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K A Roth

Publications and source records attributed to K A Roth.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocyte and enteroendocrine cell populations of fetal transgenic mice.

The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations.

Animals↗
PubMed1991

Sexually dimorphic distribution of substance P in specific anterior pituitary cell populations.

Substance P (SP) immunoreactivity is detectable in the rat pituitary by RIA; however, immunolocalization has been difficult. We used a sensitive immunogold silver-enhancement staining technique to cytochemically locate SP in the gland. SP-immunoreactive (SP-ir) cells were seen in anterior pituitary (AP), and occasional SP-ir fibers and terminals were seen in both AP and posterior pituitary. Colocalization studies showed the vast majority of SP-ir cells in the male AP to be also immunoreactive for growth hormone (GH). These GH/SP-ir cells represent approximately 23% of the somatotroph population in the male. SP-ir cells did not colocalize with lactotrophs, gonadotrophs, or corticotrophs; however, rare thyroid-stimulating hormone/SP-ir cells were found in the male AP. Comparisons of pituitaries from males and females revealed that females have 70% fewer SP-ir cells and that only approximately 6% of the somatotrophs in the female express SP. This sexual dimorphism is diminished in 6-day ovariectomized rats because this treatment increases the GH/SP-ir cell population 3-fold. This result suggests that the previously reported estrogen-induced decrease in SP gene and peptide expression in the pituitary occurs, at least in part, in a subpopulation of somatotrophs. To test this hypothesis, distribution of SP-ir cells was examined in pituitaries from estrogen- and oil-treated ovariectomized rats. Estrogen reduced the percentage of somatotrophs with SP immunoreactivity by 70% compared with ovariectomized oil-treated controls, indicating that estrogen most likely regulates SP levels in the pituitary by acting on a subpopulation of somatotrophs to suppress SP expression. Estrogen does not appear to alter SP immunoreactivity that is detected in the additional population of SP cells that colocalize with thyroid-stimulating hormone. These SP-expressing thyrotrophs were seen 6-fold more frequently in the female than in the male pituitary, regardless of steroid status. These studies reveal that males have more total SP-ir cells in the AP than do females and that there is a sexually dimorphic pattern of SP distribution in the gland. Males have a higher percentage of SP-ir GH cells, whereas females have more SP-ir thyrotrophs than do males. Identification of independently regulated SP-ir somatotroph and thyrotroph populations provides a basis for investigating the roles of SP in autocrine or paracrine regulation of pituitary hormone secretion.

Animals↗
PubMed1991

Temporal and spatial patterns of transgene expression in aging adult mice provide insights about the origins, organization, and differentiation of the intestinal epithelium.

We have used liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to explore the temporal and spatial differentiation of intestinal epithelial cells in 1- to 12-month-old transgenic mice. The intact, endogenous L-FABP gene (Fabpl) was not expressed in the colon at any time. Young adult transgenic mice containing nucleotides -596 to +21 of the rat L-FABP gene linked to the hGH gene (minus its 5' nontranscribed domain) demonstrated inappropriate expression of hGH in enterocytes and many enteroendocrine cells of most proximal and mid-colonic crypts (glands). Rare patches of hGH-negative crypts were present. With increasing age, a wave of "extinction" of L-FABP (-596 to +21)/hGH expression occurred, first in the distal colon and then in successively more proximal regions, leaving by 10 months of age only rare hGH-positive multicrypt patches. At no time during this progressive silencing of transgene expression were crypts observed that contained a mixture of hGH-positive and -negative cells at a particular cell stratum. Young (5-7 weeks) mice containing a L-FABP (-4000 to +21)/hGH transgene also demonstrated inappropriate expression of the transgene in most proximal colonic crypts. However, the additional 3.3 kilobases of upstream sequence resulted in much more rapid extinction of reporter expression, leaving by 5 months of age only scattered single crypts with detectable levels of hGH. This age-related extinction of L-FABP/hGH expression did not involve enterocytes and enteroendocrine cells in the (proximal) small intestine. These results indicate that cis-acting elements outside of nucleotides -4000 to +21 are necessary to fully modulate suppression of colonic L-FABP expression. They also define fundamental changes in colonic epithelial cell populations during adult life. Our data suggest that (i) a single stem cell gives rise to all cells that populate a given colonic crypt, (ii) stem cells represented in several adjacent crypts may be derived from a common progenitor, and (iii) such a progenitor cell may repopulate colonic crypts with stem cells during adult life. Since each colonic crypt contains the amplified descendants of its stem cell, transgenes may be powerful tools for characterizing the spatial and biological features of gut stem cells and their progenitors during life.

Aging↗
PubMed1991

Epithelial cell differentiation in normal and transgenic mouse intestinal isografts.

Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Expression of a liver fatty acid binding protein/human decay-accelerating factor/HLA-B44 chimeric gene in transgenic mice.

The intestinal epithelium is characterized by the rapid and continuous renewal of its four principal cell types and by its ability to establish and maintain remarkably complex spatial differentiation along its crypt-to-villus and duodenal-to-colonic axes. We have previously used transgenic mice containing liver fatty acid binding protein/human growth hormone (L-FABP/hGH) fusion genes to analyze the molecular mechanisms responsible for encoding positional information in this epithelium. Because these studies could not distinguish whether cis-acting sequences in the L-FABP promoter or hGH structural gene were responsible for the observed cellular and regional patterns of transgene transcription in the gut, a second model fusion gene has now been constructed. It consists of nucleotides -596 to +21 of rat L-FABP linked to a cDNA encoding a chimeric protein, human decay-accelerating factor (DAF, minus the site of attachment of its COOH-terminal glycophospholipid anchor), coupled to the transmembrane (TM) and cytoplasmic domains of human HLA-B44. RNA blot hybridization and immunocytochemical analyses revealed that the cell-specific and region-specific expressions of DAF-TM and hGH in adult mice appear identical along both axes of the gut, indicating that cis-acting elements contained within the 5' nontranscribed region of the L-FABP gene rather than in the reporter are largely responsible for these observed patterns of transgene expression. Unlike pre-hGH, a prototypical secreted protein, DAF-TM is a membrane protein. The ability to direct its expression along the length of both axes of the gut provides an opportunity to analyze in vivo the sorting pathways of membrane-associated proteins in normal epithelial cells as a function of their location and differentiation. Light microscopic studies indicate that DAF-TM is targeted to the basolateral and apical surfaces of villus-associated enterocytes.

Amino Acid Sequence↗
PubMed1990

Liver fatty acid-binding protein: a marker for studying cellular differentiation in gut epithelial neoplasms.

Human liver fatty acid binding protein is a 127 residue cytoplasmic protein synthesized in liver and in the intestinal epithelium. Previous studies of normal and transgenic mice indicated that the liver fatty acid-binding protein gene is a sensitive marker of enterocytic differentiation. This study shows the use of immunohistochemical methods to examine liver fatty acid-binding protein gene expression in normal human colonic epithelium, colonic villoglandular adenomas, nonmucinous and mucinous adenocarcinomas, and several types of noncolonic epithelial neoplasms. Cells containing liver fatty acid-binding protein were found in normal colonic epithelium, in two thirds of colorectal villoglandular adenomas and nonmucinous adenocarcinomas, and in one third of mucinous adenocarcinomas but not in noncolonic, nonhepatic carcinomas. All liver fatty acid-binding protein-positive colonic adenomas and adenocarcinomas contained patches of immunoreactive cells distributed among histologically identical patches of cells without liver fatty acid-binding protein immunoreactivity. This "mosaicism" was also found in metastases from liver fatty acid-binding protein-positive colonic adenocarcinomas. Immunostaining of these liver fatty acid-binding protein-positive tissues for carcinoembryonic antigen did not show a mosaic cellular pattern in its expression. These data suggest that within a given neoplasm, differences exist in the differentiation programs of monoclonally-derived, malignant colonic epithelial cells and that liver fatty acid-binding protein is a useful marker for operationally defining these subpopulations. Liver fatty acid-binding protein is also a potentially useful diagnostic marker for colorectal and hepatic carcinomas.

Adenocarcinoma↗
PubMed1990

Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice.

The mammalian intestinal epithelium undergoes continuous and rapid renewal of its four principal terminally differentiated cell types. These cells arise from multipotent stem cells located at or near the base of the crypts of Lieberkühn. The differentiation process is precisely organized along two spatial dimensions (axes)--from the crypt to the villus tip and from the duodenum to the colon. The enteroendocrine cell population provides a sensitive marker of the intestine's topologic differentiation. At least 15 different regionally distributed subsets have been described based on their principal neuroendocrine products. We have used immunocytochemical methods to characterize the spatial relationships of the serotonin-, secretin-, and substance P-containing enteroendocrine cell subsets in normal adult C57BL/6J x LT/Sv mice as well as in transgenic littermates that contain rat liver fatty acid-binding protein-human growth hormone fusion genes. Our results reveal precise spatial interrelationships between these populations and suggest a differentiation pathway that may involve the sequential expression of substance P, serotonin, and secretin.

Animals↗
PubMed1990

Mapping enteroendocrine cell populations in transgenic mice reveals an unexpected degree of complexity in cellular differentiation within the gastrointestinal tract.

The gastrointestinal tract is lined with a monolayer of cells that undergo perpetual and rapid renewal. Four principal, terminally differentiated cell types populate the monolayer, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. This epithelium exhibits complex patterns of regional differentiation, both from crypt-to-villus and from duodenum-to-colon. The "liver" fatty acid binding protein (L-FABP) gene represents a useful model for analyzing the molecular basis for intestinal epithelial differentiation since it exhibits cell-specific, region-specific, as well as developmental stage specific expression. We have previously linked portions of the 5' nontranscribed domain of the rat L-FABP gene to the human growth hormone (hGH) gene and analyzed expression of the fusion gene in adult transgenic mice. High levels of hGH expression were noted in enterocytes as well as cells that histologically resembled enteroendocrine cells. In the present study, we have used immunocytochemical techniques to map the distribution of enteroendocrine cells in the normal adult mouse gut and to characterize those that synthesize L-FABP. In addition, L-FABP/hGH fusion genes were used to identify subsets of enteroendocrine cells based on their ability to support hGH synthesis in several different pedigrees of transgenic mice. The results reveal remarkable differences in transgene expression between, and within, enteroendocrine cell populations previously classified only on the basis of their neuroendocrine products. In some cases, these differences are related to the position occupied by cells along the duodenal-to-colonic and crypt-to-villus axes of the gut. Thus, transgenes appear to be sensitive tools for examining the cellular and regional differentiation of this class of intestinal epithelial cells.

Animals↗
PubMed1990

Substance-P is present in a subset of thyrotrophs in the human pituitary.

Substance-P immunoreactivity and tachykinin-like peptides are present in the pituitary gland of several mammalian species. In humans, however, the biochemical nature and cellular localization of pituitary substance-P has not been defined. We report here that substance-P-immunoreactive material is present in low concentrations in both the anterior and posterior lobes of the human pituitary gland. Gel chromatography and reverse phase high performance liquid chromatography indicate that the majority of the substance-P immunoreactivity in human pituitaries elutes as authentic substance-P and its oxidized derivative. Immunohistochemical studies showed substance-P-immunoreactive fibers and terminals in the posterior pituitary gland and occasional substance-P-immunoreactive cell bodies in the anterior lobe. The substance-P-immunoreactive cells were found to colocalize with a small subpopulation of TSH beta-immunoreactive cells (thyrotrophs). Substance-P immunoreactivity was also found in a pituitary microadenoma that contained numerous TSH beta-immunoreactive cells. These studies indicate that substance-P is present in the human pituitary gland, and they suggest a relationship between substance-P and thyroid function.

Adenoma↗
PubMed1990

Neuroaxonal dystrophy in aging human sympathetic ganglia.

Autonomic dysfunction is an increasingly recognized problem in aging animals and man. The pathologic changes that produce autonomic dysfunction in human aging are largely unknown; however, in experimental animal models specific pathologic changes have been found in selected sympathetic ganglia. To address whether similar neuropathologic changes occur in aging humans, the authors have examined paravertebral and prevertebral sympathetic ganglia from a series of 56 adult autopsied nondiabetic patients. They found significant, specific, age-related neuropathologic lesions in the prevertebral sympathetic superior mesenteric ganglia of autopsied patients. Markedly swollen dystrophic preterminal axons compressed or displaced the perikarya of principal sympathetic neurons. Ultrastructurally, these swollen presynaptic axons contained abundant disoriented neurofilaments surrounded by peripherally marginated dense core vesicles. Immunohistochemical studies demonstrated that dystrophic axons contained tyrosine hydroxylase and neuropeptide tyrosine (NPY)-like immunoreactivity but not other neuropeptides (VIP, substance P, gastrin-releasing peptide [GRP]/bombesin, met-enkephalin). Similar to the animal models of aging, lesions were much more frequent in the prevertebral superior mesenteric ganglia than in the paravertebral superior cervical ganglia. These studies demonstrate anatomic, peptidergic, and pathologic specificity in the aging human nervous system similar in many respects to that which the authors have described in experimental animal models. Neuroaxonal dystrophy in the sympathetic nervous system may underlie poorly understood alterations in clinical autonomic nervous system function that develop with age.

Adult↗
PubMed1989

Nonopiate active proenkephalin-derived peptides are secreted by T helper cells.

Recent investigations have shown that the neuroendocrine and immune systems profoundly affect each other. In part, these interactions occur via common chemical messengers and receptors. One possible shared chemical messenger is the opioid precursor preproenkephalin, for which high concentrations of messenger RNA are present in brain, adrenal, and activated T helper cells. Because the biologic action of most peptide messengers depends on the posttranslational processing of the precursor, we have examined T helper cell lines for the production of proenkephalin-derived peptides. These peptides were characterized by multiple radioimmunoassays, gel filtration chromatography, and opiate radioreceptor assays. We found that activated T helper cells secrete significant concentrations of high-molecular-weight, opiate-inactive peptides, which are distinct from the proenkephalin-derived peptides of the neuroendocrine system. These studies clearly indicate cell-specific processing of proenkephalin, and suggest that the T helper cell-secreted products may have nonopiate receptor-mediated actions.

Adrenal Glands↗
PubMed1989

Ultrastructural and immunohistochemical characterization of autonomic neuropathy in genetically diabetic Chinese hamsters.

Selected portions of the prevertebral and paravertebral sympathetic and vagal parasympathetic nervous systems have been examined in the genetically diabetic Chinese hamster, an experimental animal model of diabetic gastrointestinal disease. The prevertebral sympathetic superior mesenteric/celiac ganglia, which provide much of the sympathetic innervation of the alimentary tract, developed large numbers of markedly dilated axons, many of which had the ultrastructural features of neuroaxonal dystrophy. Dystrophic axons, many involving presynaptic axonal elements, were increased in frequency in the prevertebral superior mesenteric/celiac ganglia, but not in the paravertebral superior cervical sympathetic ganglia, of chronically diabetic hamsters in comparison with age-matched controls. Dystrophic axons contained substance P- and gastrin-releasing peptide (gastrin-releasing peptide/bombesin)-like staining but were not labeled by antisera directed against vasoactive intestinal peptide, dynorphin-B, somatostatin, leu- and met-enkephalin and neuropeptide tyrosine. Substance P and gastrin-releasing peptide/bombesin containing subpopulations of presynaptic elements in prevertebral sympathetic ganglia are thought to participate in local reflex control of bowel motility and lesions preferentially involving these elements may contribute to bowel dysfunction. Immunohistologic techniques failed to demonstrate dystrophic axons in the superior cervical ganglia. Although morphometric studies failed to show significant axon loss in the abdominal vagus of chronically diabetic Chinese hamsters, evidence of markedly diminished numbers of axons comprising each Schwann cell unit and regenerative collections of Schwann cell processes devoid of axons are consistent with the participation of parasympathetic elements in the pathogenesis of alimentary dysfunction in this model system. These results suggest that selective subpopulations of neuropeptide containing axons are vulnerable to the diabetic condition and that these abnormalities may lead to physiologic dysfunction.

Animals↗
PubMed1988

Enhancement of dopamine metabolism in rat brain frontal cortex: a common effect of chronically administered antipsychotic drugs.

Administration of 4 antipsychotic drugs, haloperidol, chlorpromazine, thioridazine and clozapine, for 21 days elicited increased dopamine metabolism in frontal cortex of rat brain. Only clozapine failed to decrease the apparent firing rate of dopamine neurons in the striatum, as indexed by [homovanillic acid]/[dopamine]. These data support the hypotheses that frontal cortex dopamine neurons may be a common site for antipsychotic action while decreased release of dopamine in the striatum may be associated with the development of extrapyramidal side effects.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1988

Differential susceptibility of prevertebral and paravertebral sympathetic ganglia to experimental injury.

To investigate the response of selected sympathetic ganglia to experimental injury, neonatal rat pups were treated with either 6-hydroxydopamine (6-OHDA), guanethidine, or antiserum to nerve growth factor (anti-NGF). When examined at one month of age, each of the treatments resulted in a significantly greater loss of neurons and tyrosine hydroxylase activity in paravertebral (superior cervical and stellate) versus prevertebral (superior mesenteric and celiac) sympathetic ganglia. Guanethidine treatment produced the largest differential in neuron loss and tyrosine hydroxylase activity between pre- and paravertebral ganglia. Histologically, the acute phase of guanethidine-induced injury in the superior cervical, paravertebral, ganglia was characterized by a prominent mononuclear cell infiltrate and extensive neuronal degeneration. Minimal histopathologic changes were seen in the superior mesenteric, prevertebral, ganglia of the same animals. Immunolocalization of tyrosine hydroxylase and neuropeptide Y (NPY) in guanethidine-treated animals showed a preferential loss of sympathetic innervation of the extramural mesenteric vasculature with relative sparing of the noradrenergic innervation of Auerbach's myenteric plexus. Differences in the susceptibility of sympathetic ganglia to various insults may underlie the selective and heterogeneous involvement of sympathetic ganglia in clinical and experimental situations.

Animals↗
PubMed1988

Effects of chronic experimental streptozotocin-induced diabetes on the noradrenergic and peptidergic innervation of the rat alimentary tract.

Immunohistologic localization of tyrosine hydroxylase (TOH), dopamine-beta-hydroxylase (DBH) and selected neuropeptides (vasoactive intestinal polypeptide, gastrin-releasing peptide (GRP)/bombesin, substance P, Leu-enkephalin, Met-enkephalin, dynorphin B, neuropeptide Y (NPY), somatostatin) was used to investigate the innervation of the small bowel in a rat model of diabetic autonomic neuropathy. Paravascular mesenteric nerves (extrinsic) and intramural nerves of chronically (12-18 month) diabetic rats were characterized by the presence of numerous, markedly swollen dystrophic axons which stained intensely for TOH and DBH. The peptidergic complement of axons, however, showed no evidence of comparable dystrophic axonopathy.

Animals↗
PubMed1988

Aberrant production and regulation of proopiomelanocortin-derived peptides in ectopic Cushing's syndrome.

A 64 year old woman with a pancreatic islet cell tumor developed Cushing's syndrome. Glucocorticoid secretion did not decrease after low or high dose dexamethasone administration, and the Cushing's syndrome was cured by removal of tumor tissue. Immunohistochemistry and radioimmunoassays revealed the presence of immunoreactive ACTH, beta-endorphin and alpha-MSH in the tumor cells. Gel-permeation chromatography confirmed that beta-endorphin was the predominant opioid peptide produced by the tumor. The tumor was shown to contain a single 1.2 kilobase RNA species which hybridized to a 32P human POMC-cDNA; this POMC RNA was identical in size to that isolated from a normal human pituitary. In dispersed monolayer culture, CRF failed to elicit ACTH release from the tumor cells, but dexamethasone caused a paradoxical increase in ACTH secretion in vitro. This study demonstrates that aberrant regulation of POMC synthesis and peptide processing can be seen in tumors which synthesize a POMC RNA identical in size to that made in the pituitary gland.

Adrenocorticotropic Hormone↗