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Biomedical subjects

S L Meyer

Publications and source records attributed to S L Meyer.

At least 19 recordsLinked to original sources

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Animals↗

Nematode-antagonistic trichothecenes from Fusarium equiseti.

A strain of the fungus Fusarium equiseti isolated from soybean cyst nematode secretes nematode-antagonistic compounds. Bioassay-guided fractionation of an extract of the culture broth was undertaken to identify the compounds. Fractions were assayed for activity against a root-knot nematode (Meloidogyne incognita), a plant pathogen that attacks the roots of numerous plant species. Two trichothecene compounds were isolated that inhibited egg hatch and immobilized second-stage juveniles of this nematode: 4,15-diacetoxy-12,13-epoxy-3,7-dihydroxytrichothec-9-en-8-one (4,15-diacetylnivalenol) and 4,15-diacetoxy-12,13-epoxy-trichothec-9-en-3-ol (diacetoxyscirpenol). This is the first published report of these compounds affecting plant-parasitic nematodes.

Animals↗

Immunolocalization of caspase proteolysis in situ: evidence for widespread caspase-mediated apoptosis of neurons and glia in the postnatal rat brain.

Activation of a member of the caspase family of cysteine proteases is thought to be required for the execution of apoptosis in neurons and other cell types. We describe here an antibody (Ab127) reactive with a neoantigenic site on caspase substrate proteins degraded during apoptosis, and its characterization as a biochemical and histochemical probe for apoptosis-associated proteolysis in growth factor-deprived neural cells in vitro and the developing postnatal rat brain. Neuronally differentiated PC12 cells became strongly Ab127 immunoreactive only during apoptosis following nerve growth factor withdrawal. Apoptosis-associated caspase proteolysis was detectable on western blots as markedly increased immunoreactivity of a approximately 46,000 mol. wt polypeptide, a product also generated by caspase-3 treatment of cell-free extracts. In the postnatal rat brain, intense immunoreactivity indicative of caspase activation was exhibited by small proportions of neurons and glia in distinct regional and temporal patterns. The degenerating nature of these cells was confirmed by their argyrophilia, cytoplasmic immunoreactivity for c-jun and fragmented processes. Combined immunofluorescence and Hoechst 33342 staining demonstrated that cells immunopositive for caspase activation have apoptotic nuclear morphologies. Caspase proteolysis was observed throughout the neuraxis in a minority of progenitor cells in germinal zones, postmitotic neurons in the parenchyma, and glia in the corpus callosum and other white matter tracts, but was observed rarely in the adult brain. These data characterize a new approach for evaluating apoptosis in physiological and pathological neurodegeneration, and demonstrate that caspase-associated apoptosis is a widespread mechanism for the programmed death of neurons and glia in the postnatal rat brain.

Animals↗

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.

Afferent Pathways↗

Carotid artery and jugular vein ligation with and without hypoxia in the rat.

A continuing concern about the use of extracorporeal membrane oxygenation (ECMO) is the cannulation of the common carotid artery or the internal jugular vein. The authors investigated the changes that might occur in the brain with neck vessel ligation in the normal and the hypoxic rat. Two groups of 60 rats each were studied. The first group was divided into three subgroups of 20 animals each. Subgroup 1 (HH) was hypoxic both 24 hours before and 24 hours after operation. Subgroup 2 (HN) (the ECMO model) was hypoxic before operation and recovered for 24 hours in room air. Subgroup 3 (NN) underwent the entire procedure in room air. For each oxygen environment, four different operations were performed: carotid artery ligation, jugular vein ligation, carotid artery and jugular vein ligation, and dissection of the vessels without ligation (sham). Thus each subgroup was further divided into four sub-subgroups based on the operation performed. Rats were again anesthetized after a 24-hour recovery period and killed using low, blunt cervical dislocation. In the first group of 60 rats, the skull was opened and the brain was carefully removed from the cranial vault and placed in a fixative. The brains were placed in a small magnetic resonance imaging (MRI) head coil in groups of five and scans were obtained to provide T1 and T2 images that correlated with histological sections. MRI scans were reviewed in random, blinded fashion by an imager unaware of how these animals had been treated. The brains were then sectioned coronally at six corresponding levels: frontal, mid and posterior cerebrum, midbrain, pons, and medulla. Histological examination was performed in blinded fashion. The number of lesions (usually ischemic as noted by a decrease in the number of neurons) was totaled for each area of the brain. There were no differences that were consistent or statistically significant in the MR images of brains removed from the head, although it would appear that rats with jugular vein and carotid artery ligation were relatively protected. In the HN group jugular vein ligation was worst, and adding carotid artery ligation was best. In the histological studies the NN group had significantly more lesions than the HH group (P < .01). The second group of 60 rats was divided and treated as the first group in all respects except that MRI was conducted immediately after death on intact heads, and no histological studies were performed. This was done to control for lesions that might have been produced by removal of the brains from the skulls. In this group all findings were right sided. One animal in the HN group showed midcerebral white matter edema after jugular and carotid ligation. Focal anterior cerebral edema was seen in another animal (HH) after isolated carotid ligation. An occipital infarct was found in one animal (HH) after both carotid and jugular ligation. The authors conclude that neck vessel ligation in the hypoxic or normoxic rat causes only occasional and sporadic brain injury much as is seen clinically in newborn ECMO patients.

Animals↗

Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue.

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

Amino Acid Sequence↗

Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system.

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

Amino Acid Sequence↗

Mutational studies of conserved residues in the dimer interface of nerve growth factor.

An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.

Animals↗

Pulsatile gonadotropin secretion determined by frequent sampling from the intercavernous sinus of the mare: possible modulatory role of progesterone during luteolysis.

Twelve horse mares were used in a repeated-measures design consisting of 3 replicates of 4 mares each. On Day 6 following ovulation, luteolysis was initiated with an i.m. injection of prostaglandin F2 alpha (PGF2 alpha; Lutalyse, 10 mg). Either 12 (-12) or 36 (-36) h before PGF2 alpha (PRE), blood samples were collected simultaneously from the intercavernous sinus (ICS) and jugular (JUG) vein at 10-min intervals for an 8-h period. Pituitary capacity to exogenous GnRH (2 micrograms/kg BW, i.v.) was evaluated at the alternate time point within this period. Frequent sampling and GnRH challenge were repeated 36 (+36) or 60 (+60) h after PGF2 alpha (POST). Progesterone reached a nadir 48 h after PGF2 alpha, while the first significant elevation in estradiol (p < 0.05) was not detected until 156 h. PRE LH and FSH pulse frequencies in ICS samples were 0.6 +/- 0.3 and 1.8 +/- 0.8 pulses/8 h, respectively. Three high-amplitude, concurrent ICS pulses of LH and FSH were observed during this period. These were associated with elevated gonadotropin levels in the corresponding JUG samples. In contrast, low-amplitude ICS pulses at this time were predominately releases of FSH alone that were not detectable in the JUG. Following luteolysis, POST gonadotropin pulse frequencies in the ICS increased (p < 0.001) to 7.4 +/- 0.4 and 6.8 +/- 0.5 pulses/8 h for LH and FSH, respectively. Concurrent LH and FSH releases of relatively low amplitude characterized 87.7% of the POST ICS pulses, none of which were measurable in corresponding JUG samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Dependency characteristics of partner assaultive men.

Men who had physically assaulted female partners (PA group; N = 24) were compared with maritally discordant nonviolent men (DNV group; N = 24) and happily married nonviolent men (HNV group; N = 24) on measures of dependency and related constructs. PA men reported higher interpersonal dependency, higher spouse-specific dependency, and lower self-esteem than both contrast groups, but did not differ significantly on a measure of jealously. Data reduction indicated two constructs underlying these measures: perceived personal inadequacy and emotional investment in the primary relationship. PA men were high on both, while DNV men had moderate perceived inadequacy and low emotional investment and HNV men had low perceived inadequacy and high emotional investment. The findings support the clinical observation that interpersonal dependency is an important factor in the motivational dynamics of relationship violence.

Adult↗

Sex-role socialization: developmental influences on wife abuse.

Themes central to sex-role socialization are traced, along with the processes by which dominance and female deference are developed. Gender differences in parent-child interactions, peer-group relations, classroom settings, children's observations of the marital dyad, and messages from television are examined. The link between sex-role socialization and the etiology and maintenance of wife abuse is discussed.

Adolescent↗

Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells.

Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.

Animals↗

K-252a and staurosporine promote choline acetyltransferase activity in rat spinal cord cultures.

The protein kinase inhibitor K-252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose-dependent manner (EC50 of approximately 100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K-252a completely prevented the marked decline in ChAT activity occurring over a 5-day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor staurosporine also increased ChAT activity (EC50 of approximately 0.5 nM). Effective concentrations of K-252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin-like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose-dependent increases in ChAT activity in spinal cord cultures. The combination of K-252a with insulin-like growth factor-I or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K-252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K-252a and staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.

Alkaloids↗

Family of origin violence and MCMI-II psychopathology among partner assaultive men.

This study examined associations between family of origin violence, self-reports of psychopathology on the MCMI-II, and current spouse abuse among partner assaultive men. Compared to nonviolent men in discordant (n = 24) and well-adjusted (n = 24) relationships, partner assaultive men (n = 24) were significantly more likely to report childhood histories of physical abuse and physical abuse of the mother in the family of origin. The partner assaultive men also reported significantly higher scores on a variety of MCMI-II personality disorder and Axis I disorder scales. When negative affectivity was controlled, however, batterers differed from contrast groups only on scales assessing antisocial and aggressive characteristics. Within the partner assaultive group, a history of severe childhood abuse was associated with higher scores on a variety of MCMI-II personality disorder and Axis I disorder scales, and higher levels of psychological and physical aggression directed toward the current relationship partner. Abuse of the mother in the family of origin among batterers was associated with higher levels of psychological and physical aggression toward the current partner, but not with self-reported psychopathology. The results support prior descriptions of a batterer subgroup with significant trauma histories, more psychological difficulties, and higher abuse levels than other batterers, suggesting continuities in social and emotional development from childhood maltreatment to adult relationship violence.

Adult↗

Effect of constant administration of a gonadotropin-releasing hormone agonist on reproductive activity in mares: induction of ovulation during seasonal anestrus.

The potential of a gonadotropin-releasing hormone (GnRH) agonist (goserelin acetate), delivered constantly for 28 days via a subcutaneous depot, to induce ovulation in seasonally anestrous mares, was investigated. Two experiments were conducted, in which a range of doses (30 to 240 micrograms/mare/d) was examined. Mares were selected on the basis of lack of substantial follicular development (follicle diameter < 20 mm determined ultrasonically) and low serum concentrations of luteinizing hormone (LH) and progesterone. Constant administration of the GnRH agonist-induced ovulation in anestrous mares, but a dose-response relation was not observed. Furthermore, with identical doses tested in consecutive or alternate years, considerable variation was observed in the ovulatory response. In general, ovulation in all treated mares was accompanied by increased circulating concentrations of LH and a decrease in follicle-stimulating hormone values. Ovulation was preceded by an increase in estradiol and LH concentrations. In mares in which ovulation did not occur, concentration of LH increased during agonist treatment, whereas that of follicle-stimulating hormone either increased or did not change. It was concluded that constant administration of GnRH agonists may induce ovulation in mares during seasonal anestrus; however, percentage of mares ovulating and the lack of reproducibility of effect indicate that this approach is inappropriate for use as a reliable method to manipulate breeding activity in commercial broodmares.

Anestrus↗

Quantitative non-radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin-labeled cRNA probes.

Non-radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non-radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol-induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin-11-UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin-alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 +/- 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low-resolution radioactive methods, showing a 30-80% increase in striatal preproenkephalin mRNA hybridization signal.

Animals↗