A spectrofluorimetric assay of calmodulin-dependent protein phosphatase using 4-methylumbelliferyl phosphate

A continuous spectrofluorimetric assay of calmodulin-dependent phosphatase is described. The assay monitors the formation of a fluorescent 4-methylumbelliferone from the dephosphorylation of 4-methylumbelliferyl phosphate and detects as little as 1 pmol of 4-methylumbelliferone. The phosphatase shows a Km of 1.3 mM for the substrate and a Vmax of 100 nmol/mg/min.     

The donor chromosome breakpoint for a jumping translocation is associated with large low-copy repeats in 21q21.3

Jumping translocations (JTs) are very rare chromosome aberrations, usually identified in tumors. We report a constitutional JT between donor chromosome 21q21.3-->qter and recipients 13qter and 18qter, resulting in an approximately 15.5-Mb proximal deletion 21q in a girl with mild developmental delay and minor dysmorphic features. Using fluorescence in situ hybridization (FISH) studies, we identified an approximately 550-kb complex inter- and intra-chromosomal low-copy repeat (LCR) adjacent to the 21q21.3 translocation breakpoint. On the recipient chromosomes 13qter and 18qter, the telomeric sequences TTAGGG were retained. Genotyping revealed that the deletion was of maternal origin. We propose that genome architecture involving LCRs may be a major mechanism responsible for the origin of jumping translocations.     

An antimelanoma immunotoxin containing recombinant human tumor necrosis factor: tissue disposition,pharmacokinetic, and therapeutic studies in xenograft models

The ability of monoclonal antibody conjugates to re-direct plant or bacterial toxins, chemotherapeutic agents and radionuclides to selected target cells has been well-documented. Recombinant human tumor necrosis factor (TNF) is a macrophage-derived, non-glycosylated (17 kDa) peptide with a broad range of biological and immunological effects including antiviral activity, cytotoxic and cytostatic effects. A conjugate of the antimelanoma antibody ZME-018 and TNF in previous studies has shown melanoma-selective cytotoxic effects in vitro. Pharmacokinetic studies of the ZME-TNF immunotoxin showed that the agent cleared from plasma biphasically with -and -phase half-lives similar to that of ZME itself (72 min and 36 h compared to 84 min and 41 h respectively). In contrast, TNF itself was cleared rapidly from plasma with a terminalphase half-life of only 2.7 h. The clearance rate of ZME-TNF from plasma (Cl_p) was almost tenfold more rapid than for ZME (1.1 versus 0.16 ml/kg x min) but was threefold slower than the clearance for TNF itself (3.4 ml/kg x min). Tissue distribution studies in nude mice bearing human melanoma xenografts showed similar tumor localization of the immunotoxin compared to the free antibody and slightly higher concentrations in liver and kidney compared to ZME itself. Treatment of nude mice bearing well-developed A375 tumors with the immunotoxin resulted in a statistically significant (P<0.002) suppression in tumor growth rate (fivefold increase) compared to saline-treated controls, which increased 20-fold over the same period. These studies demonstrate the feasibility of this approach and suggest that TNF may represent a non-antigenic alternative to immunotoxins containing plant and bacterial toxins.Research conducted, in part, by the Clayton Foundation for Research     

Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

Sprint training attenuates myocyte hypertrophy and improves Ca2+ homeostasis in postinfarction myocytes

Zhang, Xue-Qian, Yuk-Chow Ng, Timothy I. Musch, Russell L. Moore, R. Zelis, and Joseph Y. Cheung. Sprint training attenuates myocyte hypertrophy and improvesCa²⁺ homeostasis in postinfarctionmyocytes. J. Appl. Physiol. 84(2): 544-552, 1998.Myocytes isolated from rat hearts 3 wk aftermyocardial infarction (MI) had decreasedNa⁺/Ca²⁺exchange currents(I_Na/Ca; 3 Na⁺ out:1Ca²⁺ in) and sarcoplasmicreticulum (SR)-releasable Ca²⁺contents. These defects in Ca²⁺regulation may contribute to abnormal contractility in MI myocytes. Because exercise training elicits positive adaptations in cardiac contractile function and myocardialCa²⁺ regulation, thepresent study examined whether 6-8 wk ofhigh-intensity sprint training (HIST) would ameliorate some of thecellular maladaptations observed in post-MI rats with limited exerciseactivity (Sed). In MI rats, HIST did not affect citrate synthaseactivities of plantaris muscles but significantly increased thepercentage of cardiac -myosin heavy chain (MHC) isoforms (57.2 ± 1.9 vs. 49.3 ± 3.5 in MI-HIST vs. MI-Sed, respectively;P  0.05). At the single myocytelevel, HIST attenuated cellular hypertrophy observed post-MI, asevidenced by reductions in cell lengths (112 ± 4 vs. 130 ± 5 µm in MI-HIST vs. MI-Sed, respectively;P  0.005) and cell capacitances (212 ± 8 vs. 242 ± 9 pF in MI-HIST vs. MI-Sed, respectively; P  0.015). ReverseI_Na/Ca wassignificantly lower (P  0.0001) inmyocytes from MI-Sed rats compared with those from rats that were shamoperated and sedentary. HIST significantly increased reverseI_Na/Ca(P  0.05) without affecting theamount ofNa⁺/Ca²⁺exchangers (detected by immunoblotting) in MI myocytes. SR-releasable Ca²⁺ content, as estimated byintegrating forwardI_Na/Ca duringcaffeine-induced SR Ca²⁺ release,was also significantly increased (P  0.02) by HIST in MI myocytes. We conclude that the enhanced cardiacoutput and stroke volume in post-MI rats subjected to HIST aremediated, at least in part, by reversal of cellular maladaptationspost-MI.

     

Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II

Two populations of A-I-containing lipoprotein particles: A-I-containing lipoprotein with A-II (Lp (A-I with A-II], and A-I-containing lipoprotein without A-II (Lp (A-I without A-II] have been isolated from plasma of 10 normolipidemic subjects by immunoaffinity chromatography and characterized. Both types of particles possess alpha-electrophoretic mobility and hydrated density in the range of plasma high-density lipoproteins (HDL). Lp (A-I without A-II) and Lp (A-I with A-II) are heterogeneous in size. Lp (A-I without A-II) comprised two distinct particle sizes with mean apparent molecular weight and Stokes diameter of 3.01 X 10(5), and 10.8 nm for Lp (A-I without A-II)1, and 1.64 X 10(5), and 8.5 nm for Lp (A-I without A-II)2. Lp (A-I with A-II) usually contained particles of at least three distinct molecular sizes with mean apparent molecular weight and Stokes diameter of 2.28 X 10(5) and 9.6 nm for Lp (A-I with A-II)1, 1.80 X 10(5) and 8.9 nm for Lp (A-I with A-II)2, and 1.25 X 10(5) and 8.0 nm for Lp (A-I with A-II)3. Apoproteins C, D, and E, and lecithin:cholesterol acyltransferase (LCAT) were detected in both Lp (A-I without A-II) and Lp (A-I with A-II) with most of the apoprotein D, and E, and LCAT (EC 2.3.1.43) in Lp (A-I with A-II) particles. Lp (A-I without A-II) had a slightly higher lipid/protein ratio than Lp (A-I with A-II). Lp (A-I with A-II) had an A-I/A-II molar ratio of approximately 2:1. The percentage of plasma A-I associated with Lp (A-I without A-II) was highly correlated with the A-I/A-II ratio of plasma (r = 0.96, n = 10). The variation in A-I/A-II ratio of HDL density subfractions therefore reflects different proportions of two discrete types of particles: particles containing A-I and A-II in a nearly constant ratio and particles containing A-II but no A-II. Each type of particle is heterogeneous in size and in apoprotein composition.