A sensitive and inexpensive high-performance liquid chromatographic assay for tyrosine hydroxylase

Summary We describe a highly sensitive assay method for tyrosine hydroxylase (TH) using high-performance liquid chromatography with amperometric determination. This assay method could be applicable to any tissues with low enzyme activity, such as rat cerebellum. We also describe the kinetic properties of TH in rat cerebral cortex.     

Structural differences between somatic H2B and testis-specific TH2B histones of the rat

Testis-specific histone TH2B from rat testis showed a very similar tryptic map to somatic H2B from rat liver, indicating a common evolutionary origin. However, 5 peptide differences were detected between the 2 proteins, and the amino acid compositions of these distinctive peptides were determined.     

Structural differences between somatic H2B and testis-specific TH2B histones of the rat

Summary Testis-specific histone TH2B from rat testis showed a very similar tryptic peptide map to somatic H2B from rat liver, indicating a common evolutionary origin. However, 5 peptide differences were detected between the 2 proteins, and the amino acid compositions of these distinctive peptides were determined.Supported by Grant 780-0609 from the Ford Foundation.     

Synthesis of ovulation-inhibiting compounds; Structure-activity relationship

Summary We describe the synthesis of some new derivatives of benzo (4,5) cyclohepta (1,2-b) thiophene which inhibit ovulation and the secretion of luteinizing hormone (LH) in the rat. We also describe the relationship between the structure and activity of these compounds.     

Synthesis of ovulation-inhibiting compounds; structure-activity relationship.

We describe the synthesis of some new derivatives of benzo(4, 5)cyclohepta(1, 2-b)thiophene which inhibit ovulation and the secretion of luteinizing hormone (LH) in the rat. We also describe the relationship between the structure and activity of these compounds.     

Zone immunoelectrophoresis assay applied to α1 glycoprotein secretion by isolated rat hepatocytes

Summary A method for measuring proteins in low concentrations applying the zone immunoelectrophoresis assay is reported. The low detection limit makes it possible to measure ₁-acid glycoprotein in rat serum and also to quantify the secretion of this protein after concentration of the incubation media containing less than 10⁷ isolated rat hepatocytes. The method is simple and consumes very small quantities of antiserum.     

Somatic cell hybrids producing inhibitors of melanotic melanoma tyrosine hydroxylase

Summary Splenic lymphocytes from BALB/c mice pre-immunized with purified tyrosine hydroxylase (TH) were fused with murine myeloma N/1 cells. Supernatants of only 2 from large number of cloned cell hybrids contained an inhibitor of TH.     

Tyrosine hydroxylase activity and tyrosine titers during cockroach metamorphosis

Summary Tyrosine hydroxylation (TH) to dopa in vivo in nymphs and pharate adults ofPeriplaneta americana was very low, but increased over 30-fold beginning at ecdysis. Free tyrosine increased in pharate, adults and peaked at ecdysis, whereas TH activity peaked 6 h later. Both TH activity and tyrosine titers declined the next 24 h as the cuticle tanned. TH appears to regulate tanning substrate biosynthesis in cockroaches.Contribution No. 81-294-j, Department of Entomology, Kansas Agricultural Experiment Station, Manhattan KS. Research supported in part by National Science Foundation grant No. PCM-8003859. I wish to thank Dr. M.A.J. Al-Izzi for assistance with the enzyme analyses.     

Nouveau procédé original de préparation de cellules vivantes par dissociation du foie de rat

Summary The authors describe a method for the isolation of hepatocytes by dissociation of rat livers in bovine serum containing sodium citrate, ATP and manganous ions. Moreover, they communicate the results of a comparative study of the morphology (studied by electron microscopy) and the metabolism (respiration and biosynthesis of RNA) of hepatocytes isolated by different methods.     

Ranbp2 haploinsufficiency mediates distinct cellular and biochemical phenotypes in brain and retinal dopaminergic and glia cells elicited by the Parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Many components and pathways transducing multifaceted and deleterious effects of stress stimuli remain ill-defined. The Ran-binding protein 2 (RanBP2) interactome modulates the expression of a range of clinical and cell-context-dependent manifestations upon a variety of stressors. We examined the role of Ranbp2 haploinsufficiency on cellular and metabolic manifestations linked to tyrosine-hydroxylase (TH(+)) dopaminergic neurons and glial cells of the brain and retina upon acute challenge to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian neurotoxin, which models facets of Parkinson disease. MPTP led to stronger akinetic parkinsonism and slower recovery in Ranbp2 (+/-) than wild-type mice without viability changes of brain TH(+)-neurons of either genotype, with the exception of transient nuclear atypia via changes in chromatin condensation of Ranbp2 (+/-) TH(+)-neurons. Conversely, the number of wild-type retinal TH(+)-amacrine neurons compared to Ranbp2 (+/-) underwent milder declines without apoptosis followed by stronger recoveries without neurogenesis. These phenotypes were accompanied by a stronger rise of EdU(+)-proliferative cells and non-proliferative gliosis of GFAP(+)-Müller cells in wild-type than Ranbp2 (+/-) that outlasted the MPTP-insult. Finally, MPTP-treated wild-type and Ranbp2 (+/-) mice present distinct metabolic footprints in the brain or selective regions thereof, such as striatum, that are supportive of RanBP2-mediated regulation of interdependent metabolic pathways of lysine, cholesterol, free-fatty acids, or their β-oxidation. These studies demonstrate contrasting gene-environment phenodeviances and roles of Ranbp2 between dopaminergic and glial cells of the brain and retina upon oxidative stress-elicited signaling and factors triggering a continuum of metabolic and cellular manifestations and proxies linked to oxidative stress, and chorioretinal and neurological disorders such as Parkinson.     

Anomeric compositions of D-glucose in tissues and blood of rat

Summary The anomeric compositions of D-glucose in the liver, kidney, heart, blood and plasma of rat were determined by our method for the assay of D-glucose anomers and the percentages of the -anomer were found to be 61.8, 61.0, 62.4, 62.7 and 62.9, respectively.     

Hypothalamic tyrosine hydroxylase activity, plasma gonadotropin and prolactin levels after aminooxyacetic acid in ovariectomized rats

Plasma concentrations of gonadotropin, prolactin and hypothalamic tyrosine hydroxylase (TH) activity were measured in ovariectomized rats treated with aminooxyacetic acid (AOAA), a drug which elevates brain GABA levels. Hypothalamic TH activity was significantly increased with a significant decrease in prolactin (Prl) release. Plasma levels of gonadotropins were not modified by AOAA. These results support an inhibitory action of GABA on Prl release possibly mediated through hypothalamic dopamine.     

Oxytocin analogs effective as noncompetitive inhibitors in uterotonic test

Summary Deamino-1-carba-oxytocin analogs with a chemically reactive group in position 4 were demonstrated to act as noncompetitive oxytocin inhibitors in the assay on isolated rat uterus.     

In vivo and in vitro differences between leukocytic uptake of oligodeoxynucleotides

Development and application of therapeutic oligonucleotides rely on proper analysis of binding and uptake. We have used several model oligodeoxynucleotides (ODNs) to analyze binding/uptake by rat and human leukocytes. Here we describe: (1) differences between in vivo and in vitro uptake of ODNs to rat leukocytes, (2) differences after injection of lipopolysaccharide (LPS), (3) large in vitro differences between primary mononuclear cells in PBS, plasma and blood, and (4) differences of ODN uptake between rat and human leukocytes. Our data show that ODN uptake by primary blood cells was different in PBS, plasma and blood. In addition, LPS treatment increased ODN uptake by leukocytes in blood, indicating that pathological conditions may influence ODN uptake. Furthermore, ODN uptake in rat and human blood is also different, suggesting that preclinical ODN uptake data from rat blood cannot easily be extrapolated to the human condition. Received 17 December 2007; received after revision 16 January 2008; accepted 5 February 2008     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Scanning transmission electron microscopy of dendritic spines stained by the Golgi method]

Scanning transmission electron microscopy of the dendritic spines of multipolar neurons in the cat inferior Colliculus was achieved on Golgi semi-thin sections. The three basic types of dendritic spines (ST, MS, TH) were identified. Scanning transmission electron microscopy provides a reliable method for a three dimensional view of these structures at high resolution and consequently a more accurate appreciation of their size. In addition, it could prove very useful in the quantitative analysis of the dendritic spines.     

Influence of castration and testosterone replacement on hypothalamic tyrosine hydroxylase activity in the rat.

Hypothalamic tyrosine hydroxylase (TH) activity of castrate rats is modulated by testosterone propionate (TP) in vivo. Kinetic studies revealed that both Vmax and Km were virtually unaltered for substrate tyrosine in the presence of an excess of DMPH4 cofactor. TP replacement to castrate rats increased the Km for added DMPH4 cofactor, while Vmax decreased. These results suggest that TP decreases TH activity of castrate rats by inhibiting the enzyme-reduced pteridine cofactor complex.     

Stimulating determinants of the secondary mixed lymphocyte reaction (MLR-II)]

An anti-Ia immune serum (A . TL anti-A . TH) directed to the antigens of the stimulating cell, blocked the MLR-II. A significant correlation was observed between the anti-Ia reactivity of the immune serum, studied against a panel of eleven original H-2 haplotypes and the reactivity against the same panel of the in vitro primed responding cells (A . TL anti-A . TH). These results confirm the hypothesis that Ia antigens are the structures stimulating the MLR-II.