Extracellular potassium can simulate the role of serum as an activator of uridine uptake by quiescent hamster cells in culture

Replacement of ~100 mM of sodium chloride in the extracellular medium of quiescent hamster fibroblasts (Nil 8 and BHK cells) by potassium chloride causes an increase in the rate of uridine uptake. This increase is identical with that achieved by addition of 10% serum to the same cultures. The effects of serum and KCl are not additive. The dependence of the rate of uridine uptake on extracellular KCl concentration is of a sigmoid nature. The time course of the activation process is similar to that of serum activation of uridine uptake in the same cells. The high rate of uridine uptake persists for at least 30 min after return to an extracellular medium containing a high concentration of sodium.     

Identification and partial characterization of discrete apolipoprotein B containing lipoprotein particles produced by human hepatoma cell line HepG2

The purpose of this study was to test the use of human hepatocarcinoma HepG2 cells as a model for studying the formation and secretion of human hepatic lipoproteins. To this end, we determined the rate of accumulation and percent composition of neutral lipids and apolipoproteins in the culture medium of HepG2 cells and isolated and partially characterized the apolipoprotein B (ApoB) containing lipoprotein particles. The rates of accumulation in the medium of HepG2 cells, grown in minimum essential medium during a 24-h incubation, of triglycerides, cholesterol, and cholesterol esters expressed as microgram/(g of cell protein X h) were 373 +/- 55, 167 +/- 14, and 79 +/- 10, respectively; the secretion rates for apolipoproteins B, A-I, E, A-II, and C-III were 372 +/- 36, 149 +/- 14, 104 +/- 13, 48 +/- 4, and 13 +/- 1 microgram/(g of cell protein X h), respectively. The major portion of ApoB was present in very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) (84%), with the remainder occurring in high-density lipoproteins (HDL) (16%). Approximately 10-13% of ApoA-I and ApoA-II were present in VLDL and LDL, while 60% of ApoE occurred in HDL and 40% in VLDL and LDL. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated by either sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. Results showed that 60-70% of ApoB occurred in the culture medium as lipoprotein B (LP-B) and 30-40% as lipoprotein B:E (LP-B:E). Both ApoB-containing lipoproteins represent polydisperse systems of spherical particles ranging in size from 100 to 350 A for LP-B and from 200 to 500 A for LP-B:E. LP-B particles were identified in VLDL, LDL, and HDL, while LP-B:E particles were only present in VLDL and LDL. The major neutral lipid of both ApoB-containing lipoproteins was triglyceride (50-70% of the total neutral lipid content); cholesterol and cholesterol esters were present in equal amounts. The LP-B:E particles contained 70-90% ApoB and 10-30% ApoE. The ApoB was identified in both types of particles as B-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B:E particles.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. II. Isolation of apolipoprotein B-containing lipoproteins from human plasma

Monoclonal antibody ('Pan B' antibody) that binds equally to all major forms of human plasma apolipoprotein B was used in an immunoaffinity chromatography procedure to isolate apolipoprotein B-containing lipoproteins from hyperlipidemic human plasma. These lipoproteins were compared with lipoproteins in native plasma, with lipoproteins isolated by polyclonal antibodies and with lipoproteins isolated by the conventional ultracentrifugational method. Judged by the apolipoprotein and lipid composition, lipoproteins isolated with 'Pan B' antibody were virtually identical to those isolated by ultracentrifugation or polyclonal antibodies. Lipoproteins isolated by 'Pan B' antibody were comparable in size and shape to the lipoproteins in native plasma and to the lipoproteins isolated by polyclonal antibodies or ultracentrifugation. The immunoaffinity column with monoclonal 'Pan B' antibody retained all apolipoprotein B-containing lipoproteins and showed significantly higher capacity than polyclonal immunoaffinity column. The column with the highest capacity allowed the isolation from whole plasma of 0.144 mg of apolipoprotein B per ml of gel in less than 2 h.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

Spontaneous generation of adriamycin semiquinone radicals at physiologic pH.

Adriamycin semiquinone radicals are spontaneously generated by adriamycin solutions at physiologic pH. Rate of radical formation and equilibrium-state radical yield increase with increasing pH from 7.4 to 8.85. The radicals are oxygen sensitive, but the mechanism of radical formation is oxygen independent and associated with proton removal from the dihydroquinone of adriamycin. The less cardiotoxic and non-mutagenic (Ames test) anthracycline 5-iminodaunorubicin does not form semiquinone radicals spontaneously at physiologic pH.     

Errors in computing drug doses.

The 85 members of the pediatric and neonatal divisions of a medical centre were tested for their ability to calculate the appropriate volumes of drugs commonly administered to pediatric patients. Of a total of 680 computations 43 (6.3%) were wrong. Half the errors would have led to either a 10-fold overdose or a dose a 10th of that prescribed. Significantly more of the errors (p less than 0.01) were made by the nurses in the neonatal division (11.5%) than by those in the pediatric division (3.4%). A deficiency in the in-service training of the nurses in the neonatal division appeared to contribute to the higher proportion of errors in this group. There was also a trend towards a greater chance of error as the length of professional experience increased. All medical personnel involved in the ordering and administration of drugs should be taught computing skills and be evaluated routinely.     

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.     

Accumulation of cocaine in maternal and fetal hair; the dose response curve.

Cocaine and its major metabolites are incorporated into hair during the growth of the shaft and stay there for the whole life of the hair. Cocaine crosses the placenta and its metabolites for example Benzoylecgonine (BZ), have been found in neonatal urine, meconium and hair. In order to utilize hair measurements of cocaine as a biological marker of systemic exposure, we conducted both animal and human investigations on the dose response characteristics of this phenomenon. Our data suggest that both maternal and fetal accumulation of cocaine and its metabolite follow a linear pattern within the clinically used doses. Similarly, a good correlation was observed in animals between maternal dose and fetal hair accumulation.