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PubMed2005

Assessing peer entry and play in preschoolers at risk for maladjustment.

This study evaluated the psychometric properties of an observational rating system for assessing preschoolers' peer entry and play skills: Observed Peer Play in Unfamiliar Settings (OPPUS). Participants were 84 preschoolers at risk for psychopathology. Reliability and concurrent validity are reported. The 30-min paradigm yielded reliable indexes of engaging and disruptive behavior. Adequate interrater reliability and stability were obtained with minimally trained observers. OPPUS scores were related in expected ways to independent observations of disruptive and engaging behavior during parent-child interactions. OPPUS scores were associated with parent-rated self-control and internalizing behaviors. OPPUS scores were associated with parent ratings of play skills for children with school experience and assertiveness for children without school experience. The OPPUS is a useful, brief method for assessing peer entry and play behaviors in preschoolers.

Child Behavior Disorders↗
PubMed1989

Indium-111-Photofrin-II scintillation scan.

Photodynamic therapy is under intense investigation as an adjuvant treatment for malignant glial tumors of the central nervous system. Photofrin-II (HpD-II) is currently the most actively investigated photosensitizing agent. A crucial issue regarding the safe and efficacious usage of HpD-II-based photodynamic therapy is the individual in vivo kinetics of tumor uptake and retention, compared with normal brain clearance. The optimal time for photoactivation of sensitized tumor must be known to ensure a high target-to-nontarget ratio, resulting in the maximal tumor destruction while preserving normal brain. Our laboratory developed a radionuclide scan based on 111indium (111In)-labeled HpD-II to evaluate HpD-II localization and clearance noninvasively within a canine model of intracerebral gliosarcoma. Synthesis of the 111In-HpD-II complex in greater than 90% yield is achieved by a simple, rapid labeling method. Radiochemical purity and stability were verified by high-performance liquid chromatography. Using the canine model of intracerebral gliosarcoma, we followed the uptake of 111In-HpD-II in tumors with serial scintillation scanning. Localization of the tumor by 111In-HpD-II has been verified by contrast-enhanced computed tomographic scan followed by gross and histological examination of the enhancing brain region. Total body biodistribution of 111In-HpD-II at various times after injection has been evaluated. The ratio of uptake in tumor compared with surrounding brain peaked at 72 hours after injection. The knowledge of regional distribution and concentration of a photosensitizing agent within a tumor mass and surrounding brain allows for the most efficacious timing and localization of a photoactivating source.

Animals↗
PubMed1990

Preparation and properties of a mitomycin C-albumin conjugate.

Mitomycin C, an anti-neoplastic agent, was covalently attached to bovine serum albumin through various kinds of spacers such as glutaryl, succinyl, trans-aconityl, methylsuccinyl and the trimellityl group. The prior acylation of albumin not only prevented protein polymerization in the presence of carbodiimide, but also increased the extent of conjugation of the drug. The conjugate of mitomycin C-glutarylated albumin showed the best properties among the conjugates prepared in meeting the requirements for a high yield of nonpolymerized product with an adequately high mitomycin C content and stability as a macromolecular prodrug.

Animals↗
PubMed1989

Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor.

Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

MDM2 as a critical effector of the MYCN oncogene in tumorigenesis.

The MYCN oncogene plays an important role in the pathogenesis of neuroblastoma. It is amplified in more than 30% of high-risk cases and over expression induces neuroblastoma in transgenic mice. MYCN amplification/overexpression is found in many types of cancers including neuroblastoma, medulloblastoma and other aggressive tumors of neuroectodermal origin as well as in rhadomyosarcoma and small cell lung cancers. MYCN exerts its oncogenic effects through transcriptional regulation of numerous target genes. We have recently characterized the p53 inhibitor MDM2 (HDM2) as one such target in MYCN amplified neuroblastoma cell lines. Conditional expression of MYCN yields elevated MDM2 mRNA and protein. MYCN inhibition leads to diminished MDM2, stabilized p53 and apoptosis. As the primary negative regulator of p53, MDM2 is critically regulated in normal cells to ensure adequate p53 activity in response to damage or stress. Additionally, MDM2 regulates many p53 independent processes pertinent to oncogenesis. We propose that increased MDM2 levels downstream of MYCN are tumorigenic secondary to disruption of multiple p53 dependent and independent mechanisms controlling genomic stability, apoptopsis and cell cycle progression. Further research into the MYCN/MDM2 regulated pathways will provide insight into the pathogenesis of MYCN-driven tumors and provide targets for novel therapeutic interventions.

Animals↗
PubMed2002

Synthesis and characterization of metal sulfide clusters for toxicological studies.

Zinc sulfide clusters were synthesized and characterized at low micromolar concentrations to assess the effect of metal-sulfide ligands on metal toxicity to aquatic organisms in oxic environments. Recommended preparation times are greater than 2 h initial reaction of equimolar sodium sulfide and zinc nitrate, followed by oxic aeration for 3 d. Ionic strength, pH, and anoxic stabilization time were found to be relatively unimportant in controlling the final yield. Adsorptive losses of zinc sulfide (ZnS) clusters to surfaces, however, were significant for a variety of vessel materials and membrane filters. Ionic strength and pH were found to be important factors controlling the extent of adsorptive losses with minimal loss for pHs greater than 9 and for soft waters. The Ag(I), Cu(II), and Hg(II) as metal sulfides completely suppress the analysis of sulfide, whereas Pb(II), Mn(II), and Co(II) partially suppress the analysis of sulfide by the methylene blue technique. Ultraviolet and fluorescence spectra are shown for synthesized ZnS clusters.

Adsorption↗
PubMed1992

[DNA of cadaver organs: comparison of two methods of extraction].

Genomic DNA was isolated from tissue specimen by two different methods. The rapid extraction according to Higuchi provided increased product yield of DNA in a better quality than the extraction with phenol/chloroform. Comparing the organ tissues according to quantity and quality of DNA it was found that best yield was recovered from brain tissue followed by lung and liver. DNA stability is directly influenced by postmortal age and age of the bodies.

Adolescent↗
PubMed1991

Cestode vaccines.

Studies over the past 20 years have clearly shown the potential for developing vaccines against larval cestode infections of man and animals. The important larval cestode infections of man (Echinococcus granulosus--hydatidosis: Taenia solium--cysticercosis) involve domesticated animals as intermediate hosts in their natural life-cycles. These animals develop strong immunity against reinfection, and immunity can be artificially induced by vaccination with oncosphere antigens. A major stumbling block in developing commercial vaccines against cestodes has been the difficulty in obtaining adequate supplies of these antigens. Recent studies with Taenia ovis, a larval cestode causing cysticercosis in sheep, have demonstrated the feasibility of developing commercial vaccines against cestodes using recombinant DNA technology. A cDNA library prepared using mRNA obtained from T. ovis oncospheres was used to isolate a clone which expressed T. ovis polypeptide antigen 45W as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-45W). GST-45W gave up to 94% protection against challenge infection when used to vaccinate sheep with saponin as adjuvant. The vaccine antigen was shown by SDS PAGE to be unstable, a major disadvantage in subsequent attempts to obtain high yields of antigen for commercial production. The fusion protein has now been stabilized by reducing the size of GST-45W cDNA through deleting 19 carboxyl terminal hydropathic acids, and the resultant fusion protein GST-45W (B/X) was highly host-protective. Another experiment showed that the 45W T. ovis polypeptide cleaved enzymatically from GST-45W was still host-protective, suggesting that GST had no influence on the immunogenicity of GST-45W fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

[The influence of infectious bronchitis virus on egg production, fertility, hatchability and mortality rate in chickens (author's transl)].

In 35 flocks comprising almost 100,000 White Plymouth Rock birds producing hatching eggs, infectious bronchitis (IB) caused a decline in production which appeared to be connected with the age of the birds, when the outbreak started (table I). In outbreaks of IB in pullets 3-4 weeks before they are ready to lay there is a considerable risk of delay of production and a generally too low yield (fig 1). Egg laying birds, 25-34 weeks of age, suffered a drop in production from 20-80% (on an average 40%) and normal yield was never regained. After 6-8 weeks the production rate usually stabilizes around 6-12% below normal production. IB outbreaks in the middle of the production period (35-45 weeks) resulted in a decline of egg production of 40-90% (on an average 55%). Recovery of production usually occurred after 6-8 weeks (fig. 2). Observations of clinically typical outbreaks without any laboratory verification indicate that IB outbreaks at the end of the egg production period resulted in irreparable decline of production. Marked alterations in exterior and interior quality of the egg have been observed. Both mishapen appearance, rough and thin shell, bleached shell color and watery albumen have occurred. These facts did sometimes cause transient increased "sorting out" of haching eggs. Also fertility rate was noticed to be affected temporarily during and after an attack of IB. In 17 flocks drops of 1.7-22.4% (on an average 7.4%) were registered (fig. 3). Also hatchability decreased 3.0-43.8% (on an average 13.7%) (fig. 4). In most of the registered outbreaks of IB the weekly mortality remained below 1%, but in single cases the figure could rise up to about 4% (fig. 5), most often in flocks where complicating factors such as secondary infections caused by E. coli or M. gallisepticum were present.

Animals↗
PubMedDate not supplied

[Isolation of Staphylococcus aureus protoplasts using lysoamidase].

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.

Hydrolysis↗
PubMed1986

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver.

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.

Cations, Divalent↗
PubMed1997

Packing as a structural basis of protein stability: understanding mutant properties from wildtype structure.

Modeling of protein core mutations using sidechain packing can forecast their effects on stability. We have assessed the structural basis of this approach, by evaluating the accuracy of our 1991 model of a three-site mutant of lambda repressor (V36L/M40L/V47I), against the recently reported crystal structure. The three mutated residues matched the crystal structure to within 0.89A (1.11A for sidechain atoms), giving fairly accurate sidechain placement and packing (81-99th percentile rank in coordinate accuracy). However, the model used different sidechain torsional angles than seen in the crystal structure at residues 36 and 40, apparently to compensate for the backbone shifts present in the actual mutant structure, but not accounted for in our modeling method. To understand the structural basis of stability across a set of lambda repressor core mutants, we have analyzed the mutant models, revealing several simple packing effects: V36I, predicted to be stabilized by filling a hydrophobic cavity; M40V, destabilized by a steric clash with the unusual structural demands of a helix-turn transition. These effects illustrate how mutant stability can often be understood directly from scrutiny of wildtype structure. Simply adding the calculated energies of neighboring point mutations predicts the stability effect of the combined mutant relatively well, with little apparent cooperativity, yielding simple rules for each site's amino acid preferences. Our treatment of core packing indicates that it can permit a large fraction of sequences to fit the native fold, as observed experimentally, far more than indicated by rotamer hard-sphere models.

Amino Acid Sequence↗
PubMed1999

Solvent effect on kinetics of appearance of haemorphins in the course of peptic hydrolysis of bovine haemoglobin.

The effect of the composition of the solvent on the peptic hydrolysis of bovine haemoglobin was studied to improve the preparation of two opioid peptides. Peptic hydrolysis was performed for 24 h at 23 degrees C in 0.1 M sodium acetate buffer, pH 4.5. A HPLC method was reported for isolating LVV-haemorphin-7 and VV-haemorphin-7 in one step. The kinetics of appearance of haemorphins was investigated in the presence of 20% (v/v) ethanol, a stabilizing solvent of haemoglobin or urea, a denaturant agent. Ethanol improved the yield of VV-haemorphin-7, whereas urea improved the yield of the two haemorphins. Because the amounts of haemorphins observed in urea were greater than in ethanol, the denatured state of haemoglobin is more favourable to obtaining LVV-haemorphin-7 and W-haemorphin-7. The peptide bonds that give rise to haemorphins would be more accessible to the pepsin.

Animals↗
PubMed2004

Studies on stereoselective [2+2] cycloadditions between N,N-dialkylhydrazones and ketenes.

Staudinger-like cycloadditions between chiral, non-racemic N,N-dialkylhydrazones 1 and functionalized ketenes constitute an efficient methodology for the stereoselective construction of the beta-lactam ring. The potential for fine tuning of the dialkylamino auxiliary structure, the availability of a high-yielding deprotection method for the release of the free azetidinones, and the high thermal and chemical stability of hydrazones as N-dialkylamino imines are highlighted as the key elements for the success of the strategy. This last aspect is of particular importance concerning generality: even hydrazones from easily enolizable aldehydes or from formaldehyde reacted to afford the corresponding cycloadducts with high chemical and stereochemical yields. The syntheses of the beta-amino-alpha-hydroxyacids (2R,3S)-phenylisoserine (42) and (2R,3S)-norstatin (45) were accomplished as illustrative examples of the synthetic utility of this procedure. A model system for the cycloaddition of g series auxiliaries was studied by ab initio computational methods. The collected results support a two-step mechanism through zwitterionic intermediates, and explain the observed absolute and relative stereochemistry in terms of the preferred outward cycloaddition to the Re face of the hydrazone.

Computer Simulation↗
PubMed2004

Transition-state effects in acid-catalyzed aryl epoxide hydrolyses.

The hydronium ion-catalyzed hydrolyses of 5-methoxyindene 1,2-oxide and of 6-methoxy-1,2,3,4-tetrohydronaphthalene-1,2-epoxide were each found to yield 75-80% of cis diol and only 20-25% of trans diol as hydrolysis products. The relative stabilities of the cis and trans diols in each system were determined by treating either cis or trans diols with perchloric acid in water solutions and following the approach to an equilibrium cis/trans mixture as a function of time. These studies establish that the trans diol in each system is more stable than the corresponding cis diol. Thus, acid-catalyzed hydrolysis of each epoxide, which proceeds via a carbocation intermediate, yields the less stable cis diol as the major product. Transition-state effects, presumably of a hydrogen-bonding nature, selectively stabilize the transition state for attack of water on the intermediate 2-hydroxy-1-indanyl carbocation leading to the less stable cis diol in this system. Transition-state effects must also be responsible for formation of the less stable cis diol as the major product in the acid-catalyzed hydrolysis of 5-methoxy-1,2,3,4-tetrahydronaphthalene 1,2-epoxide. However, in this system steric effects at the transition state may be more important than hydrogen bonding in determining the cis/trans diol product ratio. The synthesis of 5-methoxyindene 1,2-oxide and a study of its rate of reaction as a function of pH in water and dioxane-water solutions are reported. Both an acid-catalyzed reaction leading to only diol products and a pH-independent reaction yielding 71% of 5-methoxy-2-indanone and 29% of diols are observed; the half-life of its pH-independent reaction in water is only 2.4 s.

Acids↗
PubMed1987

Advances in 99mTc-labeling of antibodies.

Several methods have been developed to label antibodies with 99mTc. Direct labeling results in 99mTc binding to multiple sites of various affinities that are often weaker than the binding to strong chelating agents. Attempts to overcome this disadvantage involve conjugation of strong chelating agents to the antibodies. While stability is usually enhanced, this approach suffers from alteration of antibody properties as well as non-specific binding of 99mTc to the antibody instead of to the conjugated chelating agent. This has been of concern for studies with DTPA as the chelating agent. In this study the loss of 99mTc by N2S2 challenge shows that a fraction of the 99mTc is nonspecifically bound to the antibody. An advantage of the approach of labeling antibodies containing a bifunctional chelating agent is the simplicity of the labeling procedure and the apparent high yields that in reality are the sum of chelating agent and non-specifically bound radioactivity. The last approach described in our work of conjugation of a preformed chelate has advantages of characterizable 99mTc complex chemistry and conjugation by standard protein derivatization chemistry. Slow chelation kinetics can be overcome in the small molecule stage and then conjugation performed under mild conditions with respect to the antibody or fragments. This approach, however, suffers from greater complexity of the labeling process including multiple steps, purifications and non-quantitative yields. The use of ligands for 99mTc in which the complexes are of high stability and predictable chemistry is likely to result in eventual optimal labeling technologies. Processes which are non-specific may work in some cases, but are likely to present difficulties in optimization and general applicability from antibody to antibody.

Animals↗
PubMed2007

Selective modification of surface-exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme.

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2,6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.

D-Amino-Acid Oxidase↗
PubMedDate not supplied

Chromatographic analysis of sagebrush monoterpenes in blood plasma.

An analytical method is required for the analysis of monoterpenes in animal plasma to support a pharmacokinetic study. Monoterpenes common to sagebrush are extracted from sheep plasma by employing solid-phase extraction (SPE), followed by analysis of the extracts by gas chromatography with flame ionization detection. The analytes are quantitated versus an external standard and by comparison with a surrogate standard added to the sample prior to extraction. In addition to comparing the two quantitative methods, the storage stability of the analytes in plasma and SPE columns is evaluated. Both methods employed for quantitation yield precision suitable for pharmacokinetic studies. However, determination of monoterpenes residues versus external standards produces improved accuracy as compared with use of the surrogate standard. Some analyte loss is observed from plasma samples stored for five weeks at -12 degrees C. However, storage of extracts on the SPE columns affords excellent stability.

Animals↗