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The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.

In six patients urinary excretion of vanillylmandelic acid and catecholamines (CA) could establish the diagnosis of pheochromocytoma. Free norepinephrine (NE) in plasma was within the normal range in two patients and plasma free epinephrine (E) was only marginally elevated in one of them. The degree of CA conjugation was not altered and scattered as in controls and was therefore not complementary to the usual determination of plasma free CA. The intermittent nature of CA secretion by the tumour could be demonstrated by multiple blood samplings during a 48-h study period in two patients, e.g. normal plasma values might be associated with pheochromocytoma if measurements are made during a trough. Thus a single peripheral CA determination cannot be of discriminative value in the diagnosis of pheochromocytoma unless it shows marked elevation. Ten patients subjected to intracardial measurements and five patients suspected of having a pheochromocytoma underwent venous catheterisation to determine their free and conjugated plasma CA. In controls CA values near the orifices of adrenal veins differed enormously and partly overlapped with corresponding levels of patients with pheochromocytoma. In one patient with surgically proven left adrenal tumour highest concentrations of CA were measured in the vena cava superior. These high CA concentrations, caused by paroxysmal release of CA by the tumour arouse suspicion of an additional, ectopic tumour. Because venous catheterisation cannot be relied on implicitly we propose computed tomographic scanning as a first step in localisation of a pheochromocytoma.

The synthesis of antigens of the haptens p-tyramine, octopamine, synephrine, dopamine, normetanephrine, beta-(3,4-dichlorophenyl)-ethylamine and 3-fluoro-tyramine is described. The coupling of the haptens to the carrier protein was performed either via the side chain by means of succinic anhydride, or through the phenolic ring via an aminomethyl group which was introduced by a Mannich reaction. Antisera to these haptens were produced in rabbits by immunization with these antigens. The antisera were characterized by determination of titer and specificity (p-tyramine, synephrine, normetanephrine). The possibility is discussed of using antisera to p-hydroxyphenylethylamine for a radioimmunological determination of catecholamines.

The serum concentration of unconjugated estriol, total estriol, cortisol in one hour intervals and the urinary excretion of total estrogens and cortisol in three hour intervals was determined in eleven apparently healthy women between 26 and 40 weeks of gestation. They were divided into two groups, in order to study the influence of Fenoterol treatment on the daily variation of the measured parameters. Six patients were treated with Fenoterol and five patients received no medication. Between the two groups no statistical differences could be detected for the measured parameters. Regular and marked diurnal variations in cortisol have been observed, whereas total and unconjugated estriol showed small and irregular daily changes. These daily variations of "estriol" were of the same magnitude as the two-fold methodical standard deviation. For this reason it is not necessary to standardize the sampling time.

Urinary excretion of alanine aminopeptidase (EC 3.4.11.2) and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) was determined for 25-70 days in five patients receiving cis-platinum and for 8-53 days in six patients receiving amikacin. This study was performed to investigate if the excretion of urinary enzymes represents a sensitive parameter for the early detection of toxic kidney damage. The determination of N-acetyl-beta-D-glucosaminidase was carried out by the method of Knoll et al. [13]. The procedure of Mondorf et al. [14] for the estimation of alanine aminopeptidase activity was adapted to the Gemsaec Fast-Analyzer. In both patient groups an increase in the excretion of the two enzyme activities could be demonstrated. In patients receiving amikacin, the excretion of alanine aminopeptidase was always higher than that of N-acetyl-beta-D-glucosaminidase, whereas in three patients receiving cis-platinum it was the opposite. In two cis-platinum patients the excretion of both enzymes was of the same size. The changes during amikacin therapy seem to be reversible, whereas in four cis-platinum patients these changes seemed to be partly irreversible. Serum creatinine concentration was less sensitive than the urinary enzyme excretion for detection of kidney damage.

In this study the synthesis of various antigens from 3,4-dimethoxyphenylethylamine (3,4-DMPEA) is described. Antigen A was formed by coupling the acylated side chain of 3,4-DMPEA to the protein, while antigen B was synthesized by introducing an acylated amino group to the benzene ring and linking this reactive group to the protein. With antigen A an antiserum with a titer of 1 : 16000 could be raised, while with antigen B coupled to human albumin and bovine gamma-globulin the antiserum had a titer of 1 :1000 and 1 : 4000. Antibodies formed by immunization with antigen A exhibited a high specificity for substituents of the benzene ring, but were less specific for substituents of the side chain. Antibodies formed by treatment with the antigen B were specific for both kinds of substituents. So far, it has not been possible to harvest any specific antibodies towards the corresponding antigens of dopamine.

Plasma free and sulfoconjugated norepinephrine (NE), epinephrine (E) and dopamine (DA) concentrations measured in patients with thrombocytopenia or thrombocytosis, in newborns and pregnant women were not statistically different from values determined in 41 healthy volunteers. The percentage free to total NE, E and DA was 30 +/- 10%, 35 +/- 11% and 1.5 +/- 1.1% (mean +/- SE) in the controls, resp; not different from the previously described patients or from patients with liver failure who showed significantly higher free and conjugated NE and E levels when compared with controls (p less than 0.01, resp.). Conjugated catecholamine (CA) levels from the femoral artery and from multiple sites in the venous system sampled in patients undergoing intracardiac measurements were identical. The data suggest that sulfation of CA may not be simply ascribed to platelets, to the liver, to vascular beds, or to organs along the vena cava including the adrenal glands. The parallel increase of free and conjugated NE with age in healthy controls, as well as the unchanged degree of conjugation in patients with increased spillover of NE and E caused by a pheochromocytoma or by a heart attack, suggest that there is a balance between free and sulfated CA. A normal ratio of free to conjugated NE and E observed in patients receiving high dosage DA infusion further indicates that there is an adequate sulfate supply and no apparent substrate inhibition of the conjugation process. Because the percentage free of total NE, E and DA were significantly lower in patients in the hypothyroid state when compared with controls (p less than 0.01, resp.), hypothyroidism may affect the balance of free to conjugated CA in a yet unknown way.

The circadian rhythm of the excretion of the catecholamines and some main metabolites is investigated in five normal subjects and two patients with a high excretion. In the normal subjects there are remarkable differences in the amplitudes of the variables. At high catecholamine excretions these differences seem to disappear.

A specific and sensitive radioimmunoassay for the determination of 3,4-dimethoxyphenylethylamine (3,4-DMPEA) in urine was developed. In 5 fasting healthy volunteers excretion of 3,4-DMPEA was not diminished, indicating that this substance can be regarded as an endogenous metabolite. In addition, the time profile of the excretion of 3,4-DMPEA was measured in one patient during total fasting. In 25 patients with schizophrenic psychoses, 9 psychiatric patients without schizophrenia and 80 control patients excretion of 3,4-DMPEA was compared. No significant differences could be observed between these three groups.