Sub-micro-Newton resolution thrust balance.

Herein is described a sensitive vacuum balance for measuring the thrust produced by small (∼0.5 kg) thrusters typically employed in microsat station-keeping. The balance is based on a torsion design but incorporates jewel-pivot bearings instead of the more typical torsion spring bearings. Novel tilt control allows maintenance of true verticality of the bearing axis even while under vacuum. The low moment of inertia design allows it to measure small thrusts from high-voltage devices without direct wire conductor connections. Calibration by several means is described including use of a previously calibrated dielectric barrier discharge thruster.

Application of a coupled reducing system to Edman sequencing.

Addition of low levels of solid tris-(2-carboxyethyl)-phosphine hydrochloride to Applied Biosystems' R4A (2 mM) and R5 (0.17 mM) resulted in a coupled, phosphinedithiothreitol reducing system. Coupling the two reductants eliminated or greatly decreased interference from the oxidation product of dithiothreitol and allowed quantitation of phenylthiohydantoin (PTH)-Asp below the 400-fmol level. Use of the system resulted in a measurable increase in repetitive yield for beta-lactoglobulin, and instrument PTH standards were stable for more than 3 months.

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry.

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.

A parallel method for desalting and concentrating microliter volume samples for mass spectrometry.

A method is described for constructing spin columns for reverse-phase centrifugal desalting of proteolytic digests. The technique employs small, self-packed, reusable cartridges and required less than 30 minutes to process six samples, making the procedure useful as a parallel technique. Up to 15 microL of sample could be loaded and eluted with 2 to 7 microL of a solvent compatible with electrospray ionization. The method was not limited to large-diameter resins or short column heights; a relative centrifugal driving force as low as 30 (500 rpm) applied for 1 to 3 minutes usually was sufficient for sample loading. Subsequently, data were obtained with 1 5-mm columns of 3- 5-, and 10-microm silica resins. The efficiently of recovery in the range of 0.5 to 250 pmol of peptides was measured as 60% to 90%, depending on resin type and sample load. Successful nanospray data were obtained with peptides that had been adulterated with 2 M urea and processed with a spin column. Matrix-assisted laser desorption and ionization/time-of-flight mass spectrometry data greatly improved after desalting of an in-gel digest of a 280-kd protein. Data are presented on the preparation of columns, optimization of procedures, the use of various types of C18 resins, and the efficiency of peptide recovery. The effect of rotor speed and the rate of sample processing are discussed.

Comparison of the properties of native and pentaammineruthenium(III)-modified xylanase.

Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were purified and then modified by the attachment of pentaammineruthenium, thereby resulting in the generation of a xylanase with veratryl alcohol oxidase activity. Hydrolytic activity of T. reesei xyn II on soluble xylans was unchanged by modification with pentaammineruthenium; however, modification of B. pumilus xynA greatly reduced xylan hydrolysis unless the active site of the xylanase was protected with xylose during the modification. The presence of histidine, cysteine, or reduced glutathione during xylan hydrolysis greatly increased the xylanase activity of the pentaammineruthenium-modified B. pumilus xylanase. Glycine, glutamic acid, methionine, or oxidized glutathione had no effect on xylanase activity.

Characterization of modified and normal deoxyoligonucleotides by MALDI, time-of-flight mass spectrometry.

A commercially available, matrix-assisted, laser desorption/ionization (MALDI), time-of-flight mass spectrometer has been used for the successful characterization of picomole quantities of modified deoxyoligonucleotides. The procedure was found to be applicable to the analysis of a variety of modified synthetic structures that included oligos with an intact dimethoxytrityl-blocking group, with omega-aminohexyl- and omega-hexyl-modified bases and with alterations to the normal phosphate backbone: 2'-5' and phosphorothioate linkages. Use of an alpha-cyano-4-hydroxycinnamic acid/ammonium formate based matrix gave sharp peaks, sensitivity to 1 pmol, accuracy to 0.06% on average and avoided the necessity for cryogenic technique and/or specialized equipment. Mixtures of normal and modified oligonucleotides worked well without any special precautions as long as the modified oligonucleotides contained a polyanionic backbone. Modified oligonucleotides that contained an amide linkage in place of the usual phosphate ester link required a sinapinic acid- or esculetin-based matrix with no ammonium formate. Crude oligonucleotides could be rapidly analyzed as a quality-control measure using from 10 to 20 pmol of a typical 0.2-mumol scale synthesis.

Long distance related stressors and coping behaviors in parents of children with cancer.

This descriptive comparative study addresses long distance related stress and coping behaviors of 53 parents of children with cancer. The purpose of the study was to determine the differences in the stress and coping behaviors of parents living 100 miles from the tertiary treatment center compared with those who live less than 100 miles from the center. The theoretical framework used was Lazarus' theory on stress and coping. The study participants were from several Pediatric Oncology Group member institutions. The parents completed Hymovich's Parent Perception Inventory and a demographic data sheet. Parametric (one-tailed t-test) and nonparametric (Mann-Whitney and chi-squared tests) were included in the statistical analysis. The results showed significant differences in demographic data, concerns, beliefs/feelings, and coping. Implications for the pediatric oncology treatment team include specific interventions that will improve the quality of care for the children/parents who live a long distance from the tertiary treatment center.

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.

State-of-the-art biomolecular core facilities: a comprehensive survey.

A survey of 124 protein and/or nucleic acid chemistry facilities has provided a basis for estimating the resources needed to establish a facility, the financial support needed to keep it operating, and the technical capabilities it might reasonably be expected to achieve. Based on these data, an average core facility occupied 870 ft2, was staffed by three full-time personnel, and was equipped with 4-5 major instrument systems. Because user fees generated an average of about $101,000/year in income compared with an average operating budget of about $197,000/year, even a facility that charged user fees would, on average, still require an annual subsidy of about $96,000. Although most government and industrial core facilities did not assess user fees, at least 83 of the 124 respondents did have a preestablished schedule of service charges that enabled a compilation to be made of the average cost of providing a number of typical facility analyses and syntheses. The greater than 100-fold range in charges assessed in core facilities for seemingly identical services was shown to result from the equally large range in the degree of subsidization of these laboratories. Although an average facility might be expected to offer four or five of the following six major services--amino acid sequencing, amino acid analysis, HPLC peptide isolation, peptide synthesis, fragmentation of proteins and DNA synthesis--less than 10% of the responding laboratories provided mass spectrometry, capillary zone electrophoresis, or RNA synthesis. With the exception of peptide synthesis, which had an average turn-around time of about 24 days, all other major services had turn-around times that averaged in the range of 4-9 days. Additional data are summarized regarding average sample throughput in core laboratories and the amount of protein that is needed for hydrolysis/amino acid analysis and sequencing.

An experience with an implanted port system in 66 children with cancer.

Totally implanted port catheter systems have a lower incidence of infection and are more easily used in home care that external catheters in adult cancer patients. Experience with this method in children has been limited. During the past 2 years, we have implanted 71 ports in 66 children with cancer. Our experience demonstrates an infection rate (0.15 episodes of bacteremia per 100 patient days) slightly lower than that reported for children with Broviac or Hickman catheters, but not as low as that seen in adults with implanted systems. Patients and families have been extremely satisfied with the devices. Our experience supports further use of implanted systems in children with cancer.

The child with sickle cell anemia: implications and management.

Sickle cell anemia is a chronic illness affecting eight percent of the black population in the United States. It is an autosomal recessive, genetic disease. The clinical manifestations include anemia and vaso-occlusion. If these manifestations are properly managed, life threatening side effects can be prevented. Comprehensive medical care can prevent and minimize these crises and identify problem areas of concern before major damage is done to the organ systems of the body.

Interaction of polyamines and magnesium with casein kinase II.

In reticulocytes, polyamines appear to be physiologically relevant activators of casein kinase II [Hathaway, G. M. and Traugh, J. A. (1984). J. Biol. Chem. 259, 7011-7015]. The mechanism by which polyamines and Mg2+ interact to activate casein kinase II has been investigated. These studies were conducted by holding ionic strength constant at 0.10 M. At low Mg2+ (2.5 mM), activation by spermine resulted in a 33% decrease in the apparent Km for casein. Under these conditions, a 2.3-fold increase in the maximum velocity of the reaction was observed, and half-maximal stimulation was obtained with 275 microM spermine. At a kinetically optimal Mg2+ concentration of 12.5 mM, the effects of spermine on Km and Vmax were reduced, and the concentration of spermine required to give 50% of maximal stimulation was increased to 750 microM. Kinetic data obtained at the two Mg2+ concentrations indicated that Mg2+ and spermine competed for the same form of the enzyme. Double-reciprocal plots of velocity versus Mg2+ concentration showed downward curvature at Mg2+ concentrations higher than 1 mM, and these results were interpreted as evidence for two binding sites on the enzyme with an apparent Km of 0.5 and 2.5 mM. Experiments carried out with ATP-Mg2+ in the absence of excess MgCl2 gave results consistent with an absolute requirement of the enzyme for the metal ion which could not be replaced by spermine. These results are consistent with the formation of an enzyme-activator complex. A model is proposed where spermine activates casein kinase II at one site on the enzyme at which MgCl2 can also bind, while a second, high-affinity site exists exclusively for the metal ion.

Kinetics of activation of casein kinase II by polyamines and reversal of 2,3-bisphosphoglycerate inhibition.

The effects of polyamines on the catalytic activity of casein kinase II have been studied. Of the three polyamines tested (putrescine, spermidine, and spermine), spermine was the most effective at stimulating the enzyme. When physiological concentrations of potassium and magnesium were utilized, 50% activation was observed at 0.28 mM spermine or 0.70 mM spermidine. With mixtures of spermine and spermidine at physiological concentrations for the reticulocyte (0.04 and 1.06 mM, respectively), a 2.5-fold stimulation of casein kinase II activity was observed. In general, stimulation of the enzyme was dependent on salt concentration and it was necessary to hold ionic strength constant in order to separate specific activation by the polyamines from general salt activation. Optimum activation by polyamines was observed at low ionic strength and physiological concentrations of Mg2+. When beta-casein and eukaryotic initiation factors 2 or 3 were used as substrates, up to 3.5-fold stimulation of casein kinase II was observed. In the absence of polyamine, half-saturation of the enzyme by Mg2+ was observed at 1-3 mM MgCl2, a concentration much higher than required for ATP-Mg2+ complex formation. This dependence of the enzyme on Mg2+ was greatly diminished in the presence of spermine. Spermine decreased the apparent Km for casein and increased the maximum velocity of the reaction. Spermidine and spermine also effectively reversed inhibition by 2,3-bisphosphoglycerate. The significant activation by polyamines observed under conditions similar to those measured for the red cell suggest that the polyamines, spermidine and to a lesser extent spermine, function to regulate casein kinase II in vivo.

Regulation of casein kinase II by 2,3-bisphosphoglycerate in erythroid cells.

The hemoglobin regulator, 2,3-bisphosphoglycerate (glycerate-2,3-P2) has been shown to modulate the activity of casein kinase II from rabbit reticulocytes. Kinetic results were obtained with the exogenous substrate, beta-casein and the endogenous substrates, initiation factors (eIF-) 2 and 3. Experiments carried out to determine the interaction between glycerate-2,3-P2, Mg2+, substrate, and casein kinase II led to the following conclusions: 1) glycerate-2,3-P2 inhibition was competitive with respect to the protein substrate and noncompetitive with respect to ATP; 2) inhibition was not caused by depletion of ATP-Mg2+ as a consequence of Mg2+ complexation with glycerate-2,3-P2; 3) the response curve for glycerate-2,3-P2 was cooperative, but the cooperativity decreased as salt concentration increased; 4) glycerate-2,3-P2 inhibition was dependent on Mg2+ concentration up to about 5 mM MgCl2 but did not parallel glycerate-2,3-P2 X Mg2+ complex formation indicating that the Mg2+ dependence was not due to the formation of a glycerate-2,3-P2 X Mg2+ complex; 5) experiments with analogs of glycerate-2,3-P2 showed that the binary phosphate grouping was important in determining inhibition by glycerate-2,3-P2 while the presence of the carboxylate-phosphate pair was much less important; 6) low levels of glycerate-2,3-P2 stimulated phosphorylation of beta-casein, eIF-2, and eIF-3; the extent of stimulation was dependent on the affinity for casein kinase II and the level of the substrate. These effects were observed in the range of glycerate-2,3-P2 concentrations predicted for intracellular fluctuations in this metabolite. Therefore, it was concluded that glycerate-2,3-P2 could function both as an activator and an inhibitor of casein kinase II in the erythroid cell by binding at the substrate binding site.