Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes. (1/95)

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.  (+info)

Interaction of glutamine and arginine on cerebrovascular reactivity to hypercapnia. (2/95)

Glutamine is purported to inhibit recycling of citrulline to arginine and to limit nitric oxide release in vitro. However, vasoactive effects of glutamine have not been clearly demonstrated in vivo. During hyperammonemia, impaired cerebrovascular reactivity to CO(2) is related to glutamine accumulation. We tested the hypotheses that 1) glutamine infusion in the absence of hyperammonemia impairs cerebrovascular CO(2) reactivity and 2) arginine infusion preserves CO(2) reactivity during glutamine infusion and during hyperammonemia. Pentobarbital sodium-anesthetized rats were equipped with a closed cranial window for measuring pial arteriolar diameter. Intravenous infusion of 3 mmol. kg(-1). h(-1) of L-glutamine for 6 h produced threefold increases in plasma and cerebrospinal fluid concentrations. Dilation to hypercapnia was reduced by 45% compared with that of a time control group at 6 h but not at 3 h of glutamine infusion. Coinfusion of 2 mmol. kg(-1). h(-1) of L-arginine with glutamine maintained the hypercapnic vasodilation at the control value. Infusion of ammonium acetate at a rate known to produce threefold increases in cortical tissue glutamine concentration resulted in no significant hypercapnic vasodilation. Coinfusion of arginine with ammonium acetate maintained hypercapnic vasodilation at 60% of the control value. Arginine infusion did not augment hypercapnic vasodilation in a control group. We conclude that glutamine modulates cerebrovascular CO(2) reactivity in vivo. Glutamine probably acts by limiting arginine availability because the vascular inhibitory effect required >3 h to develop and because arginine infusion counteracted the vascular effect of both endogenously and exogenously produced increases in glutamine.  (+info)

Biosynthesis of cladospirone bisepoxide, a member of the spirobisnaphthalene family. (3/95)

The biosynthesis of cladospirone bisepoxide (1) was investigated by feeding 13C-labeled acetate to growing cultures of the fungus Sphaeropsidales sp. (strain F-24'707). 13C NMR spectral analysis demonstrated the polyketide origin of both naphthalene units. The origin of two epoxide oxygens was confirmed as from air by cultivation of the strain in an 18O2-enriched atmosphere. The [18O]incorporation pattern into palmarumycin C12 (11), the putative precursor of 1 led to the hypothesis that the carbonyl oxygen of 1 is derived from water by exchange of an oxygen atom. Inhibition of the biosynthesis of 1 with tricyclazole, an inhibitor of the 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis, confirmed the connection of both biosynthetic pathways.  (+info)

Effects of various arterial infusion solutions on red blood cells in the newborn. (4/95)

AIM: To examine in vitro the effects of brief contact with various infusion solutions on red blood cells from newborn infants, as occurs in the "waste" syringe during routine blood sampling from umbilical artery catheters. The mixture of blood and solution in the "waste" syringe is usually reinfused into the baby. Reinfused red blood cells may be damaged by the infusion solution. It is hypothesised that an isotonic amino acid solution would cause no red blood cell agglutination and no more haemolysis than many commonly used solutions. METHODS: Blood was obtained from the placentas of 15 normal term babies. Haemolysis was estimated by measuring plasma (free) haemoglobin after mock blood sampling. Agglutination was measured semiquantitatively by direct observation. RESULTS: A 0.25% normal saline solution caused 5.4% haemolysis, significantly more than all the other fluids tested. There was less haemolysis with 0. 25% normal saline when there was complete mixing of blood and solution within the "waste" syringe. Normal saline and isotonic sodium acetate solutions caused < 0.1% haemolysis, significantly less than all the other fluids tested. The isotonic amino acid solution caused 0.8% haemolysis, which is similar to that caused by the remaining solutions tested. Agglutination was seen with isotonic dextrose and with the two isotonic amino acid solutions containing cysteine. CONCLUSIONS: Isotonic amino acid solution (without added cysteine) caused no agglutination and the same or less haemolysis than many commonly used solutions and may offer advantages in nutrition and fluid balance.  (+info)

Comparison of deuterium incorporation and mass isotopomer distribution analysis for measurement of human cholesterol biosynthesis. (5/95)

To compare endogenous cholesterol biosynthesis measured by deuterium incorporation (DI) and mass isotopomer distribution analysis (MIDA), cholesterol fractional and absolute synthetic rates were measured simultaneously by both techniques under identical physiological conditions. Twelve subjects (22 to 39 years of age) underwent a dual stable isotope protocol, involving oral deuterium oxide administration and measurement of incorporation of deuterium into cholesterol coincident with constant infusion of sodium [1-(13)C]acetate and measurement of the mass isotopomer distribution pattern of newly synthesized cholesterol. Synthesis was determined over 24 h with a 7-h feeding period. Both methods yielded similar measurements of fractional cholesterol synthesis (7.8 +/- 2.5% day(-)(1) for DI vs. 6.9 +/- 2.2% day(-)(1) for MIDA). Correlation of fractional synthesis across techniques was strong (r = 0.84, P = 0.0007). Absolute synthesis rates were also not different at 24 h (13.4 +/- 4.3 mg kg(-)(1) day(-)(1) for DI vs. 11.9 +/- 3.6 mg kg(-)(1) day(-)(1) for MIDA, r = 0.79, P < 0.002). We conclude that despite different assumptions and analytical requirements, deuterium incorporation and MIDA yield similar rates of cholesterogenesis in humans when measurements are made over 24 h. The decision as to which method to adopt depends on available clinical and analytical facilities  (+info)

Effect of intracellular pH on spontaneous Ca2+ sparks in rat ventricular myocytes. (6/95)

1. A fall of intracellular pH (pHi) typically depresses cardiac contractility. Among the many mechanisms underlying this depression, an inhibitory effect of acidosis upon the sarcoplasmic reticulum (SR) Ca2+ release channel has been predicted, but not so far demonstrated in the intact cardiac myocyte. In the present work, pHi was manipulated experimentally while confocal imaging was used to record spontaneous 'Ca2+ sparks' (local SR Ca2+ release events) in rat isolated myocytes loaded with the fluorescent Ca2+ indicator fluo-3. In other experiments, whole cell (global) pHi or [Ca2+]i was measured by microfluorimetry (using, respectively, intracellular carboxy SNARF-1 and indo-1). 2. Reducing pHi (i) increased whole cell intracellular [Ca2+] transients induced either electrically or by addition of caffeine, whereas (ii) it decreased spontaneous Ca2+ spark frequency. Conversely, raising pHi increased spontaneous Ca2+ spark frequency. 3. Blocking sarcolemmal Ca2+ influx with 10 mM Ni2+, or reducing external pH by 1.0 unit, had no effect on the pHi-dependent changes in spontaneous Ca2+ spark frequency. 4. Decreasing pHi over the range 7.78-7.20, decreased Ca2+ spark frequency exponentially as a function of pHi, with frequency declining by approximately 33 % for a 0.2 unit fall in pHi. In contrast, over the same pHi range, Ca2+ spark amplitude was unaffected. Intracellular acidosis produced a slight slowing of Ca2+ spark relaxation. 5. The results indicate that, in the intact myocyte, a reduced pHi decreases the probability of opening of the SR Ca2+ release channel. This phenomenon may contribute to the negative inotropic effects of acidosis.  (+info)

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells. (7/95)

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.  (+info)

Lack of functional and morphological susceptibility of the greater superficial petrosal nerve to developmental dietary sodium restriction. (8/95)

Restriction of dietary sodium during gestation has major effects on taste function and anatomy in the offspring. The chorda tympani nerve of offspring that are maintained on sodium-reduced chow throughout life (NaDep) has reduced neurophysiological responses to sodium and altered morphology of its terminal field in the nucleus of the solitary tract. There are many anatomical and physiological similarities between the chorda tympani nerve that innervates taste buds on the anterior tongue and the greater superficial petrosal nerve (GSP) that innervates taste buds on the palate. To determine if the GSP is similarly susceptible to the effects of dietary sodium restriction, the present study examined neurophysiological responses and the terminal field of the GSP in NaDep and control rats. Neurophysiological responses of the GSP to a variety of sodium and non-sodium stimuli did not differ between NaDep and control rats. Furthermore, the volume and shape of the GSP terminal field in the nucleus of the solitary tract did not differ between the groups. Therefore, despite the high degree of functional and anatomical correspondence between the chorda tympani nerve and the GSP, the GSP does not appear to be susceptible to the effects of lifelong dietary sodium restriction.  (+info)