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An unreactive cardiotocograph late in the mid-trimester led to a sequence of investigations that revealed an appropriately grown fetus, with hepatic calcification, and evidence of intrauterine infection. Ultrasound observation and stimulation of the fetus with vibration and glucose revealed altered neurophysiological responses. The profound neurological impairment was confirmed by detailed neonatal investigations. [ABSTRACT FROM AUTHOR]

This investigation aims to explore the relationship between the academic backgrounds of youth soccer coaches (U10 and U12 age groups) in Spain and the type of verbal behavior used during training sessions. The sample consisted of 70 coaches divided into two groups, depending on whether or not they had engaged with a university-level academic studies related to Physical Education and or Sport Sciences. A modified version of the " Coach Analysis and Intervention System " (CAIS), developed by Cushion et al. (2012), was used to collect data. A total of 32,886 verbal behaviors were noted and analyzed. Our results suggest that the coaches with university academic backgrounds frequently use more verbal behaviors and that these could be associated with positive effects on the players' learning and development processes. We suggest it is important to develop specific training programs aimed at optimizing the coaches' communicative and socio-affective skills in order to maximize their impact in youth athletes' learning process., (Copyright © 2020 Agustí, Ballester, Juan-Blay, Taylor and Huertas.)

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.

Crude methanol extracts from four cultivated varieties of mature lentil seeds (Lens culinaris Medik.) were found to possess antifeedant and insecticidal properties in laboratory tests with the rice weevil (Sitophilus oryzae L.), an insect pest of stored products. Flash chromatography with silica gel on active Diaion HP-20 methanol extracts gave flavonol, lysolecithin, soyasaponin, and peptide fractions, as determined by HPLC and electrospray ionization LC/MS. The flavonol fraction was shown by high-resolution NMR experiments to contain a mixture of kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and, tentatively, kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside. These inactive tetraglycosides, although inseparable under the reported HPLC conditions, were detected by NMR spectroscopy in nearly equal proportions. Three lysolecithins were identical to those previously identified in pea extracts. Soyasaponin I (soyasaponin Bb) and soyasaponin VI (soyasaponin betag) were found in Diaion HP-20 methanol extracts. An insecticidal lentil peptide with a mass of 3881 Da, isolated from an Eston variety in small quantities by anion exchange chromatography, was related to the cysteine-rich pea albumin 1b class of botanical insecticides. Binary mixtures of the insecticidal lentil peptide and soybean soyasaponin I were synergistic in tests with S. oryzae.

Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.

Methanol soluble insecticidal peptides with masses of 3752, 3757, and 3805 Da, isolated from crude extracts (C8 extracts) derived from the protein-enriched flour of commercial field peas [Pisum sativum (L.)], were purified by reversed phase chromatography and, after reduction and alkylation, were sequenced by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry with the aid of various peptidases. These major peptides were variants of pea albumin 1b (PA1b) with methionine sulfoxide rather than methionine at position 12. Peptide 3752 showed additional variations at positions 29 (valine for isoleucine) and 34 (histidine for asparagine). A minor, 37 amino acid peptide with a molecular mass of 3788 Da was also sequenced and differed from a known PA1b variant at positions 1, 25, and 31. Sequence variants of PA1b with their molecular masses were compiled, and variants that matched the accurate masses of the experimental peptides were used to narrow the search. MALDI postsource decay experiments on pronase fragments helped to confirm the sequences. Whole and dehulled field peas gave insecticidal C8 extracts in the laboratory that were enriched in peptides with masses of 3736, 3741, and 3789 Da, as determined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry. It was therefore concluded that oxidation of the methionine residues to methionine sulfoxide occurred primarily during the processing of dehulled peas in a mill.

Extracts from field peas (Pisum sativum L.) have previously been shown to have a utility to control insect pests. To identify potentially new bioinsecticides in field crops, we describe the fractionation of impure extracts (C8 extracts) derived from protein-rich fractions of commercial pea flour. The activity of separated fractions was determined by a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)], an insect pest of stored products. Bioassay-guided fractionation showed that the triterpenoid saponin fractions were partly responsible for the antifeedant activity of C8 extracts. Soyasaponin I (soyasaponin Bb), isolated from peas and soybeans, and mixtures of soyasaponins, comprised of soyasaponins I-III and isolated from soybeans, were inactive antifeedants, but dehydrosoyasaponin I (the C-22 ketone derivative of soyasaponin I), a minor component found in C8 extracts, was shown to be an active component. Dehydrosoyasaponin I (soyasaponin Be) and soyasaponin VI (soyasaponin betag) coeluted under conditions of silica gel thin-layer chromatography and C18 high-performance liquid chromatography. However, dehydrosoyasaponin I could be isolated from saponin-enriched fractions with a reversed phase column of styrene/divinylbenzene operated at alkaline pH. Phospholipids of the lysolecithin type were also identified in saponin fractions of C8 extracts from peas. Three of the lysolecithins were inactive alone against rice weevils, but mixtures of these phospholipids enhanced the insecticidal activity of dehydrosoyasaponin I.

A collection of 10 accessions of fenugreek (Trigonella foenum-graecum L.), an annual legume, was grown during two summers at three plot locations in western Canada to assess whether genetic (accession) and environmental factors (site and year of production) influenced levels of diosgenin, a steroidal sapogenin. The 60 harvested seed samples, each analyzed by single determinations on three subsamples of defatted and dried seed material, were hydrolyzed by a microscale procedure in water containing 2-propanol (70%) and sulfuric acid (1 M). The extracts were analyzed by capillary gas chromatography with 6-methyldiosgenin as internal standard. Diosgenin levels from mature seeds ranged from 0.28 to 0.92% (28-92 microg/10 mg). Analysis of variance on combined diosgenin levels from the three sites and two years revealed that accession, accession x year, and site x year effects were significant for diosgenin content, whereas site, year, and site x accession effects were not. Four accessions, CN 19062, CN 19067, CN 19070, and CN 19071, were identified with high levels of diosgenin on the basis of the 2-year data set. In these accessions, mean levels of diosgenin plus yamogenin from seven site years were estimated at 0.70, 0.98, 0.84, and 0.87%, respectively.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

Sulfuric acid hydrolysis of steroidal glycosides of Amber fenugreek was studied by capillary gas chromatographic analysis of diosgenin [(25R)-spirost-5-en-3-ol] and isomeric spirostadiene artifacts from 100 mg samples of seed material. Following extraction with 80% ethanol, highest recoveries of diosgenin occurred when hydrolyses were conducted in sulfuric acid, prepared at 1 molar (M) concentration in water containing 60-80% 2-propanol. Compared to a previous method with aqueous hydrochloric acid, the selected conditions of hydrolysis at 100 degrees C for 2 h with sulfuric acid in 70% 2-propanol reduced diene formation but did not completely eliminate these artifacts. Extraction of steroidal saponins with various alcohol/water mixtures prior to sulfuric acid hydrolysis gave similar recoveries of diosgenin. Application of the quantitative method to experimental samples of Amber, Quatro, and ZT-5 fenugreek, using 10 mg subsamples of crushed seed that had been defatted with petroleum ether and dried at 60 degrees C, gave diosgenin levels of 0.55, 0.42, and 0.75%, respectively. Levels of smilagenin and sarsasapogenin were very low in hydrolyzed seed extracts from ZT-5, a Canadian breeder line of fenugreek.

The ect2 oncogene was originally identified as a transforming complementary DNA (cDNA) from mouse epithelial cells in an expression cloning approach and encodes a product related to Rho-specific exchange factors and yeast cell cycle regulators. To explore the potential role of ect2 in the cell cycle, we examined the expression of the ect2 proto-oncogene in a liver regeneration model in mice after partial (two thirds) hepatectomy. We found that the expression of the ect2 transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases. The timing of ect2 expression matched that of proliferating cell nuclear antigen (PCNA) and partially overlapped cell division cycle 2 (Cdc2) expression. In situ hybridization analysis showed that ect2 was expressed at a high level in cells undergoing mitosis in regenerating liver. Moreover, expression of a dominant negative or an oncogenic mutant of ect2 in cultured mouse hepatocytes resulted in a large increase in the number of binucleated cells. These findings showed that Ect2 is expressed in a cell cycle-dependent manner during liver regeneration, and suggest that it has an important role in the regulation of cytokinesis.

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

Hepatocyte growth factor (HGF/SF), is a heparin-binding polypeptide which stimulates DNA synthesis in a variety of cell types and also promotes cell migration and morphogenesis. HGF/SF mRNA has been found in a variety of tissues, including brain. In a previous study, we showed that basic fibroblast growth factor (bFGF), another heparin-binding protein is increased in Alzheimer's disease (AD), and appears to be associated with the heparan-sulfate proteoglycans bound to B/A4 amyloid (Biochem. Biophys. Res. Commun. 171 (1990) 690-696). In the present study, we examined the distribution of HGF/SF in 4% paraformaldehyde fixed samples of prefrontal cortex from control and Alzheimer patients, in order to assess the possibility that HGF/SF may be found in association with the pathologic changes which occur in Alzheimer's disease. A specific polyclonal antibody directed against HGF/SF revealed widespread HGF/SF-like immunoreactivity in both the cerebral cortex and white matter. Confocal microscopy confirmed that HGF/SF could be found in both GFAP positive astrocytes and LN3 positive microglia cells, as well as rare scattered cortical neurons. In the AD cases studied, the immunoreactivity was increased within both the astrocytes and microglial cells surrounding individual senile plaques. No staining was seen within the neurofibrillary tangles. Western blot analysis confirmed the normal molecular form of HGF/SF in Alzheimer's disease. Quantitative ELISA assay demonstrated a significant increase in HGF/SF in AD relative to age matched controls. These studies confirm the presence of HGF/SF immunoreactivity within neurons, astrocytes and microglial cells. They also indicate that HGF/SF may be increased within senile plaques as a function of the gliosis and microglial proliferation which occurs in association with these structures in Alzheimer's disease.

Keratinocyte growth factor (KGF) is a secreted member of the fibroblast growth factor (FGF) family of heparin-binding proteins. Studies reported to date indicate that it functions primarily as an important paracrine mediator of epithelial cell growth and differentiation. KGF appears to act via binding to a specific FGF receptor-2 isoform generated by an alternative splicing mechanism. To determine whether KGF may play a role in vascular smooth muscle cell (SMC) biology, we investigated KGF and KGF receptor gene expression in human SMC cultured in vitro as well as in several human nonatherosclerotic artery and atheroma specimens. KGF mRNA but not KGF receptor mRNA was expressed by SMCs, as determined by Northern blot hybridization analysis or reverse transcription-polymerase chain reaction assays, respectively. Additional experiments demonstrated that 1) human SMCs produce and secrete mitogenically active KGF and that 2) the cytokine interleukin-1 increases KGF mRNA and protein levels in human SMCs. We also found that KGF transcripts but not KGF receptor transcripts were expressed in control and atherosclerotic human arteries. Taken together, these results indicate that KGF is unlikely to be involved in SMC growth regulation unless it can function intracellularly or interact with a presently unidentified KGF receptor.

Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2). We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF. All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps. Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations. Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays. These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.

A qualitative method based on thin-layer chromatography (TLC) is described that reliably detects ivermectin as a fluorescent derivative in extracts of cattle dung. The limit of detection (LOD) was < or = 40 ng/g of wet dung. These observations were statistically documented and shown visually using digital imagery. Residues were detected in fresh dung deposited by animals treated 10 days previously with a topical dose (500 microg/kg body weight) of ivermectin.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF consists of a series of structural units, including an amino-terminal segment with a hairpin loop, four kringle domains, and a serine protease-like region. In this study, we demonstrate that the amino-terminal (N) domain retains the heparin-binding properties of full-length HGF/SF. In contrast to a previous hypothesis, selected basic amino acid residues in the hairpin loop are not critical for heparin binding, although alanine substitution at a subset of these sites markedly reduced the biological activity of the HGF/SF isoform, HGF/NK1. Covalent cross-linking experiments performed with wild-type and heparan sulfate glycosaminoglycan (HSGAG)-deficient Chinese hamster ovary (CHO) cells revealed that Met-HGF/NK1 binding was strongly dependent on HSGAG. Addition of heparin to HSGAG-deficient CHO cells not only restored ligand binding, but also increased ligand-dependent Met tyrosine phosphorylation and c-fos expression. Moreover, our results showed that heparin stimulated ligand oligomerization through an interaction with the N domain. These findings establish the importance of the N domain for heparin-ligand and ligand-ligand interactions, and demonstrate a crucial role for HSGAG in receptor binding and signal transduction.

A sensitive method was developed to determine permethrin extracted from phosphate buffer and cattle plasma by potassium cyanide catalyzed transesterification of this insecticide with refluxing ethanol and detection of the resulting ethyl esters by capillary gas chromatography with an electron capture detector. With a reflux time of 2 h and with 3-phenoxybenzyl 2-chlorobenzoate as an internal standard, linear calibration curves from buffer (5-250 ng) and plasma (5-100 ng) were obtained. Precision and accuracy of the method were < or = 15%. The limit of detection was approximately 2.5 ng/ml (cis) and 1 ng/ml (trans) from buffer. In cattle sprayed along the back at 2 mg/kg, the concentration of cis- and trans-permethrin in plasma was below the detection limit (5 ng/ml).

Both platelet-derived growth factor (PDGF) and interleukin-4 (IL-4) play major roles in cell proliferation, differentiation, chemotaxis, and other functional responses. Here, we demonstrate that Stat6, previously shown to be activated by only IL-4 and IL-3, becomes activated after PDGF stimulation of NIH 3T3 fibroblasts. PDGF BB, and to a lesser extent PDGF AA, rapidly induced DNA binding activity from NIH 3T3 cell lysates utilizing the immunoglobulin heavy chain germ line epsilon promoter (Iepsilon) that specifically binds to Stat6 in an electrophoretic mobility shift assay. DNA binding activity could be detected within 5 min and reached maximum levels at approximately 20 min in parental NIH 3T3 cells. An identical mobility shift and time course of PDGF-mediated Iepsilon binding activity was more pronounced in lysates of NIH 3T3 transfectants overexpressing human Stat6 (NIH 3T3-Stat6). The observed radiolabeled Iepsilon mobility shift was competed by unlabeled Iepsilon as well as by the beta-casein gene promoter but not by the interferon-alpha-stimulated response element or the interferon-gamma response region of the guanylate-binding protein gene. A Stat6-specific polyclonal antisera also supershifted the PDGF-induced Iepsilon mobility shift. After PDGF BB treatment, a 100-kDa tyrosine phosphorylated species was detected in anti-Stat6 immunoprecipitates. Cycloheximide had little effect on Stat6 tyrosine phosphorylation. In addition to Stat6, Stat5a, and Stat5b, PDGF BB also induced Jak1 tyrosine phosphorylation suggesting a potential pathway for Stat activation. Strikingly, the concurrent addition of IL-4 enhanced PDGF BB-induced Iepsilon binding activity, Jak1 tyrosine phosphorylation, and [3H]thymidine incorporation. These results provide evidence that Stat6 and Jak1 are common elements in PDGF and IL-4 signaling pathways and suggest that IL-4 could play a role in potentiating certain known PDGF-induced biological responses.

The potential relationships between chlorinated hydrocarbon contamination in human serum and red/white blood cell profiles were investigated by multivariate techniques to assess the cellular response patterns to high and low organochlorine levels in the serum. Twenty-three healthy control subjects and fourteen patients with unexplained and persistent fatigue were divided on the basis of (a) high or low total organochlorine content, (b) high or low DDE (1,1-dichloro-2,2-bis(p-chlorophenyl) ethene) content, and (c) high or low HCB (hexachlorobenzene) content. Discriminant function analysis revealed that the groups with high organochlorine content had significantly different red/white blood cell profiles compared with the low organochlorine groups ((a) P < 0.017, (b) P < 0.015, and (c) P < 0.0002). As a variable, the percentage of neutrophils was the most important discriminant parameter for differentiating between the high and low total organochlorine groups. Thirteen of the fourteen fatigued patients were characterized as "high total organocholorine content" (P < 0.04). The red cell distribution width was elevated in the high DDE group (P < 0.04) and was the most important discriminant parameter for differentiating between the high and low DDE groups. The percentage of eosinophils and the hemoglobin content were both reduced in the high HCB group (P < 0.009,P < 0.003, respectively) and the percentage of eosinophils was the most important discriminant parameter for differentiating between the high and low HCB groups. Those patients with unexplained and persistent fatigue had significantly higher levels of DDE compared with the controls and had different specific blood cell responses to organochlorines compared with control subjects.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF is structurally related to plasminogen, including an amino-terminal hairpin loop, four kringle domains, and a serine protease-like region. A truncated HGF/SF isoform, designated HGF/NK2, which extends through the second kringle domain and behaves as a competitive HGF/SF antagonist, was previously shown to be encoded by an alternative HGF/SF transcript. In this study, we describe a second naturally occurring HGF/SF variant, HGF/NK1, consisting of the HGF/SF amino-terminal sequence and first kringle domain. This product is encoded by a 2-kilobase alternative transcript containing intronic sequence that was contiguous with exon K1b. Analysis of baculovirus-expressed HGF/NK1 revealed that this isoform possesses the heparin binding properties of HGF/SF and modest mitogenic and scattering activity relative to HGF/SF. However, at a 40-fold molar excess, HGF/NK1 inhibited HGF/SF-dependent DNA synthesis. HGF/NK1 stimulated tyrosine phosphorylation of Met, and covalent affinity cross-linking demonstrated a direct HGF/NK1-receptor interaction. These findings establish that the HGF/SF gene encodes multiple alternative products, which include not only a mitogenic agonist (HGF/SF) and a pure antagonist (HGF/NK2) but also a molecule with partial agonist/antagonist properties.

Epidermal cell proliferation is required for re-epithelialization during wound repair. Re-epithelialization of partial thickness excisional wounds in pigs is complete by 6 days after injury. The presence of insulin-like growth factor-I (IGF-I) and heparin-binding molecules that are mitogenic for keratinocytes was examined in wound fluid obtained daily from these wounds. Two significant heparin-binding growth factor activities for Balb/MK keratinocytes were detected, a major one that was eluted from a heparin affinity column with 1.1 M NaCl and a minor one with 0.5 M NaCl. These activities appeared 1 day after injury, were maximal by 2-3 days later, and disappeared by 6 days after injury. The molecule eluting with 1.1 M NaCl was heparin-binding EGF-like (HB-EGF). The levels of IGF-I in wound fluid were 45-90 ng/ml during the first 3 days following injury, decreased thereafter, and were not detectable 6 days after injury. IGF-I at 100 ng/ml, increased HB-EGF mitogenic activity for Balb/MK keratinocytes by 40-50 fold. We conclude that the synergism between IGF-I and HB-EGF and their relative concentration at the various days after injury may be important variables for regulating re-epithelialization during wound repair.

In rats treated ip with N-ethylbenzamide (EB) at doses of 250-500 mg/kg, this amide was metabolized by hydrolysis to ethylamine and benzoic acid, the latter detected in the urine as hippuric acid. Proposed metabolic pathways were investigated by treatment of male Wistar rats with EB and N-ethyl-alpha-13C-benzamide followed by examination of urine samples by high-resolution NMR spectroscopy. Ethylamine was detected by 1H and 13C NMR techniques. Estimation of metabolite levels in 24-hr posttreatment urine by 1H NMR spectroscopy with an internal standard of 3-(trimethylsilyl)-1-propanesulfonic acid and correction for background levels of hippuric acid in control urine showed that the percentage of the dose excreted as hippuric acid was 52-76%. The corresponding range for the excretion of ethylamine (detected as an ethylammonium cation) was 27-41%. Signals for EB, benzamide, and benzoic acid were not above the background in urine of treated animals. A similar NMR study with unlabeled p-chloro-N-ethylbenzamide (p-Cl EB) resulted in the detection of p-chlorohippuric acid and ethylamine. Treatment of rats with N,N-diethylbenzamide and p-chloro-N,N-diethylbenzamide gave the same detectable metabolites in the urine as those found in experiments with EB and p-Cl EB. These observations could be rationalized with a metabolic pathway involving an initial oxidative mono-N-deethylation reaction followed by enzymatic hydrolysis of the secondary amides to ethylamine and benzoic acids and conjugation of the latter with glycine. N-Methylbenzamide was also found to be eliminated in the urine as hippuric acid.

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex steroid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.

A study was conducted on the detection of ivermectin (22,23-dihydroavermectin B1) in cattle serum by thin-layer chromatography (TLC) after derivatization of this parasiticide by the known reaction with trifluoroacetic anhydride-1-methylimidazole and visual examination of the chromatograms under long-wavelength ultraviolet light. By derivatization of reference samples of ivermectin in acetonitrile, approximately 0.1 ng of a highly fluorescent material, tentatively identified as 2,5,6-tetradehydro-5,7-dideoxy-22,23-dihydroavermectin B1, could be detected on silica-gel thin layer plates. Extraction of fortified serum samples with methyl tert.-butyl ether followed by derivatization, hydrolysis, partitioning between water-hexane and TLC gave a limit of detection of 1-2 ng/ml. With these simple techniques ivermectin could be detected in cattle serum for 3-4 weeks after subcutaneous treatment of Hereford heifers.

Model-independent pharmacokinetic methods based on statistical moments were applied to investigate the plasma disposition characteristics of N,N-diethyl-m-toluamide (DEET insect repellent) after single-dose treatment of experimental cattle by rapid intravenous injection (2.5-2.7 mg/kg) and by dermal application (10 mg/kg) to the back. DEET was determined in jugular blood samples by capillary GC with a nitrogen-selective detector and an internal standard of N,N-dipropyl-m-toluamide. Using weighted least squares linear regression analysis, the assay was validated over the concentration range of 19-1910 ng/ml of plasma. Comparison of areas under the plasma concentration-time curves after intravenous and dermal treatments of four Hereford heifers indicated that 72.9 +/- 8.3% (mean +/- SD) of the dermally applied dose was absorbed into the systemic circulation. The time-to-peak plasma concentrations following dermal treatments was 37.5 +/- 8.7 min. Apparent elimination rate constants were not significantly different between the two routes of administration. Linear pharmacokinetics was demonstrated with four additional cattle by comparing systemic clearance after intravenous infusion to steady-state plasma levels of approximately 0.5 and 2.5 micrograms/ml. The rapid and extensive dermal absorption of DEET observed in this study will probably contribute to a short duration of insect repellent action if ethanol-based sprays are used to protect cattle under field conditions.

Pharmacokinetic variables of phenolsulfonphthalein (PSP) were determined in sheep after rapid IV injection and IV infusion to steady state. In Suffolk wethers, an average of < 75% of an IV administered dose was eliminated in urine, indicating that measures of systemic clearance overestimate renal clearance in this species. Furthermore, PSP elimination from plasma was more rapid in Suffolk than Rambouillet wethers and, in Suffolk ewes, systemic clearance decreased from mean +/- SD 7.8 +/- 0.3 ml/min/kg of body weight to 4.7 +/- 1.1 ml/min/kg at steady-state plasma concentration of 2.4 +/- 0.3 and 151.3 +/- 31.8 micrograms/ml, respectively. These observations indicate that, similar to that in other species, systemic clearance of PSP in sheep is concentration-dependent and that significant differences may exist between breeds.

The O2 and NADPH-dependent metabolism of N,N-diethylbenzamide (DEB), a topically applied insect repellent, has been investigated in liver microsomal suspensions from phenobarbital-pretreated male Wistar rats. Incubation conditions for the enzymatic production of N-ethylbenzamide (EB) from DEB (0.2 mM) were studied by use of HPLC with UV detection. With the microsomal enzymes suspended in 10 mM phosphate buffer (pH 7.4) and the substrate delivered in acetone (10 microliter), the yield of EB was 81.9 +/- 2.9% in five replicated experiments with NADPH (2 mM) and MgCl2 (4 mM). Conversion of DEB to EB was strongly inhibited by carbon monoxide, a cytochrome P-450 inhibitor. A sample of N,N-diethyl-alpha,alpha'-13C-benzamide was synthesized and was used, in conjunction with high-resolution 13C NMR spectroscopy, to identify the metabolites arising from oxidation of the ethyl group. Using the distortionless enhancement by polarization transfer pulse sequences and a substrate concentration of 0.4 mM, 13C-labeled acetaldehyde and glycolaldehyde, both detected as the hydrates, were found in the microsomal incubates. N-Ethyl-N-(alpha-hydroxyethyl)benzamide, the presumed metabolic progenitor of acetaldehyde, was not detected in these metabolic experiments. A pathway is proposed to account for the metabolic generation of glycolaldehyde by hydroxylation of both carbons of the N-ethyl group.

Hepatocyte growth factor is a plasminogen-like molecule with diverse biological effects. Although it is synthesized as a single chain polypeptide, it was originally purified as a disulfide-linked heterodimer which was generated by an internal proteolytic event. Subsequent work indicated that preparations consisting largely of the monomeric form also exhibited potent activity. By using a combination of protease inhibition and site-directed mutagenesis, we established that conversion of the single chain polypeptide to the heterodimer occurred during the bioassay and was required for mitogenic and motogenic activity.

Commercial porcelain refinishing kits are claimed to restore the surface finish on porcelain after adjustments in circumstances that preclude reglazing. This study investigates the efficacy of one such kit in restoring a Vitadur N porcelain surface finish after grinding with fine (30 microns grit-red band) and extra-fine (15 microns grit-yellow band) high-speed diamond burs. The production of the porcelain test samples, the refinishing procedures, and the refinishing times were standardized throughout. Techniques were selected to simulate clinical practice. Randomly selected examples of surfaces created during refinishing were subjected to scanning electron microscopy, using standard kV and tilt angle settings, and to surface profilometry tracings. Roughness average (Ra) readings were recorded for each test specimen for every surface finish. Ra readings were analyzed statistically using a two-way unbalanced model for analysis of variance. Although refinishing after grinding with a 15 microns grit bur produced surfaces significantly smoother than on specimens previously ground with the 30 microns grit burs, the surfaces remained significantly rougher than when originally glazed. It is concluded that, using the type of kit tested, burs of a grade finer than the existing 15 microns grit yellow band types would be appropriate for porcelain adjustments to permit subsequent refinishing to a surface smoothness comparable to the original glaze.

The study examined the accuracy of stone casts produced from impressions taken in stock polycarbonate trays, some of which had been strengthened with autopolymerizing polymethyl methacrylate resin and four designs of custom-made trays. Impressions of an acrylic master model of the mandibular arch on which two extracoronal crown preparations and one intracoronal inlay preparation had been carried out were taken using a medium-bodied polyvinyl siloxane material. Each preparation had been intended with a reference point for later measurement. Five impressions were taken for each type of tray and these were cast in die-stone after 24 h. The distances between the points were measured with a reflex microscope and the means determined for each design of tray. The mean difference between casts produced from the various tray designs and the acrylic master model were determined for each of the distances between the three measuring points. Statistical analysis showed that, with the stock trays, there were no significant differences between any of the modifications, or between them and the acrylic model, for any of the three distances (P greater than 0.1). When the results from the custom-made trays were analysed there were statistical differences between the acrylic model and the casts from the unperforated tray with no stops (P = 0.02), and between the unperforated tray with no stops and the perforated with stops for the distance A-B (P less than 0.01). There were no other significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examined the accuracy of stone casts produced from impressions taken in stock polycarbonate trays, some of which had been strengthened with autopolymerizing polymethyl methacrylate resin. Three techniques were used to make the impression of an acrylic master model of the mandibular arch on which two extracoronal preparations for bridgework and one intracoronal inlay preparation had been carried out. Each preparation had been indented with a reference point for later measurement. The impression material was a putty-wash polyvinyl siloxane material. Five impressions were taken for each type of tray for each impression technique and these were cast in die-stone after 24 h. The distances between the points were measured with a reflex microscope and the means determined for each design of tray. The mean difference between casts produced from the various tray designs and the acrylic master model were determined for each of the distances between the three measuring points for the various impression techniques. Statistical analysis showed that, with the polycarbonate stock trays, there were significant differences between some of the modifications and between them and the acrylic model, for the three distances (P less than 0.05). These differences were limited to one measurement for one design of tray for each of the two-stage impression methods. With the one-stage technique the unreinforced tray and those reinforced with acrylic, over the heels and anteriorly, and the barred design were statistically significantly different from the acrylic model for measurement A-B.(ABSTRACT TRUNCATED AT 250 WORDS)

Scatter factor, a stimulant of epithelial cell motility, and Hepatocyte Growth Factor (HGF) were compared by cross-biological studies using naturally occurring and recombinant proteins in four bioassays. Both scatter factor and HGF produced similar effects in cell motility and DNA synthesis assays. Antibodies to scatter factor or HGF neutralized the biological activities of each cytokine, and in immunoblotting reacted with species of the same Mr. These results, together with the available sequence data, suggest that scatter factor and HGF are the same protein.

Dental porcelain occasionally requires adjustment in circumstances that preclude reglazing. Porcelain refinishing kits are now available commercially. The effects of using one such kit on Vita VMK bonded porcelain was investigated qualitatively and quantitatively using scanning electron microscopic and surface profilometric techniques, respectively. The kit proved incapable of restoring a surface glaze to porcelain adjusted using a fine (red band) diamond bur, but was capable of reducing significantly the surface roughness (Ra) of adjusted porcelain. The importance of distinguishing between the integrity of the surface glaze and measurements of surface roughness is discussed. Confining the application of refinishing procedures to the surface adjusted is important to avoid unnecessary removal of the original surface glaze.