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Biomedical subjects

L E Schneider

Publications and source records attributed to L E Schneider.

At least 19 recordsLinked to original sources
PubMed2001

Gprk2 controls cAMP levels in Drosophila development.

G protein-coupled receptors mediate their biological responses through the generation of second messengers, such as cAMP. The down-regulation of their activity (desensitization) is carried out, in part, by the family of G protein-coupled receptor kinases, which phosphorylate activated receptors. The Gprk2 gene in Drosophila melanogaster is a putative member of this family. The GPRK2 protein is expressed most abundantly in the ovaries and in the mushroom bodies, the brain region that is implicated in learning and memory in insects. Many of the genes that are involved in learning in Drosophila are members of a cAMP-signaling pathway and are also expressed in the mushroom bodies. These observations suggest that the Gprk2 gene may be involved in a cAMP-mediated pathway. To investigate this possibility, we tested for a genetic interaction between Gprk2 and dunce (which encodes cAMP-specific phosphodiesterase). A mutant allele of Gprk2, called gprk2(6936), has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2(6936). Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2(6936) background suppressed all of these defects to a significant degree. Suppression was also observed when a single copy of gprk2(6936) was introduced into a dunce background. Like mutants of rutabaga (which encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2(6936) has reduced levels of cAMP. Ovaries from gprk2(6936) females contain about one third of the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

The Drosophila G-protein-coupled receptor kinase homologue Gprk2 is required for egg morphogenesis.

G protein signaling is a widely utilized form of extracellular communication that is mediated by a family of serpentine receptors containing seven transmembrane domains. In sensory neurons, cardiac muscle and other tissues, G protein-coupled receptors are desensitized through phosphorylation by a family of kinases, the G protein-coupled receptor kinases (GRKs). Desensitization allows a cell to decrease its response to a given signal, in the continued presence of that signal. We have identified a Drosophila mutant, gprk2(6936) that disrupts expression of a putative member of the GRK family, the G protein-coupled receptor kinase 2 gene (Gprk2). This mutation affects Gprk2 gene expression in the ovaries and renders mutant females sterile. The mutant eggs contain defects in several anterior eggshell structures that are produced by specific subsets of migratory follicle cells. In addition, rare eggs that become fertilized display gross defects in embryogenesis. These observations suggest that developmental signals transduced by G protein-coupled receptors are regulated by receptor phosphorylation. Based on the known functions of G protein-coupled receptor kinases, we speculate that receptor desensitization assists cells that are migrating or undergoing shape changes to respond rapidly to changing external signals.

Amino Acid Sequence↗
PubMed1993

An immunocytochemical study of the FMRFamide neuropeptide gene products in Drosophila.

We have mapped protein expression of the FMRFamide neuropeptide gene in Drosophila with polyclonal antisera against three small peptides whose sequences were derived from the Drosophila proFMRFamide precursor. One antiserum was affinity-purified and extensively characterized. The enriched antibodies labeled 15-21 bilaterally symmetric pairs of neurons in a pattern that corresponded very closely to the pattern of in situ hybridization that was determined previously (Schneider et al. [1991] J. Comp. Neurol. 304:608-622; O'Brien et al. [1991] J. Comp. Neurol. 304:623-638). The other antisera produced complementary results. These findings suggest that the antisera specifically label cells that express the FMRFamide gene. In larvae we consistently observed strong staining in identified interneurons and neuroendocrine cells, and moderate to weak staining in neurons of unknown function. The adult pattern of expression included both larval neurons whose immunoreactivity persisted through metamorphosis and adult-specific neurons. During metamorphosis, we observed transient staining in a small number of neurons and in specific neuropil regions that included the central body, the protocerebral bridge, and the optic ganglia. Based on these morphological features, we suggest that the FMRFamide-like neuropeptides in Drosophila play a number of functional roles, perhaps affecting both physiological and developmental phenomena. Such roles include general modulation throughout all post-embryonic stages, via the blood, and also more stage- and region-specific modulation within the CNS.

Amino Acid Sequence↗
PubMed1993

Cell type-specific transcriptional regulation of the Drosophila FMRFamide neuropeptide gene.

We have used lacZ reporter gene constructs to study the promoter/enhancer regions of the Drosophila FMRFamide neuropeptide gene in germ line transformants. FMRFamide is normally expressed in approximately 60 diverse neurons of the larval CNS that represent approximately 15 distinct cell types. An 8 kb FMRFamide DNA fragment (including 5 kb of 5' upstream sequence) was sufficient to direct a pattern of lacZ expression that mimicked nearly all spatial aspects of the normal pattern. This result indicates that the cell-specific regulation of FMRFamide expression is largely generated by transcriptional mechanisms. Reporter gene expression was lost from selected cell types when smaller fragments were tested, suggesting that multiple control regions are included in the FMRFamide promoter. One region (a 300 bp fragment from -476 to -162) acted as an enhancer for 1 of the approximately 15 FMRFamide-positive cell types, the OL2 neurons. These results suggest that, in the mature nervous system, the complex pattern of FMRFamide neuropeptide gene expression derives from the activity of discrete, cell type-specific enhancers that are independently regulated.

Animals↗
PubMed1991

In situ hybridization analysis of the FMRFamide neuropeptide gene in Drosophila. I. Restricted expression in embryonic and larval stages.

We have used in situ hybridization techniques to describe the cellular distribution of transcripts from a Drosophila gene that encodes multiple FMRFamide-related neuropeptides. The Drosophila FMRFamide gene consists of two exons and is expressed predominantly as a approximately 1.7 kb RNA throughout postembryonic stages (Nambu et al., '88; Schneider and Taghert, '88, '90). We used exon-specific oligonucleotide probes to assay transcription in both embryonic and larval stages and found a pattern of hybridization signals that was restricted to the central nervous system and, within that tissue, was cell-specific. The pattern included 36 distinct signals distributed throughout both the brain and segmental nerve cord (ventral ganglion). These observations suggest that the cell-specific pattern of FMRFamide-like neuropeptide expression in the Drosophila CNS (White et al., '86; Taghert and Schneider, '90) is due to the restricted expression of specific gene transcripts. The results also indicate that, with few exceptions, all previously identified FMRFamide-immunoreactive neurons in Drosophila larvae express FMRFamide gene transcripts. The 36 hybridization regions of the CNS could be divided into three categories, based on their signal intensities (strong, moderate, and weak). The differences in intensity were reproducible and suggest that steady-state levels of specific neuropeptide RNA differ among individual neurons. The two exon-specific probes produced patterns that were indistinguishable both in pattern and in intensity. This result supports the previous conclusion that the one detectable FMRFamide transcript contains both exons (Schneider and Taghert, '90). A single identifiable signal was detected during embryogenesis (beginning at stage 16), but the mature complement of signals was not fully established until the final larval stages.

Animals↗
PubMed1991

In situ hybridization analysis of the FMRFamide neuropeptide gene in Drosophila. II. Constancy in the cellular pattern of expression during metamorphosis.

We have studied changes in the pattern of specific neuropeptide gene expression during the metamorphosis of the Drosophila nervous system. Prior to metamorphosis, the Drosophila FMRFamide gene is expressed exclusively within the central nervous system in a stereotyped pattern that comprises roughly 60 neurons (Schneider et al., '91). Using in situ hybridization, we found that the FMRFamide gene was continuously expressed throughout all stages examined: at each of 15 stages of adult development and through at least the first 10 days of adult life. There were no differences between the results observed with 2 exon-specific hybridization probes, thus indicating little if any alternative splicing during postembryonic development. Despite many changes in the positions of individual hybridization signals due to the large-scale reorganization of the nervous system, the continuous pattern of gene expression through adult development permitted many adult signals to be identified as larval signals. We concluded that the adult pattern of FMRFamide gene expression was largely derived from persistent larval neurons. Adult-specific hybridization signals in the brain and ventral ganglion were also detected and these corresponded to many of the approximately 40 adult-specific FMRFamide-immunoreactive neurons. One specific larval signal was lost during adult development and the intensities of other signals fluctuated in reproducible manners. These stereotyped differences in hybridization signal intensity resemble similar observations made in larval stages (Schneider et al., '91) and support the hypothesis that the steady-state levels of FMRFamide transcripts are differentially regulated among the diverse neurons that express the gene.

Animals↗
PubMed1990

Organization and expression of the Drosophila Phe-Met-Arg-Phe-NH2 neuropeptide gene.

We have studied a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide). Using a combination of genomic sequencing, Northern blot analysis, primer extensions, and nuclease protection assays, we have described its organization and expression. The results indicate that this Drosophila neuropeptide gene contains two exons separated by an intron of approximately 2.5 kilobase pairs, and that its expression is limited to a single predominant RNA species of approximately 1.7 kilobases. The FMRFamide gene contains a 5' untranslated region that is 106 nucleotides long and is encoded in its entirety by exon I. The open reading frame (encoding a 347-amino acid neuropeptide precursor) begins with the first nucleotide of exon II.

Amino Acid Sequence↗
PubMed1990

Interspecific comparison of a Drosophila gene encoding FMRFamide-related neuropeptides.

In order to identify functionally important regions of a neuropeptide gene in Drosophila melanogaster, we have studied its occurrence in related species and have characterized the structure of a homologous gene in Drosophila virilis. The melanogaster gene encodes a precursor that contains 13 neuropeptides related to the molluscan tetrapeptide FMRFamide (Nambu et al., 1988; Schneider and Taghert, 1988). Using the melanogaster gene as a probe in Southern blot analysis, related sequences were detected in DNA from each of 7 species tested. D. virilis, which is estimated to have diverged from D. melanogaster between 60 and 80 million years ago (Throckmorton, 1975), was chosen for more detailed study. Immunocytochemical staining using an antibody to authentic FMRFamide revealed a similar set of immunoreactive neurons in the CNS of larvae from the 2 Drosophila species. Using a melanogaster gene probe, overlapping clones were isolated from a virilis genomic library; DNA sequence analysis indicated the presence of a homologous gene. Comparisons of the genes and deduced proteins between the 2 species revealed the following points. (1) Both genes are divided into 2 exons: in D. melanogaster the exons are 106 and 1352 bp long; in D. virilis, they are 169 and at least 1232 bp long; in both species, the intron is approximately 2.5 kb long. (2) The sequence of exon I has largely diverged, and in neither species are exon I sequences translated. In this vicinity of the gene, sequence conservation is limited to a 67 bp region that spans the TATA box and the RNA start site. (3) The deduced neuropeptide precursors have very similar sizes (347 vs 339 amino acids) and the presumed signal sequences are perfectly conserved. (4) While the melanogaster precursor contains 13 FMRFamide-related peptides, the virilis precursor contains only 10. (5) The sequences of some but not all of the FMRFamide-like peptides are perfectly conserved. (6) In the rest of the precursor, significant sequence conservation is found only in the N-terminal portion; immediately downstream of the final FMRFamide-like peptide, the protein sequences are highly divergent. (7) 5' to the RNA start sites (1.2 kb of melanogaster DNA and 1.8 kb of virilis DNA), 17 small (9-52 base pairs) regions are evolutionarily conserved (greater than 80% sequence conservation). We discuss neuropeptide biosynthesis, the functions and evolution of FMRFamide-like neuropeptides in insects, and the cell-specific regulation of neuropeptide gene expression in the contexts of these results.

Animals↗
PubMed1988

Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide).

A Drosophila gene that encodes neuropeptides related to molluscan Phe-Met-Arg-Phe-NH2 (FMRFamide) was isolated by screening a genomic library with a fragment of an Aplysia Phe-Met-Arg-Phe-NH2 cDNA and with synthetic oligonucleotides. This gene was used to isolate a cDNA from a Drosophila adult head cDNA library. The cDNA was defined by sequence analysis to encode 13 peptides that have Phe-Met-Arg-Phe-NH2 or related sequences at their carboxyl termini. Other putative neuropeptides, including one that has homology to mammalian corticotropin-releasing factor, are present in the deduced approximately equal to 39-kDa precursor. Southern blot analysis suggested the presence of a single Phe-Met-Arg-Phe-NH2-like gene within the haploid genome. RNA blot analysis indicated the expression of at least two transcripts of approximately equal to 1.7 and approximately equal to 0.7 kilobases. Both transcripts are evident throughout larval, pupal, and adult developmental stages. In situ hybridization was used to localize this neuropeptide gene to band 46C on the right arm of the 2nd chromosome. These data provide the basis for utilizing the advanced genetics and molecular techniques of Drosophila to address complex aspects of neuropeptide expression and function.

Amino Acid Sequence↗
PubMed1977

Experimental diabetes reduces circulating 1,25-dihydroxyvitamin D in the rat.

Duodenal calcium absorption and a vitamin D-dependent duodenal calcium-binding protein are depressed in rats with alloxan- or streptozotocin-induced diabetes. To test for possible abnormal vitamin D metabolism in diabetes we measured serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in control, streptozotocin diabetic, and insulin-treated diabetic rats. The serum concentration of 1,25-dihydroxyvitamin D was depressed in untreated diabetic rats to one-eighth of the level in controls and was restored to control levels by insulin treatment. The serum concentration of 25-hydroxyvitamin D was the same in all three groups. Hence, effects of diabetes on duodenal calcium transport can be explained by reduced concentrations of 1,25-dihydroxyvitamin D resulting either from failure of renal 1alpha-hydroxylation of 25-hydroxyvitamin D or increased catabolism of 1,25-dihydroxyvitamin D.

Animals↗
PubMed1977

Renal calcium binding protein in the diabetic and vitamin D-depleted rat.

A calcium binding protein has been purified 220 fold from rat kidney. The molecular weight of this protein (26 000-28 000) is more than double that of the duodenal calcium binding protein of the rat. In response to the stimuli of both streptozotocin diabetes and depletion and repletion with vitamin D, changes in the renal protein are minimal. This contrasts markedly with responses of the duodenal protein to the same stimuli: (a) there was marked depression of duodenal calcium binding protein by vitamin D depletion and diabetes; (b) duodenal calcium binding protein was restored by vitamin D treatment of depleted rats. The renal protein appears to be identical with a previously described 28 000 molecular weight protein from the kidney purified by a different technique (Hermsdorf, C.L. and Bronner, F. (1975) Biochim. Biophys. Acta 379, 553-561). In contrast to findings of the current study, previous investigators were unable to isolate the protein from vitamin D-deficient rats and postulated vitamin D dependence. The protein activator of cyclic AMP phosphodiesterase is a calcium binding protein found in many tissues including kidney. Based on lack of response to stimuli we used and similarity in method of isolation and properties, our renal calcium binding protein may be this protein activator.

Animals↗
PubMed1977

Insulin-treatment of diabetic rats: effects on duodenal calcium absorption.

We tested the hypothesis that depressed duodenal calcium absorption in the streptozotocin diabetic rat is the consequence of diabetes rather than nephrotoxicity of the diabetogenic agent causing abnormal renal vitamin D metabolism. We treated streptozotocin diabetic rats with insulin and compared their duodenal calcium transport response with that of untreated diabetics and matched controls. Insulin treatment restored depressed calcium transport of diabetics to control levels in in vivo studies and significantly increased calcium transport in vitro. Previous studies showed that even in uncontrolled diabetes the mucosa retains the ability to respond to an end organ stimulus enhancing calcium transport: 1,25-dihydroxycholecalciferol corrects the defect, but vitamin D and 25-hydroxycholecalciferol are ineffective. Since 1,25-dihydroxycholecalciferol is synthesized in the kidney, these findings, in conjunction with the current study, are consistent with the association of experimental diabetes with a renal defect depressing synthesis of 1,25-dihydroxycholecalciferol. Since insulin treatment restores duodenal calcium transport, the renal defects is unlikely to be caused by streptozotocin nephrotoxicity.

Animals↗
PubMed1977

Effects of Solanum malacoxylon on duodenal calcium binding protein in the diabetic rat.

Duodenal calcium absorption and calcium binding protein (CaBP) are depressed in uncontrolled experimental (alloxan and streptozotocin) diabeties in the rat. Administration of an aqueous extract of the South American plant Solanum malacoxylon to diabetic rats restores duodenal calcium absorption to control levels. Since CaBP is thought to play a role in intestinal calcium transport, we isolated CaBP from duodenal mucosa of control, diabetic, and S. malacoxylon-treated diabetic rats. CaBP, whose concentration is about half normal in mucosal extracts from diabetic rats by treatment of diabetics with Smalacoxylon extracts. Hence these studies provide a further correlation between duodenal calcium absorption and levels of duodenal CaBP. In addition, a new purification procedure is described which produces a 17-fold increase in purity of CaBP above that attainable by our previously reported method.

Animals↗
PubMed1976

Effects of vitamin D and its metabolites on calcium transport in the diabetic rat.

We studied diabetic rats, 5 days after streptozotocin injection, and matched controls to determine whether depressed duodenal calcium absorption associated with uncontrolled diabetes in the rat would respond to vitamin D or its metabolites. At the appropriate time following the intravenous injection of 0.25 mug of either vitamin D3, 25-hydroxycholecalciferol (25-OHD3), 1,25-dihydroxycholecalciferol (1,25-OH)2D3), or 1alpha-hydroxycholecalciferol (1alpha-OHD3) to half of each diabetic and control group, calcium transport was evaluated using everted duodenal sacs with 0.4 mM40Ca and tracer 45Ca on both mucosal and serosal surfaces. All agents stimulated duodenal calcium absorption in controls. Diabetics responded only to 1,25-(OH)2D3, the metabolite that acts directly on the duodenum, and to its synthetic analog, 1alpha-OHD3. 1alpha-OHD3 is activated to 1,25-(OH)2D3 by 25-hydroxylation in the liver; 25-OHD3 must be 1alpha-hydroxylated in the kidney to be active. The stimulation of duodenal calcium absorption in diabetic rats by 1alpha-OHD3, but not by either vitamin D3 or 25-OHD3, is most consistent with a defect in vitamin D metabolism at the 1alpha-hydroxylation step in the kidney.

Animals↗
PubMed1975

Depressed duodenal calcium absorption in the diabetic rat: restoration by Solanum malacoxylon.

Duodenal calcium absorption is depressed in alloxan and streptozotocin diabetic rats taking normal amounts to dietary vitamin D. Depression of absorption appears to be at least in part the result of altered metabolism of vitamin D with failure to form 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), the vitamin D metabolite that acts directly on duodenum to stimulate calcium absorption. The South American plant Solanum malacoxylon causes extensive soft tissue calcification when ingested by cattle. An extract of this plant restores calcium absorption depressed by dietary strontium blockage of 1,25-(OH)2D3 formation in chicks. We gave an aqueous extract of S. malacoxylon to diabetic rats and restored duodenal calcium absorption to normal. These findings provide further evidence of the ability of a factor in the S. malacoxylon extract to mimic the actions of 1,25-(OH)2D3 on duodenal calcium transport and reinforce the hypothesis that abnormal vitamin D metabolism is an important determinant of depressed duodenal calcium absorption in diabetes.

Animals↗