Stephen Lentz | University of Michigan (original) (raw)

Papers by Stephen Lentz

Endocrinology, Jan 21, 2015

A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) ... more A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR) and (IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the non-canonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunoflourescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without ...

Endocrinology, 2015

A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) ... more A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR) and (IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the non-canonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunoflourescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα while it enhanced nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Further, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.

Advances in experimental medicine and biology, 1993

This chapter has attempted to describe and integrate some of the clinical and basic research that... more This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.

Advances in Experimental Medicine and Biology, 1993

Advances in Experimental Medicine and Biology, 1993

Neurochemistry International, 1996

GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopt... more GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. A quantitative in situ hybridization technique was used to study the expression of GTP cyclohydrolase I mRNA in the rat brain at the cellular level. Coronal sections between the diencephalon and myelencephalon were exposed to a 35S-labelled antisense GTP cyclohydrolase I cRNA probe. Sections serial to these were hybridized with a 35S-labelled antisense cRNA probe complementary to tyrosine hydroxylase mRNA. Tyrosine hydroxylase and GTP cyclohydrolase I mRNAs were found to colocalize within catecholamine neurons located throughout the brain. The overall distribution of neurons expressing GTP cyclohydrolase I mRNA was observed to correspond exactly to the known distribution of the dopamine, norepinephrine/epinephrine and serotonin-containing cell groups. Overall, a 30-fold range of GTP cyclohydrolase I mRNA expression was observed, with the transcript being significantly more abundant in serotonin than in dopamine or norepinephrine/epinephrine neurons. Comparisons across serotonin cell groups indicated that neurons of the median raphe nucleus, caudal linear nucleus raphe (B8) and the dorsal raphe (B6/B7) expressed the highest levels of GTP cyclohydrolase I mRNA. Comparisons across dopamine cell groups indicated that the transcript was more abundant in neurons of the ventral tegmental area (A10) than in neurons of the substanti~ nigra pars compacta (A9) and that both A9 and A10 dopamine neurons exhibited higher levels of expression than the DA neurons of the hypothalamus (A11-AM). Norepinephrine neurons of the locus coeruleus (A6) and subcoeruleus (A6v) exhibited significantly higher levels of GTP cyclohydrolase I mRNA than did neurons in other norepinephrine (A1 and A2) or epinephrine (C1 and C2) cell groups. GTP cyclohydrolase I mRNA could not be detected unequivocally in neurons known to contain nitric oxide synthase. Heterogeneity in the level of expression of GTP cyclohydrolase I mRNA by monoamine-containing neurons may play an important role in determining steady state levels of tetrahydrobiopterin and, ultimately, the regulation of monoamine biosynthesis.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 2, 2016

Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX),... more Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX), the final biosynthetic precursor to heme. Hepatic PP-IX accumulation may lead to end-stage liver disease. We tested the hypothesis that systemic administration of porphyrin precursors to zebrafish larvae results in protoporphyrin accumulation and a reproducible nongenetic porphyria model. Retro-orbital infusion of PP-IX or the iron chelator deferoxamine mesylate (DFO), with the first committed heme precursor α-aminolevulinic acid (ALA), generates high levels of PP-IX in zebrafish larvae. Exogenously infused or endogenously produced PP-IX accumulates preferentially in the liver of zebrafish larvae and peaks 1 to 3 d after infusion. Similar to patients with protoporphyria, PP-IX is excreted through the biliary system. Porphyrin accumulation in zebrafish liver causes multiorganelle protein aggregation as determined by mass spectrometry and immunoblotting. Endoplasmic reticulum stress and i...

Journal of Diabetes and its Complications, 2014

To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN... more To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN) status in patients with type 1 or type 2 diabetes mellitus (DM). In this cross-sectional study, we examined 25 diabetic patients without DN, 10 patients with mild DN, 8 patients with severe DN, and 9 controls without diabetes. DN status was assigned based on a combination of clinical symptoms, signs, and electrophysiological testing. Patients underwent corneal confocal microscopy (CCM) of the sub-basal nerve plexus. Post-hoc analysis of the CCM images was performed to quantify the average CNFL, and ANOVA was used to assess for differences in CNFL. All 25 subjects without DN had type 1 DM, and subjects with DN had type 2 DM. Participants with severe DN had significantly lower CNFL (12.5±6.1mm/mm(2)) compared to controls (20.7±2.2mm/mm(2)) (p=0.009). However, lower CNFL was also found in participants with type 1 DM who did not have DN (15.1±4.7mm/mm(2)) relative to controls (p=0.033). CCM of the sub-basal nerve plexus may be an indicator of early peripheral nerve degeneration in type 1 DM. Type of diabetes, in addition to degree of neuropathy, may influence the extent of corneal nerve damage.

The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently... more The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substanti...

Laminin trimers composed of a , b , and g chains are major components of basal laminae (BLs) thro... more Laminin trimers composed of a , b , and g chains are major components of basal laminae (BLs) throughout the body. To date, three a chains ( a 1-3) have been shown to assemble into at least seven het- erotrimers (called laminins 1-7). Genes encoding two additional a chains ( a 4 and a 5) have been cloned, but little

The Journal of Physiology, 2013

Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activ... more Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation.

The Journal of Cell Biology, 1997

Laminin trimers composed of ␣ , ␤ , and ␥ chains are major components of basal laminae (BLs) thro... more Laminin trimers composed of ␣ , ␤ , and ␥ chains are major components of basal laminae (BLs) throughout the body. To date, three ␣ chains ( ␣ 1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional ␣ chains ( ␣ 4 and ␣ 5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant ␣ 4 and ␣ 5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that ␣ 4 and ␣ 5 assemble into four novel laminin heterotrimers (laminins 8-11: ␣ 4 ␤ 1 ␥ 1, ␣ 4 ␤ 2 ␥ 1, ␣ 5 ␤ 1 ␥ 1, and ␣ 5 ␤ 2 ␥ 1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of ␣ 1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, ␣ 4 and ␣ 5 exhibited the broadest patterns of expression, while ex-pression of ␣ 1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the ␣ chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one ␣ chain, all ␣ chains were present in multiple BLs, and some BLs contained two or three ␣ chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different ␣ chains and two different ␤ chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five ␣ chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length ␣ 3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Gastroenterology, 2010

Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secre... more Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2 + i -dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)activation.

Gastroenterology, 2010

tumor angiogenesis through a regulation of BM-derived pro-angiogenic cells (BMPCs). Cyclopamine s... more tumor angiogenesis through a regulation of BM-derived pro-angiogenic cells (BMPCs). Cyclopamine significantly attenuated the homing of BM-derived cells into the tumor vasculature in human PDAC xenografts, suggesting that Hh signaling may play a role during migration and differentiation of BMPCs. The effect of the Hh ligand Shh in the tumor microenvironment was considered to be cell non-autonomous, and IGF-1 production in the tumor stroma played a key role during the process. In Vitro co-culture experiments demonstrated that KP-1N human PDAC cells induced Gli1 and IGF-1 in c-Kit+ BM cultured mononuclear cells utilized as BMPCs, and the induction was significantly attenuated either by cyclopamine or lentiviral shRNA targeting Smo. Tube formation assay with the mouse endothelial line MS-1 supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of the BMPCs, and the enhancement of the capillary morphogenesis was blocked by an anti-IGF-1 neutralizing antibody. This "paracrine" effect of Hh seems to be a late event during pancreatic tumorigenesis, as full length Gli2 expression in the neovasculature was detected within PDAC lesions in Pdx1-Cre;LSL-KrasG12D;p53lox/+ mice, but not in precursor PanIN lesions in Pdx1-Cre;LSL-KrasG12D mice. Collectively, Hh derived from cancer cells can have a profound effect on neovascularization through the regulation of BMderived cells during late stages of pancreatic tumorigenesis, and targeting Hh would be a novel therapeutic approach to inhibit tumor angiogenesis.

Developmental Biology, 2005

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebr... more Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with g-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Mqller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis. D

Biochemical and biophysical research communications, 2016

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating... more Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of di...

PloS one, 2015

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory ... more The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (...

Journal of Visualized Experiments, 2010

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential c... more Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

Endocrinology, Jan 21, 2015

A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) ... more A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR) and (IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the non-canonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunoflourescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without ...

Endocrinology, 2015

A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) ... more A relationship between the actions of thyrotropin (TSH) and insulin-like growth factor-1 (IGF-1) was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR) and (IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the non-canonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunoflourescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα while it enhanced nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Further, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.

Advances in experimental medicine and biology, 1993

This chapter has attempted to describe and integrate some of the clinical and basic research that... more This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.

Advances in Experimental Medicine and Biology, 1993

Advances in Experimental Medicine and Biology, 1993

Neurochemistry International, 1996

GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopt... more GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. A quantitative in situ hybridization technique was used to study the expression of GTP cyclohydrolase I mRNA in the rat brain at the cellular level. Coronal sections between the diencephalon and myelencephalon were exposed to a 35S-labelled antisense GTP cyclohydrolase I cRNA probe. Sections serial to these were hybridized with a 35S-labelled antisense cRNA probe complementary to tyrosine hydroxylase mRNA. Tyrosine hydroxylase and GTP cyclohydrolase I mRNAs were found to colocalize within catecholamine neurons located throughout the brain. The overall distribution of neurons expressing GTP cyclohydrolase I mRNA was observed to correspond exactly to the known distribution of the dopamine, norepinephrine/epinephrine and serotonin-containing cell groups. Overall, a 30-fold range of GTP cyclohydrolase I mRNA expression was observed, with the transcript being significantly more abundant in serotonin than in dopamine or norepinephrine/epinephrine neurons. Comparisons across serotonin cell groups indicated that neurons of the median raphe nucleus, caudal linear nucleus raphe (B8) and the dorsal raphe (B6/B7) expressed the highest levels of GTP cyclohydrolase I mRNA. Comparisons across dopamine cell groups indicated that the transcript was more abundant in neurons of the ventral tegmental area (A10) than in neurons of the substanti~ nigra pars compacta (A9) and that both A9 and A10 dopamine neurons exhibited higher levels of expression than the DA neurons of the hypothalamus (A11-AM). Norepinephrine neurons of the locus coeruleus (A6) and subcoeruleus (A6v) exhibited significantly higher levels of GTP cyclohydrolase I mRNA than did neurons in other norepinephrine (A1 and A2) or epinephrine (C1 and C2) cell groups. GTP cyclohydrolase I mRNA could not be detected unequivocally in neurons known to contain nitric oxide synthase. Heterogeneity in the level of expression of GTP cyclohydrolase I mRNA by monoamine-containing neurons may play an important role in determining steady state levels of tetrahydrobiopterin and, ultimately, the regulation of monoamine biosynthesis.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 2, 2016

Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX),... more Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX), the final biosynthetic precursor to heme. Hepatic PP-IX accumulation may lead to end-stage liver disease. We tested the hypothesis that systemic administration of porphyrin precursors to zebrafish larvae results in protoporphyrin accumulation and a reproducible nongenetic porphyria model. Retro-orbital infusion of PP-IX or the iron chelator deferoxamine mesylate (DFO), with the first committed heme precursor α-aminolevulinic acid (ALA), generates high levels of PP-IX in zebrafish larvae. Exogenously infused or endogenously produced PP-IX accumulates preferentially in the liver of zebrafish larvae and peaks 1 to 3 d after infusion. Similar to patients with protoporphyria, PP-IX is excreted through the biliary system. Porphyrin accumulation in zebrafish liver causes multiorganelle protein aggregation as determined by mass spectrometry and immunoblotting. Endoplasmic reticulum stress and i...

Journal of Diabetes and its Complications, 2014

To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN... more To examine the relationship between corneal nerve fiber length (CNFL) and diabetic neuropathy (DN) status in patients with type 1 or type 2 diabetes mellitus (DM). In this cross-sectional study, we examined 25 diabetic patients without DN, 10 patients with mild DN, 8 patients with severe DN, and 9 controls without diabetes. DN status was assigned based on a combination of clinical symptoms, signs, and electrophysiological testing. Patients underwent corneal confocal microscopy (CCM) of the sub-basal nerve plexus. Post-hoc analysis of the CCM images was performed to quantify the average CNFL, and ANOVA was used to assess for differences in CNFL. All 25 subjects without DN had type 1 DM, and subjects with DN had type 2 DM. Participants with severe DN had significantly lower CNFL (12.5±6.1mm/mm(2)) compared to controls (20.7±2.2mm/mm(2)) (p=0.009). However, lower CNFL was also found in participants with type 1 DM who did not have DN (15.1±4.7mm/mm(2)) relative to controls (p=0.033). CCM of the sub-basal nerve plexus may be an indicator of early peripheral nerve degeneration in type 1 DM. Type of diabetes, in addition to degree of neuropathy, may influence the extent of corneal nerve damage.

The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently... more The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substanti...

Laminin trimers composed of a , b , and g chains are major components of basal laminae (BLs) thro... more Laminin trimers composed of a , b , and g chains are major components of basal laminae (BLs) throughout the body. To date, three a chains ( a 1-3) have been shown to assemble into at least seven het- erotrimers (called laminins 1-7). Genes encoding two additional a chains ( a 4 and a 5) have been cloned, but little

The Journal of Physiology, 2013

Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activ... more Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation.

The Journal of Cell Biology, 1997

Laminin trimers composed of ␣ , ␤ , and ␥ chains are major components of basal laminae (BLs) thro... more Laminin trimers composed of ␣ , ␤ , and ␥ chains are major components of basal laminae (BLs) throughout the body. To date, three ␣ chains ( ␣ 1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional ␣ chains ( ␣ 4 and ␣ 5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant ␣ 4 and ␣ 5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that ␣ 4 and ␣ 5 assemble into four novel laminin heterotrimers (laminins 8-11: ␣ 4 ␤ 1 ␥ 1, ␣ 4 ␤ 2 ␥ 1, ␣ 5 ␤ 1 ␥ 1, and ␣ 5 ␤ 2 ␥ 1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of ␣ 1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, ␣ 4 and ␣ 5 exhibited the broadest patterns of expression, while ex-pression of ␣ 1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the ␣ chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one ␣ chain, all ␣ chains were present in multiple BLs, and some BLs contained two or three ␣ chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different ␣ chains and two different ␤ chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five ␣ chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length ␣ 3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Gastroenterology, 2010

Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secre... more Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2 + i -dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)activation.

Gastroenterology, 2010

tumor angiogenesis through a regulation of BM-derived pro-angiogenic cells (BMPCs). Cyclopamine s... more tumor angiogenesis through a regulation of BM-derived pro-angiogenic cells (BMPCs). Cyclopamine significantly attenuated the homing of BM-derived cells into the tumor vasculature in human PDAC xenografts, suggesting that Hh signaling may play a role during migration and differentiation of BMPCs. The effect of the Hh ligand Shh in the tumor microenvironment was considered to be cell non-autonomous, and IGF-1 production in the tumor stroma played a key role during the process. In Vitro co-culture experiments demonstrated that KP-1N human PDAC cells induced Gli1 and IGF-1 in c-Kit+ BM cultured mononuclear cells utilized as BMPCs, and the induction was significantly attenuated either by cyclopamine or lentiviral shRNA targeting Smo. Tube formation assay with the mouse endothelial line MS-1 supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of the BMPCs, and the enhancement of the capillary morphogenesis was blocked by an anti-IGF-1 neutralizing antibody. This "paracrine" effect of Hh seems to be a late event during pancreatic tumorigenesis, as full length Gli2 expression in the neovasculature was detected within PDAC lesions in Pdx1-Cre;LSL-KrasG12D;p53lox/+ mice, but not in precursor PanIN lesions in Pdx1-Cre;LSL-KrasG12D mice. Collectively, Hh derived from cancer cells can have a profound effect on neovascularization through the regulation of BMderived cells during late stages of pancreatic tumorigenesis, and targeting Hh would be a novel therapeutic approach to inhibit tumor angiogenesis.

Developmental Biology, 2005

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebr... more Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with g-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Mqller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis. D

Biochemical and biophysical research communications, 2016

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating... more Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of di...

PloS one, 2015

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory ... more The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (...

Journal of Visualized Experiments, 2010

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential c... more Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.