Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.

[The antistreptolysin reaction with and without dextran sulfate addition in various diseases]. / Die Antistreptolysinreaktion mit und ohne Dextransulfatzusatz bei verschiedenen Erkrankungen.

It is reported on the results of determinations of the antistreptolysin titre in patients with infectious hepatitis, renal and essential hypertension, mitral stenosis, acute tonsillitis, scarlet fever, rheumatic fever as well as rheumatoid arthritis according to the usual and the dextran sulphate absorption method in altogether 739 patients. Here as in a former publication in 1,700 normal persons partly considerable significant differences between the antistreptolysin titre with and without dextran sulphate were found. The two techniques are discussed, also the influence of the beta-lipoproteins with their proportion of phosphatide on the result of the antistreptolysin titre. It is possible to absorb beta-lipoproteins and perhaps also phosphatides with the help of the addition of dextran sulphate. The present investigations show unequivocally that the absorption the dextran sulphate is a necessary demand which saves the clinician from diagnostic and therapeutic errors. The antistreptolysin reaction is to be regarded only as one constituent for the clinical diagnosis and must not be overvalued in its importance.

Biologically active macromolecular forms of oxytocin. [8-Lysine]oxytocin as a suitable ligand.

[8-Lysine]oxytocin was synthesized on a solid support and possessed an oxytocic activity of 100 +/- 6 units mumol on the isolated rat uterus. The epsilon-carbamoyl, epsilon-3-carboxypropionyl and epsilon-3-carboxybutryl derivatives were prepared and had uterotonic activities of 400, 55 and 50 units/mumol respectively. [8-Lysine]oxytocin was coupled unambiguously through the epsilon-amino group to the carboxyl groups of carboxymethylated dextrans or epsilon-3-carboxypropionly-gelatin. The macromolecular oxytocins were water-soluble and retained signigicant oxytocic activity. [8-Lysine]oxytocin should prove a useful ligand for affinity chromatography of oxytocin-binding proteins.

Mechanisms of the puromycin-induced defects in the transglomerular passage of water and macromolecules.

To investigate the mechanism(s) of increased filtration of serum proteins after glomerular injury, polydisperse samples of uncharged [(3)H]dextran (D) or anionic [(3)H]dextran sulfate (DS) were infused into 14 control and 16 puromycin aminonucleoside- (PAN) treated Munich-Wistar rats. Fractional clearances of D or DS ranging in radius from 18 to 42A were determined in these rats, together with direct measurements of the forces governing the glomerular filtration rate of water. Whole kidney and single nephron glomerular filtration rates were approximately 40% lower in PAN-treated rats, relative to controls, due mainly to a marked reduction in the glomerular capillary ultrafiltration coefficient and, to a lesser extent, to a small reduction in glomerular plasma flow rate as well. In PAN-treated rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and both D and DS were shown to be neither secreted nor reabsorbed. Fractional clearances of uncharged D were reduced after PAN administration, falling significantly for effective D radii from 22 to 38A. Utilizing a theory based on macromolecular transport through pores, these results indicate that in PAN-treated rats, effective pore radius is the same as in controls, approximately 44A. In PAN nephrosis, however, the ratio of total pore surface area/pore length, a measure of pore density, is reduced to approximately one-third that of control, due very likely to a reduction in filtration surface area. In contrast to the results with uncharged D, fractional clearances of DS were found to increase after PAN administration for all DS radii studied. These results with D and DS suggest that proteinuria in PAN nephrosis is due, not to an increase in effective pore radius or number of pores, but rather to a diminution of the electrostatic barrier function of the glomerular capillary wall, thereby allowing increased passage of polyanions such as DS and albumin.

[Purification of phospholipase C from Bacillus cereus by chromatography on aminoalkylpolysaccharide adsorbents]. / Khromatograficheskaia ochistka fosfolipazy s iz Bacillus cereus na aminoalkilpolisakharidnykh sorbentakh.

Purification of phospholipase C from Bac. cereus by chromatography on aminoalkylpolysaccharide adsorbents is described. The dependence of the degree of enzyme purification on the amount of ligant and effect of pH and buffer systems on the adsorption-desorption of phospholipase have been studied. At a pH below 9.0 phospholipase C is not retained by the adsorbents and is purified 4-5-fold and up to 23-fold, when aminoalkyl-Sepharose and hexamethylenediamine Sephadex are used respectively. With an increase in the pH value up to 10.0, the enzyme is bound by the adsorbent and is eluted with a 40-90% yield of activity and 7-10-fold purification. The resulting phospholipase C is highly purified and electrophoretically homogeneous. A mechanism of the enzyme-adsorbent interaction is discussed.

Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles.

A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually +/- 5%. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesions.

Treatment of hypercholesterolemia with Secholex. A long-term clinical trial and comparison with cholestyramine.

The efficacy of an anion-exchange gel, Secholex, as a hypocholesterolemic agent was assessed in 46 patients in 4 different studies and the effects were compared with those of cholestyramine. All patients had severe Type II-a or II-b hyperlipoproteinemia. In short-term metabolic studies Secholex (15 g/day) and cholestyramine (16 g/day) decreased serum cholesterol levels and increased total fecal sterol output and serum methyl sterol concentration to a similar extent, but cholestyramine was more effective than Secholex in increasing fecal bile acid excretion. In crossover studies, the two drugs appeared to be equally effective in lowing serum cholesterol levels but the patients mostly preferred Secholex. Twenty patients were treated with Secholex over a two-year period. The average decrease in serum cholesterol levels from the mean pretreatment value of 406 mg/100 ml was 15% during the first year, and 13% during the second year. In 5 patients the serum cholesterol was permanently lowered by more than 20% (good responders), while in 7 patients the average reduction of serum cholesterol level during Secholex administration was less than 10% (non-responders). The serum triglyceride level was slightly decreased by Secholex in Type II-b patients but was unaltered in Type II-a patients. At the end of the treatment period, serum iron and vitamin B12 levels were normal but the serum folic acid concentration was reduced in eight of 20 patients. A dose--response study indicated that a similar cholesterol-lowering effect was obtained with daily doses of 9 and 15 g of Secholex. It is concluded that Secholex is a relatively safe drug which effectively reduces serum cholesterol levels in two-thirds of patients with severe hypercholesterolemia.

Polycation-induced assembly of purified tubulin.

Several different polycations have been found that can substitute for the microtubule-associated proteins, or tau factor, in facilitating assembly of tubulin that has been purified by ion exchange chromatography. In low concentrations of the polycation diethylaminoethyl-dextran, 7 mg of tubulin is pelleted per 1 mg of polycation added. Under conditions favorable to microtubule assembly the entire pellet is seen by electron microscopy to consist of "double wall microtubules", which are essentially identical to normal microtubules in subunit structure and arrangement. When assembly is inhibited approximately the same amount of tubulin is pelleted, but it is in the form of clusters of curved sheets or filaments apparently related to tubulin rings. When conditions are changed to favor assembly, the tubulin within these clusters appears to reassemble to form the double wall microtubules.

The in vitro effect of diethylaminoethyl dextran on stimulation of mouse lymphocytes, and on the mitogenic activity of concanavalin A and lipopolysaccharide.

Unpurified lymphocytes from spleen, lymph nodes, thymus, and T and B cell-enriched fractions of spleen cells from NMRI mice as well as lymphocytes of nude mice were cultured and stimulation of the lymphocytes by DEAE-D, Con A and LPS and combinations thereof was measured by incorporation of 3H-thymidine. It was demonstrated that DEAE-D is a rather weak mitogen for mouse lymphocytes, acting apparently on B cells as well as on T cells. Frequency and intensity of lymphocyte stimulation by the T cell mitogen Con A and the B cell mitogen LPS was much better. When combinations of DEAE-D with Con A or LPS, respectively, were used significant enhancement of lymphocyte stimulation occurred, as compared to the mitogens and DEAE-D alone. This enhancement was more prominent with the DEAE-D/Con A combination than with the DEAE-D/LPS combination, and more pronounced with the T cell-rich lymphocyte preparations than with the B cell-rich suspensions. The results are discussed and it is concluded that DEAE-D enhances both T cell and B cell functions, however, T cell functions are favoured. This enhancement is assumed to be mediated by a membrane effect of DEAE-D, and to be responsible for th immunological adjuvant effect of DEAE-D.

Adjuvant effects of diethylaminoethyl-dextran.

Diethylaminoethyl-dextran exhibited a significant effect on the primary immune response of rhesus monkeys to formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (IVEE). Antibody formed to IVEE and adjuvant followed a classic immunoglobulin M-immunoglobulin G pattern; however, as compared with vaccine alone, use of this adjuvant with IVEE reduced the time required for onset of immunoglobulin G synthesis.

Soluble dextran-hemoglobin complex as a potential blood substitute.

A complex between soluble dextran and human hemoglobin has been synthesized by two different methods. In the alkylation method, hemoglobin was allowed to react with bromoacetyl groups incorporated into the dextran; the yield of the complex was about 80% in terms of the hemoglobin used. In the dialdehyde method, hemoglobin was allowed to react with dialdehyde groups on the dextran generated by periodate oxidation; the yield of the complex was about 60%. Both soluble dextran-hemoglobin complexes could bind and release oxygen reversibly, but the oxygen-binding curves were shifted to the left relative to that of free hemoglobin. In the rabbit, the complex obtained by the alkylation method was excreted by the kidneys and cleared from the circulation much more slowly than free hemoglobin.

Preparation and enzymatic properties of subtilisin Novo chemically attached to soluble DEAE-dextran and insoluble DEAE-sephadex.

Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.