Significant Differences In Labeling Research Articles

Enteric glia regulates the expression of neutrophil inflammatory factors during acute inflammation

Abstract Inflammation is an event in functional gastrointestinal disorders that has detrimental effects in the enteric nervous system. Recent advances in our understanding of the immune and enteric nervous system indicate that enteric glial cells modulate immune cells in the gastrointestinal tract. Previous single-nuclei multiomic analysis has shown that genes that encode for and influence neutrophil function, Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5, are changed in enteric glia during peak colitis. We hypothesize that enteric glia regulate the expression of neutrophil modulating factors during acute inflammation. We tested this using the acute dinitrobenzene sulfonic acid colitis model. Animal body weight and gross macroscopic damage were recorded as indications of overall inflammation and tissue was harvested for immunohistochemical analysis. Enteric glial cells were labeled with antibodies against Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5. Results suggest no significant difference in labeling for immunological markers in enteric glial cells of dinitrobenzene sulfonic acid treated mice. Results also show labeling in some neurons that may or may not be specific. Further experimentation is needed to elucidate the mechanisms of enteric neurons and glia in gut inflammation. These studies will contribute to further understand how immune responses are regulated in the gut and impact the general knowledge of gut disease processes. This work was supported by the National Institute of Health grants R01DK103723 and R01DK120862 to BDG and DPR was supported by the National Institute of Health Bridge to the Ph.D. in Neuroscience grant 2R25NS090989.

A Partial C4 Photosynthetic Biochemical Pathway in Rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.

A method for 13C‐labeling of metabolic carbohydrates within French bean leaves (Phaseolus vulgaris L.) for decomposition studies in soils

The molecular composition of plant residues is suspected to largely govern the fate of their constitutive carbon (C) in soils. Labile compounds, such as metabolic carbohydrates, are affected differently from recalcitrant and structural compounds by soil-C stabilisation mechanisms. Producing (13)C-enriched plant residues with specifically labeled fractions would help us to investigate the fate in soils of the constitutive C of these compounds. The objective of the present research was to test (13)C pulse chase labeling as a method for specifically enriching the metabolic carbohydrate components of plant residues, i.e. soluble sugars and starch. Bean plants were exposed to a (13)CO(2)-enriched atmosphere for 0.5, 1, 2, 3 and 21 h. The major soluble sugars were then determined on water-soluble extracts, and starch on HCl-hydrolysable extracts. The results show a quick differential labeling between water-soluble and water-insoluble compounds. For both groups, (13)C-labeling increased linearly with time. The difference in delta(13)C signature between water-soluble and insoluble fractions was 7 per thousand after 0.5 h and 70 per thousand after 21 h. However, this clear isotopic contrast masked a substantial labeling variability within each fraction. By contrast, metabolic carbohydrates on the one hand (i.e. soluble sugars + starch) and other fractions (essentially cell wall components) on the other hand displayed quite homogeneous signatures within fractions, and a significant difference in labeling between fractions: delta(13)C = 414 +/- 3.7 per thousand and 56 +/- 5.5 per thousand, respectively. Thus, the technique generates labeled plant residues displaying contrasting (13)C-isotopic signatures between metabolic carbohydrates and other compounds, with homogenous signatures within each group. Metabolic carbohydrates being labile compounds, our findings suggest that the technique is particularly appropriate for investigating the effect of compound lability on the long-term storage of their constitutive C in soils.

In Vivo Functional Analysis of Polyglutamic Acid Domains in Recombinant Bone Sialoprotein

Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSP(E)) within an actual in vivo environment. Full-length prBSP(E) and prBSP(E) in which the poly [E] domains were replaced by polyalanine (prBSP(A)) were tagged with dinitrophenol (DNP). Tagged preparations comprised intact molecules and some fragmented forms. They were infused through a surgically created hole in the bone of rat hemimandibles and detected using immunogold labeling with anti-DNP antibodies. prBSP(E)-DNP was consistently immunodetected along exposed mineralized bone surfaces and osteocyte canaliculi at the surgical site. Few gold particles were observed on these surfaces when prBSP(A)-DNP was infused. Quantitative analyses showed significant differences in labeling between prBSP(E)-DNP (5.04 +/- 0.73 particles/micro m2) and prBSP(A)-DNP (1.37 +/- 0.35 particles/micro m2). These results indicate that poly [E] domains influence binding of prBSP(E) to surfaces presenting a mixture of mineral and proteins bathed by tissue fluids and suggest that they may similarly mediate the interaction of native BSP in the bone environment.

Expression of the kynurenine pathway enzyme tryptophan 2,3-dioxygenase is increased in the frontal cortex of individuals with schizophrenia

Markers of the kynurenine pathway were studied in postmortem frontal cortex obtained from individuals with schizophrenia and controls. Quantitative endpoint RT-PCR was used to measure mRNA transcripts. Of the two enzymes capable of catalyzing the first step in the pathway, tryptophan 2,3-dioxygenase (TDO2) and indoleamine dioxygenase (IDO), the concentration of mRNA for TDO2 was found to be elevated 1.6-fold in the schizophrenia group ( P = 0.03), whereas the concentration of the mRNA for IDO was not significantly different between the schizophrenia and control groups. Immunohistochemistry showed an increased density of TDO2-immunopositive astroglial cells in the white matter of patients with schizophrenia ( P = 0.04). Neurons and vessels were also immunopositive for TDO2, but there were no significant differences in labeling of these structures between the two groups. These results add to the evidence that kynurenine pathway changes might be involved in the pathogenesis of schizophrenia and the schizophrenia-like psychoses of other disorders.

Role of sphingolipids in the transport of prosaposin to the lysosomes

Prosaposin is the precursor of four lysosomal saposins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fumonisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the lysosomes. Fumonisin B1 interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumonisin B1 produced a 59–85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an expression vector containing a human prosaposin cDNA. To examine whether the mannose 6-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody. The results showed no significant differences in labeling of the lysosomes, suggesting that the effect of fumonisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process.▪In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.—Lefrancois, S., L. Michaud, M. Potier, S. Igdoura, and C. R. Morales. Role of sphingolipids in the transport of prosaposin to the lysosomes. J. Lipid Res. 1999. 40: 1593–1603.

Student Interpretations of Teacher Verbal Praise in Selected Seventh- and Eighth-Grade Choral Classes

This study was an investigation of the effect that familiarity with a teacher and rehearsal context might have on seventh- and eighth-grade choral students' interpretations of teacher verbal praise. Teachers (N = 4) labeled randomly selected videotaped examples of their praise according to the purpose it was intended to serve. Students (N = 80) viewed 16 brief (30-second) examples of the praise and labeled it as deserved (directed at the performance) or one of three instructional uses (i.e., to encourage, to gain student cooperation, or to send a message to other members of the class). Results showed students across choirs could separate deserved from instructional praise, but in 9 examples, familiarity with a context made a significant difference in labeling praise as deserved and in detecting a specific instructional purpose. Results suggest the importance of further investigations that determine how teachers intend praise to function and how their students interpret its use.

Cochlear ablation in deafness mutant mice: 2-deoxyglucose analysis suggests no spontaneous activity of cochlear origin

Deafness mutant mice show no stimulus-related cochlear potentials as well as abnormal electrically-evoked responses recorded from the inferior colliculus. Abnormal spontaneous activity in the auditory periphery could result in abnormal development and/or maintenance of the central auditory pathways. We therefore assessed spontaneous activity of cochlear origin in the central nuclei of the mutants by ablating one cochlea and subsequently using the 2-deoxyglucose (2 dg) technique to study metabolic activity. Any asymmetries in labeling in a given nucleus should be due to spontaneous activity in the cochlear nerve on the unoperated side. In control animals (+ /dn mice undergoing unilateral cochlea ablation), statistically significant decreased 2 dg labeling was observed in the ipsilateral PVCN and AVCN, and contralateral MNTB and IC; all receive primary excitatory input from the ablated ear. No significant differences in labeling between right and left sides were observed in any of the nuclei studied in the mutant animals. These findings suggest that there is no spontaneous activity of cochlear origin in these mutants, even though many cochlear nerve fibers and spiral ganglion cells survive.

Role of neural retina and vitreous body during lens regeneration: transplantation and autoradiography.

The trophic influence of neural retina in regeneration of the lens from dorsal iris is well known. The first part of this study involved transplantation of dorsal iris pieces into various intra-ocular positions in order to determine the field of action of the neural retina. The most favorable location for lens regeneration was the pupillary space, closely followed by the vitreous chamber. Lens regeneration was retarded from implants lying in the anterior chamber; this was most evident when grafts were placed in front of the iris and not in contact with the swollen vitreous body. Incorporation of 3H-leucine in the neural retina and vitreous body was also investigated using autoradiography. No significant difference in labeling was seen between the control and lentectomized neural retina at all time intervals studied. However, increased labeling above that in the controls was observed over the vitreous body between 8 to 20 days after lentectomy. There was also an increase in the number of silver grains over the vitreous body with increasing time between injection of isotope and sacrifice of the animal. These results provide evidence that vitreous body plays an important role in the process of lens regeneration, perhaps serving as a preferential pathway for transfer of the neural retinal factor to the dorsal iris.

Incorporation of leucine-14C into proteins of the placenta and liver of mice

Incorporation in vivo of leucine-14C into proteins of the placenta and maternal liver of mice is highest on the fifteenth day of gestation. The two tissues show significant differences in labeling of proteins as a function of time after the amino acid administration. The pattern of leucine incorporation into hepatic proteins of gravid and nongravid mice is different.