Sign cancellation and scaling in the vertical component of velocity and vorticity in rotating turbulence.

We study sign changes and scaling laws in the Cartesian components of the velocity and vorticity of rotating turbulence, in the helicity, and in the components of vertically averaged fields. Data for the analysis are provided by high-resolution direct numerical simulations of rotating turbulence with different forcing functions, with up to 1536(3) grid points, with Reynolds numbers between ≈1100 and ≈5100, and with moderate Rossby numbers between ≈0.06 and ≈8. When rotation is negligible, all Cartesian components of the velocity show similar scaling, in agreement with the expected isotropy of the flow. However, in the presence of rotation, only the vertical components of the fields show clear scaling laws, with evidence of possible sign singularity in the limit of an infinite Reynolds number. Horizontal components of the velocity are smooth and do not display rapid fluctuations for arbitrarily small scales. The vertical velocity and vorticity, as well as the vertically averaged vertical velocity and vorticity, show the same scaling within error bars, in agreement with theories that predict that these quantities have the same dynamical equation for very strong rotation.

Dynamic change in phosphorylated platelet-derived growth factor receptor in peripheral blood leukocytes following docetaxel therapy predicts progression-free and overall survival in prostate cancer.

In a placebo-controlled randomised study of the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib mesylate and docetaxel in metastatic prostate cancer with bone metastases (n=116), no significant differences in progression-free and overall survival were observed. To evaluate pharmacodynamic correlates of outcomes, we assessed the association of plasma platelet-derived growth factor (PDGF) isoform kinetics and PDGFR inhibition with progression-free and overall survival by individual treatment arm. We found that in the docetaxel-placebo arm alone, the probability of decrease in PDGFR phosphorylation (Pr-Decr-pPDGFR) above 0.5 (vs 30 months (HR 3.1; P=0.04 in log-rank test). By contrast, in the docetaxel plus imatinib arm, the association of Pr-Decr-pPDGFR >0.5 with a rise in plasma PDGF isoform concentrations and inferior survival was not observed. The data suggest that dynamic changes in PDGFR phosphorylation in peripheral blood leukocytes predict docetaxel efficacy. Rising plasma PDGF concentrations may explain and/or mark docetaxel resistance. Validation and mechanistic studies addressing these unexpected findings should anticipate a confounding influence of concurrent PDGFR inhibitor therapy.

Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles.

Follicular atresia is associated with the presence of increased macrophages within the follicle. What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event. As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion. Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus. Prolactin and LPS increased the number of atretic vs. healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups). Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei. Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin. In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin. Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH.

Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail.

BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.

Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain.

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.

Cloning and characterization of human estrogen receptor beta isoforms.

Multiple transcripts which arise from the human estrogen receptor beta (ER beta) gene have been characterized. Three full length isoforms of the hER beta gene, designated hER beta 1-3, were identified in a testis cDNA library. An additional two isoforms, designated hER beta 4 and hER beta 5, were identified by PCR amplification from testis cDNA and from the MDA-MB 435 cell line. hER beta 1 corresponds to the previously described hER beta. All five isoforms diverge at a common position within the predicted helix 10 of the ligand binding domain of hER beta, with nucleotide sequences consistent with differential exon usage. The hER beta isoform mRNAs displayed a differential pattern of expression in human tissues and in tumor cell lines when analyzed by RT-PCR. Further characterization of the three full length isoforms, hER beta 1-3, by in vitro band shift studies indicated that the isoforms were able to form DNA-binding homodimers and heterodimers with each other and with the ER alpha subtype.

A novel sorbent for the determination of clenbuterol in bovine liver.

The use of three C18 sorbents in matrix solid phase dispersion (MSPD) for the determination of clenbuterol in bovine liver fortified at 5 ng g-1 is described. MSPD grade C18 sorbents give rise to more efficient blending and packing of the material for subsequent washing and analyte elution in comparison with a non-MSPD grade C18 sorbent. Following enzymatic deconjugation of the liver extracts, radioimmunoassay is used as the method of determination. The mean recovery of clenbuterol with all sorbents is comparable and within the range 86-96% in two intra-assay studies (n = 3). The liver extracts in each case are highly coloured. The variation in recovery is observed to be lowest with MSPD grade C18 (end-capped). This sorbent was used in further studies to evaluate the use of solid phase extraction (SPE), post MSPD, with normal phase aminopropyl or mixed mode cation exchange columns for extract purification. The mean recovery of clenbuterol (n = 4, inter-assay study) following MSPD and normal phase SPE clean-up was 95 +/- 15% and 89 +/- 9% at fortification levels of 1 and 2.5 ng g-1, respectively.

Matrix solid-phase dispersion technique for the determination of moxidectin in bovine tissues.

An HPLC method has been developed for the determination of moxidectin in bovine tissues. The extraction and clean-up procedure is based on the matrix solid-phase dispersion technique. Control and moxidectin-fortified bovine tissue samples (0.25 g) are blended with octadecyl (C18 end-capped) packing material. A column made from the C18-bovine tissue blend is washed with hexane (2 ml); when all the hexane has eluted, an Alumina-B SPE cartridge is attached below the C18-tissue column and, after washing, moxidectin is eluted with methanol (6 ml). Moxidectin is derivatized and determined by HPLC with fluorescence detection. The recovery from fortified samples was greater than 80% in the concentration range 1-100 ng g-1 of tissue. This method permits the determination of moxidectin at levels as low as 1 ng g-1 (1 ppb).

Analysis of protein-bound metabolites of furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and 5-morpholino-methyl-3-amino-2-oxazolidinone (AMOZ), respectively. These side-chains have molecular characteristics in common with the parent compounds and may be released from liver tissue under mild acidic conditions. Derivatization with 2-nitrobenzaldehyde (NBA) serves to isolate the released side-chains and the derivatives NPAOZ and NPAMOZ are chromophoric, thereby permitting UV detection. This paper reports the introduction of an extract clean-up step to the existing procedure which eliminates or decreases interference from NBA in the HPLC-UV determination of NPAOZ. The modified procedure was also applied to the determination of AMOZ. The development of an LC-MS method for the quantitative and confirmatory determination of AOZ and AMOZ extracted and derivatized according to the same procedure as that for HPLC-UV is described. The methods were validated for AOZ and AMOZ in fortified (intra- and inter-assay studies) and incurred (inter-assay studies) pig liver samples. The limit of determination for fortified control liver samples was 5 ng AOZ g-1 and 10 ng AMOZ g-1 by HPLC-UV and 10 ng AOZ or AMOZ g-1 by LC-MS. In addition, a study to determine the ratio of released AOZ to the total bound residues present in incurred liver samples from pigs treated with furazolidone is described.

Transition Metal Ion Complexation and Extraction by Hydroxamate Functionalised p-Tert-Butylcalix[4]Arenes.

The functionalisation of macrocyclic p-tertbutylcalix[4]arenes at the lower rim with hydroxamic acid and proline hydroxamic acid groups gives the calixarenes chelating properties similar to siderophores. The (1)H NMR spectrum of p-tertbutylcalix[4]arene tetrahydroxamic acid shows a broad band at 10.8 ppm in DMSO-d(6) attributed to NHOH protons. Diffuse reflectance spectral analysis of PVC membranes containing the calixarene hydroxamic acids show absorption bands at 485 nm and 504 nm following contact with aqueous solutions of Fe(III) and V(V) respectively. The pH dependency of this extractive selectivity is examined for Fe(III), Co(II), Pb(II), Mn(II), Cu(II) and Ni(II) using a solid phase extraction approach. Spectrophotometric evidence for the complexation of Cu(II) in methanol by p-tertbutylcalix[4]arene tetraproline acid is also provided.

Dosing guidelines for common neonatal and pediatric emergency drugs.

In some instances, pediatric emergency drug use will be necessary in areas besides the neonatal intensive care unit, or emergency rooms of hospitals which treat children. It is essential in the non-specialized areas that an easily accessible, relevant, source of drug information be available for emergency drug use. Drug dosing guidelines and monitoring parameters for the pediatric population for 14 emergency drugs have been compiled into an easy-to-read compendia in order to assist physicians, residents, nurses, and pharmacists in instances when time is crucial.

Weaning--a first step to independence.

There are no hard and fast rules when it comes to weaning babies, but it can be an emotional time for both parents and child. The information given by health professionals can have a radical effect on the success of this stage.