de l'hyperglycémie orale. Neuf ont une insulinémie dans les limites de la normale alors que leurs courbes glycémiques sont diabétiques. Le pic insulinémique lors de l'épreuve à l'arginine n'est pas diminué chez ces derniers. Un malade enfin a une réponse insulinémique très faible au cours des deux épreuves. L'hétérogénéité des troubles de l'insulino-secrétion observée au cours du syndrome de Cushing est discutée à la lumière des données expérimentales et des effets physiologiques connus des glucocorticoïdes. Bei 15 Cushing-Patienten wurden die Seruminsulinspiegel während einer oralen Glucosebelastung und einer Arginin-Infusion untersucht. 12 der Patienten zeigten eine eingeschränkte, 2 eine leicht gestörte und nur 1 Patient eine normale Glucosetoleranz. 5 Patienten hatten während der oralen Glucosebelastung eine statistisch signifikante Hyperinsulinämie; bei 9 lag die Insulinkonzentration des Blutes innerhalb normaler Grenzen, obgleich sich eine pathologische Blutzuckerkurve fand. Die Maximalwerte der Seruminsulinspiegel unter Arginin-Infusion waren bei diesen 9 Patienten nicht verringert. Bei einem Patienten führten beide Belastungen nur zu einem minimalen Anstieg der Seruminsulinspiegel. Diese heterogenen Befunde zur Insulininkretion beim Cushing-Syndrom werden unter Berücksichtigung experimenteller Befunde und der bekannten physiologischen Effekte der Glucocorticoide besprochen.