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The plasma cell myeloma (multiple myeloma, myelomatosis) is a progressive disease, characterized by bone marrow plasmacytomas and the presence of monoclonal antibodies (IgG, IgA, IgD, IgE), or free kappa or lambda immunoglobulin side chains. The monoclonal antibodies or Bence-Jones protein may precipitate in the tubuli and impair kidney function. In addition, the plasma protein concentration may increase at the expense of plasma water level causing unrealistically low electrolyte levels. Since the isoelectric points of immunoglobulins are higher than those of most other plasma proteins, the net charge of plasma proteins may change causing new electrolyte balance. In addition, some monoclonal antibodies are more hydrated than others, and their high concentration may cause not only increased plasma viscosity but further electrolyte imbalance. In the present work the relationship between plasma protein and electrolyte levels is studied in samples of 100 multiple myeloma patients.

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Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.

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In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.

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If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks' balanced salt solution in either absence or presence of non-ionic detergents (0.2% Triton X-100 or 0.2% Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K+, Mg2+, Na+, Ca2+, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 degree C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K+, Mg2+, water and cytomatrix proteins.

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The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K+ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K+, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values).

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A silver-staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase-Ni-diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver-intensification has been reached. In this way a 16- to 64-fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.

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This study was designed to test how extended exposure of lenses to sera with different ionic strengths influences the distribution of ions and water in the lens. Pig lenses were incubated in cold sera (0 degree C), which were adjusted to variable concentrations of NaCl, and their K+, Na+, Cl-, and water contents were measured. Incubation at 0 degree C inhibits active transport processes and thereby allows equilibration of the mobile ions and water. The hypothesis was that lens water content (volume) would follow the ion-induced protein changes predicted by a model derived from previous osmotic studies on proteins. As expected, exposure of the lens to cold caused a gain of sodium and a partial loss of potassium. However, the potassium concentration in the lens remained several fold higher than that in the bathing solution (about 41 vs. 1.8-4.6 mM/kg H2O), indicating that a portion of the potassium within the cold-exposed lens was not free to diffuse. That the water content of the lens showed a negative rather than a positive relationship with the concentration of NaCl within the lens was explained by the idea that an increase in NaCl within the lens (up to at least 250 mM/kg H2O) causes a decrease in the osmotically unresponsive water volume associated with lens proteins.

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The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.

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Ultrastructural observations in combination with electron probe X-ray microanalysis on detergent (Brij 58) permeabilized (disruption of the plasma membrane) nucleated chicken erythrocytes support the view that a large fraction of cytoplasmic and nuclear K+ is not freely diffusible and that adsorption of K+ on detergent released mobilizable proteins exists within the cell. The data also suggest that the detergent proteins are normally immobilized by a detergent-resistant cytoskeleton so that they are not immediately free to diffuse from the cell for several minutes after detergent disruption of the plasma membrane.

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Crystalline lenses provided good material to study and measure the properties of cellular water. Different methods were used to establish the extent and properties of nonbulk water in mammalian lenses. These methods include: NMR titration analysis, a test of the osmotic properties, a test of dye exclusion In lenses with intact cell membranes and in lenses with disrupted cell membranes, and the water-holding capacity of lenses subjected to 40,000 x g for 1 hour with intact cell membranes and in lenses with disrupted cell membranes. The data from these methods, as well as other data from the literature, lead to the conclusion that most, if not all, of the water in lens cells (up to 2.2 g water/g dry mass) has motional and osmotic properties that distinguish it from bulk water. These findings call into question the common and convenient assumption that all but a small proportion of cellular water is like that in dilute solution.

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The platelets and leukocytes of human peripheral blood were separated and cultured for 24 h in the same medium. The culture medium was completed with S-methionine. The overall protein synthetic activity of the platelets, compared with leukocytes, was studied with a two-dimensional gel electrophoresis technique combined with fluorographic detection of labelled proteins. Our findings confirm the platelet-origin of a few newly synthesized proteins. The most pronounced newly synthesized platelet protein with a molecular weight of approximately 35 Kda and with a more acidic isoelectric point than that of actin was identified as a cytoskeleton-associated protein.

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Rat liver cell nuclei isolated in the presence of Ca++ and Mg++ ions were exposed to citrate buffer at different pH levels. Their morphology was studied with the electron microscope and the water content measured. At a low pH the chromatin remained compact, but at neutral pH an intense fibrillarization of the chromatin was observed; and, seemed to be reversible, because of deoxyribonucleoproteins (DNP) filaments which dispersed at a pH of 7 were repacked to their previous site at a pH of 3. The ribonucleo-proteins (RNP)-containing structures did not show the same reversible fibrillarization. In addition to the morphological changes, a large increase in the water content ("water-holding capacity") of the chromatin structures was observed.

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Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins and K+ were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brij 58. In Triton X-100-treated cells, the loss of K+ was complete within 2 min. The loss of K+ from the Brij 58-treated cells was complete only after 10 min and the mobilization of K+ showed sigmoid-type release kinetics. These results support the view that most of K+ and "diffusible" proteins not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell.

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Rats maintained on a normal diet supplemented with 10 microCi 65Zn/day for a period of 10 days were given allylisopropylacetamide (AIA), 400 mg/kg body weight: 24 h later they were killed. Cytosol was prepared from liver cells and analysed by gel filtration on a Sephadex G-75 column. The AIA treatment caused an alteration in the chromatographic pattern of Zn-binding proteins, and a low-molecular-weight fraction appeared.

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In experiments reported here electrophoretic mobility of washed platelets and platelets suspended in diluted plasma obtained from adults and newborns was practically the same. No significant difference was observed in the pH-mobility relationship of the two types of platelets. These comparative studies indicate that the actual charge density, i.e. the number and sign of the charged groups at the newborn and adult platelet surfaces are essentially identical. However, a significant difference was found between the two platelet populations in the mobility changes induced by ADP. On the basis of cross over experiments between the platelets and plasma of adults and newborns it seems likely that the different behaviour of neonatal platelets arises from a difference between adult and newborn plasma. Preliminary results indicate that this may be due to the presence in adult plasma of a component with molecular weight about 10 000.

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Zn2+, Fe2+, Cu2+, Ca2+, and Mg2+ were determined by means of atomic absorption techniques in tetramethylammonium hydroxide digests of cell organelles isolated from normal rat liver tissue. The metal content of the cell organelles was expressed with reference to their protein content. The results showed that digestion with tetramethy la ammonium hydroxide can be applied for the determination of metal to protein ratios in the cell organelles and that these ratios appear to be suitable for the characterization of cell organelles.

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65Zn2+ was given to rats per os and intraperitoneally for in vivo labelling of Zn2+-metalloproteins of cytosol. At the chromatographic analysis of the cytosol, two 65Zn2+-containing protein peaks were found in per os, while an additional low molecular weight third peak appeared in the intraperitoneal dosing samples. The chromatographic fractions were further separated by a non-denaturing gradient polyacrylamide electrophoresis technique. At the electrophoretic analysis of the chromatographic fractions the recovery of 65Zn2+ was the highest in samples taken from a second chromatographic peak (88%), which indicates the presence of proteins with exceptionally strong Zn2+-binding character in the 30-40 000 molecular weight fraction of the cytosol.

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The amount of Zn, Fe, Cu, Mg and Ca in rat liver samples was analysed by atomic-absorption spectrophotometry and the association of Zn, Fe and Mg to cytosol proteins was studied by ultrafiltration. Nearly all of the Zn and Fe attached tightly to the protein molecules, while more than 30% of Mg was found to be "free" (ultrafilterable) in the cytosol.

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TSCM-(0.01 M Tris-HCl, 0.25 sucrose, 3.8 mM CaCl2, 12 mM MgCl2, pH 7.2) and TS-(0.01 M Tris-HCl, 0.25 M sucrose, pH 7.2)-soluble proteins of isolated rat liver nuclei were analyzed by a two-dimensional polyacrylamide gel electrophoresis method. Light microscopy showed that treatment with the TSCM solution did not alter the morphology of isolated nuclei. After treatment with the divalent cation-free TS solution, the isolated nuclei were swollen; some were disrupted and clumped together. The TSCM solution extracted 20 additional proteins including histones. These results suggest that Ca++ and Mg++ play an intracellular role in the interactions between nuclear proteins and chromatin.

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