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The ability of a number of antipsychotic drugs such as phenothiazines to bind to calmodulin with high affinity in a calcium-dependent manner was applied to the study of the nature of their interactions with calmodulin. Thus, a series of phenothiazine derivatives and analogues were immobilized on agarose and examined for their binding characteristics to calmodulin. The binding of calmodulin to fluphenazine, perphenazine and 7-aminotriflupromazine involved on the one hand non-specific electrostatic interactions which are abolished by increasing the eluent salt concentration, and on the other hand, Ca2+-dependent interactions which are reversed by EGTA addition. However, the Ca2+-dependent binding of calmodulin was less specific with phenothiazine structural analogues (neutral Red, diphenylamine) and was suppressed with other phenothiazine derivatives (thionine, Azure C, Toluidine Blue) or analogues (Brilliant Cresyl Blue). It is suggested that the calcium-dependent interactions between calmodulin and drugs involve a charge transfer pi-pi interaction which may be modulated by the electron donor-acceptor properties of the substituents of the aromatic ring. Affinity chromatography using immobilized fluphenazine was also used as the basis for the purification of calmodulin from a number of tissues in a rapid one-step procedure.

The pharmacokinetics of vincristine (VCR) after an intravenous bolus dose of 2 mg were studied in patients with cancer with and without a concomitant treatment with the calcium-entry blocker nifedipine (NIF). VCR concentrations were determined by a sensitive radioimmunoassay. Pharmacokinetic data were analyzed by a nonlinear weighted least-square regression program (SAS-NLIN). A tri-exponential model fitted the raw data better than a bi-exponential model in five of 14 (35%) patients treated with VCR alone and in seven of 12 (58%) patients treated with VCR plus NIF (P = NS). The T1/2 alpha was shorter in NIF-treated patients, whereas the T1/2 gamma was longer in the NIF-treated group. The NIF-treated group showed an increase in the AUC O-infinity and AUC 1 to 96 hours, and a decrease in the AUC 0 to 1 hour. Total plasma clearance of VCR and 7-day urinary excretion of VCR was reduced in the NIF-treated patients. These data suggest that, when VCR is administered to NIF-treated patients with cancer, there is a decrease in VCR clearance from the body. Theoretically, a greater cytotoxicity may be anticipated.

Ten patients with congestive heart failure (CHF) (NYHA II-IV) on adjusted doses of digitalis and diuretics underwent a careful clinical assessment including an evaluation of exertion dyspnoea and the usual echocardiographic indices of cardiac performance. A cardiopulmonary exercise test with an increment of 20W every 3 minutes was prolonged until exhaustion. Systemic arterial pressure, ECG, VO2, VCO2 and VE were monitored throughout. Gas tensions, plasma catecholamines and lactate were measured in blood samples taken at the first and third minute of each exercise stage. The above measurements were carried out before and after 3 months of treatment with Captopril, 50 mg b.i.d. or t.i.d. A highly significant correlation between arterial lactate and plasma norepinephrine (NE) was observed in each patient during both exercise tests (r = 0.77 to 0.99; p less than 0.05 at least). Left ventricular end-diastolic dimensions were reduced by Captopril (from 69.9 +/- 1.7 to 65.2 +/- 1.4 mm, p less than 0.01) along with a concomitant increase in percent fractional shortening. Most of the patients were reclassified at a lower NYHA class and a significant decrease in dyspnoea score was observed. The exercise time was significantly increased (from 11.2 +/- 1.8 to 12.9 +/- 1.9 min; p less than 0.05), but the peak values of NE, arterial lactate and VO2 were not affected by the treatment. The predicted value of VE at a VCO2 of 1 L/min, regarded as an index of dyspnoea, was significantly decreased by Captopril (from 41.4 +/- 2.9 to 38.9 +/- 2.7 L/min; p less than 0.05). The positive effects of long-term treatment with Captopril on cardiac performance in CHF are confirmed. Sympathetic activity is linked to anaerobic muscular metabolism during exercise and seems to be independent of pharmacological ACE inhibition. The discrepancy between the exercise tolerance and the peak VO2 might be explained by a better utilization of the available energy.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.

A new type of drug acid-labile arm-carrier is described. A hydrophilic polymer [polytrisacryl, or poly(acryloyl 2-amido-2-hydroxymethyl-1,3-propanediol)], used as a model, is substituted with p-nitrobenzyl groups. The nitrobenzyl groups are reduced to aminobenzyl and transformed into benzylisothiocyanate groups which are allowed to react with aminoacyl daunorubicin. The excess of benzylisothiocyanate is transformed into benzylthiocarbamoyl N-methyl glucamine. A conjugate containing benzylthiocarbamoyl-aspartyl daunorubicin is stable at neutral pH, and releases free daunorubicin when it is exposed to pH 5 or below. This conjugate is about 200-fold less toxic than free daunorubicin, on a molecular basis, when it is added to the medium of Lewis lung carcinoma (3LL) cells in culture.

Biological glue is obtained by mixing different specific plasma proteins including a serine protease, thrombin. Surprisingly at present the thrombin used in such a mixture is from equine or bovine origin while all other components are from human. In this paper we described a particular efficient and specific chromatographic method for the purification of human thrombin usable as a serine protease in the preparation of biological glue. A pure and active thrombin is obtained from a plasma fraction after adsorption on benzamidine-Spherodex followed by an elution with non specific (sodium chloride gradient) or biospecific competitors (arginine methylester or benzamidine). The obtained thrombin with a yield close to 80% and a purification factor close to 160, showed good properties in the replacement of animal thrombin in the condition of biological glue.

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.

Cell walls from Nocardia opaca induce the production of mitogenic factors by mouse peritoneal macrophages in vitro. These factors stimulate thymocytes from C3H/HeJ mice. Supernatants of peritoneal cell culture exhibiting this activity were fractionated by chromatographic procedures such as gel filtration and metal chelate affinity chromatography and the biological activities assayed. These fractionation studies indicate that several biologically active products occur in the supernatant. Four factors monokines (M) with different apparent molecular masses M1 (100,000), M2 (50,000), M3 (16,000) and M4 (7000) were obtained, one of which (M3) was identical to interleukin 1 (IL1). Several of the biochemical parameters of one of these factors, M2, were analyzed. It was found that this monokine had many properties in common with IL1: stimulation of proliferation of thymocytes from C3H/HeJ mice, similar amino acid composition and mobility during isoelectric focusing.

A new methodology for cell separation by affinity chromatography is described. We have conjugated the organomercurial mersalyl to trisacryl beads bearing primary amino groups. Thiolated ligands can be immobilized on this matrix through cleavable Hg-S bonds. Two model studies of cell separation are reported: (i) concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacryl were eluted from the support by short thiol treatment which preserved cell viability; (ii) anti-dinitrophenyl antibodies modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-trisacryl; sheep erythrocytes, previously labelled with trinitrobenzene sulfonic acid, bound to this support and were easily recovered by thiol treatment without hemolysis. This methodology should overcome difficulties frequently encountered in cell affinity chromatography. Cell support multivalent interactions or high affinity of cell-ligand bindings often require drastic elution conditions which prevent viable cell recovery.

A study of the physical properties of paraprotein required until now, the use of fairly complicated techniques, such as analytical ultracentrifugation. The use of chromatography on acrylamide agar pearls and electrophoresis on indubiose plates, coupled with the classical techniques of electrophoresis and immunoelectrophoresis, permitted a more simple approach to these problems. The study of IgA paraproteins in several polymerised forms, is an example of the possibilities of this new method of investigation.

The present study was carried out to test the hypothesis of a possible effectiveness of captopril--an enzymatic inhibitor of both angiotensin II formation and bradykinin degradation--on hypoxic pulmonary hypertension. In 6 patients with this clinical condition, captopril-induced changes in pulmonary hemodynamics were observed after the acute administration of the drug with and without a short period of oxygen therapy at a flow rate sufficient to keep the PaO2 over 60 mm Hg. In our patients, captopril significantly lowered pulmonary arterial pressure and vascular resistance only when combined with oxygen, suggesting that an increase in bradykinin availability and/or a decrease in angiotensin II synthesis might be important factors in reversing pulmonary arterial hypertension only after blunting of the hypoxic stimulus on pulmonary circulation. Moreover, the authors suggest that the employment of vasodilators in the setting of hypoxic pulmonary hypertension should be considered not only as a means of relieving vasoconstriction but also as a possible tool for maintaining cardiac output and, in turn, peripheral oxygen delivery.

We have undertaken the present study to evaluate the acute effects of propranolol and verapamil on the diastolic function in patients with hypertrophic cardiomyopathy. We used a non imaging isotope device, the nuclear stethoscope, that has been so far mainly employed for assessment of systolic function, although fitted for detecting diastolic filling rates and times as well. Relative cardiac output, ejection fraction, rapid and slow filling times and rates were measured in five patients suffering from hypertrophic cardiomyopathy in basal conditions and 10.20 and 30 minutes after i.v. administration for either propranolol or verapamil, with a time interval of at least 24 hours between the two acute drug studies. With respect to the baseline values only verapamil showed a significant improvement in rapid and maximum filling rates, suggesting that diastolic function is more beneficially affected by Ca-entry blockers rather than beta-blockers in hypertrophic cardiomyopathy.

Acid-base parameters were studied in 10 adult patients with chronic respiratory failure during spontaneous acute exacerbations of chronic hypercapnia and after return and stabilization of PaCO2 values at previous levels with improvement of clinical conditions. All factors interfering with acid-base equilibrium were carefully avoided. The results of this investigation confirm qualitatively those obtained by GOLDSTEIN et al. [7] in dog studies and those of INGRAM et al. [9]in human studies: increasing degrees of chronic hypercapnia tend to minimize the pH reduction induced by acute PACO2 changes. From a quantitative standpoint INGRAM's data in humans are considerably different from GOLDSTEIN's data obtained in dogs. Our data, although numerically limited, confirm the acute deltaH+/deltaPaCO2 slope obtained from dogs by GOLDSTEIN et al. As data are not yet sufficient, definitive conclusions cannot be drawn. However the pattern of acid-base equilibrium changes in man in this situation seems to be similar to that observed by GOLDSTEIN et al. in the dog and could be described with reasonable accuracy by the theoretical model of LEUENBERGER et al.[13].