Methods in clinical hemorheology: the continuous measurement of arterial blood density and blood sound speed in man

Both blood density and sound speed are closely related to total protein concentration in blood and, as a consequence, to rheologically important parameters of blood. Two methods that permit continuous measurement of these properties, the mechanical oscillator technique and the new ultrasonic technique, were used for measuring blood protein concentration over a continuous period of time in a group of hemodialysis patients and in volunteers. It was seen that the concentration of the components of blood varies considerably. This variability is related to transport phenomena within as well as to the flow of masses across the cardiovascular compartment. From the continuous measurement of concentrations during hemodialysis treatment, relative changes in blood volume can be recorded in order to control the fluid balance of the patient. Rapid fluctuations at the macroscopic scale with periods of 5 to 30 seconds are due to heterogeneities at the microscopic scale and to the particular rheological behaviour of the red blood cells at the level of the capillaries and the small blood vessels. The amplitude of rapid oscillations increased up to 1.2% in terms of hematocrit values when there was rhythmic, spontaneous breathing at various frequencies. The measurement of concentrations at an accessible measuring site may be used to investigate the rheology of blood in the human microvasculature.     

Purification of an antitrypanosomal factor fromPseudomonas fluorescens by high-pressure liquid chromatography

Studies on the purification of an antitrypanosomal factor (ATF-II) obtained fromPseudomonas fluorescens disclosed that high-pressure liquid chromatography (HPLC) with a Rad-pak porasil (10 silica) column and a GPC-60 Å column was an efficient procedure for the separation of the active components. Extraction of the factor with absolute ethanol prior to elution significantly enhanced the lytic activity of the HPLC eluates, as shown by marked pathologic changes followed by lysis in bioassays performed withTrypanosoma equiperdum. HPLC provided an increase of purification 30 times that obtained with gel filtration of the crude bacterial product. The lipopolysaccharide content of the purified fractions was markedly reduced and indicated an additional advantage for further in vivo tests in experimental infections withT. cruzi.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Interaction of opioid peptides and other drugs with multiple delta ncx binding sites in rat brain: further evidence for heterogeneity.

Recent pharmacological data strongly support the hypothesis of δ receptor subtypes as mediators of both supraspinal and spinal antinociception (δ₁ and δ₂ receptors). In vitro ligand binding data, which are fully supportive of the in vivo data, are still lacking. A previous study indicated that [³H][ -Ala², -Leu⁵]enkephalin labels two binding sites in membranes depleted of μ binding sites by pretreatment with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole-HCI (BIT). The main goal of the present study was to develop a ligand-selectivity profile of the two δ_ncx binding sites. The data indicated that naltrindole and oxymorphindole were relatively selective for site 1 (20-fold). [ -Ser²,Thr⁶]Enkephalin and deltorphin-II were only 2.7-fold and 2.2-fold selective for site 1. [ -Pen², -Pen⁵]Enkephalin and deltorphin-I were 80-fold and 38-fold selective for site 2.3-Iodo-Tyr- -Ala-Gly-Phe- -Leu was 52-fold selective for site 1. Morphine had moderate affinity for site 1 (K_i = 16 nM), and was about 11-fold selective for site 1. Thus, of the 10 drugs studied, only DPDPE and DELT-I were selective for site 2. Viewed collectively with other data, it is likely that the δ₁ receptor and the δ_ncx binding site are synonymous.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.