Mapping of glycine distributions in gliomas.

BACKGROUND AND PURPOSE: Increased glycine concentration in the brain is associated with altered metabolism in cancer and can be detected by using in vivo MR spectroscopy. This has been proposed as a marker for grade IV gliomas; however, little is known about the potential significance and frequency of in vivo glycine observation. The purpose of this study was to examine the rate of occurrence and spatial distribution of glycine observation with respect to other MR imaging parameters. MATERIALS AND METHODS: Data from volumetric whole-brain MR spectroscopic imaging of 59 subjects with glioma were analyzed with glycine included in the spectral model. The associations of the signal amplitude and spatial distributions of glycine with findings from contrast-enhanced T1, perfusion, and diffusion MR imaging were then examined. RESULTS: Glycine was detected in 24% of all studies, though with a wide range of signal amplitude and extent of the spatial distributions. While more commonly seen in grade IV tumors (42% of studies), relatively large concentrations were also detected in grade II and III gliomas. Coanalysis with other metabolites indicated a strong association with choline and that glycine was frequently seen to be overlapping with, and adjacent to, areas of high lactate concentration. Increased glycine was always associated with contrast enhancement and areas of increased cerebral blood flow, but without any clear association with other image parameters. CONCLUSIONS: Detection of increased glycine in gliomas appears to identify a subgroup of tumors and areas of increased proliferation.

Internalization of cloned pancreatic polypeptide receptors is accelerated by all types of Y4 agonists.

Internalization of cloned rat or human Y4 receptors expressed in Chinese hamster ovary (CHO) cells increased with concentration of all types of Y4 agonists, including human and rat pancreatic polypeptides, the Y1 receptor group co-agonists possessing C-terminal TRPRY.NH2 pentapeptide, and a C-terminally amidated dimeric nonapeptide related to neuropeptide Y, GR231118. These peptides also inhibited forskolin-stimulated adenylyl cyclase activity in Y4 receptor-expressing cells, and stimulated the binding of 35S-labeled GTP-gamma-S to pertussis toxin-sensitive G-proteins in particulates from these cells. Peptide VD-11 (differing from GR231118 only by C-terminal oxymethylation) acted as a competitive antagonist in all of the above processes. Agonist-induced stimulation of the Y4 receptor internalization persisted in the presence of allosteric inhibitors of hPP binding, N5-substituted amilorides, which also were relatively little active in G-protein stimulation and cyclase inhibition by Y4 agonists. Acceleration of Y4 receptor internalization by agonists apparently is related to relaxation of allosteric constraints to ligand attachment and sequestration of the receptor-ligand complex.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Regulation of corticotropin-releasing hormone in vitro.

Studies examining regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Most in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Furthermore, clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. CRH levels in the amygdala in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated the signal transduction mechanisms responsible for CRH expression. These mechanistic studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Clearly much more investigative work in better understanding CRH regulation in vitro is needed.

Glucagon-like peptide-2 stimulates gut mucosal growth and immune response in burned rats.

Major burn trauma often leads to reduced gut barrier function, immunosuppression, and increased bacterial translocation. We hypothesized that treatments that maintain normal gut after burn trauma will also reduce immunosuppression and bacterial translocation. Recent studies suggest that treatment with glucagon-like peptide-2 (GLP-2), which is synthesized in the intestine and released after food intake, elicits mucosal hyperplasia in the small intestine of rodents and prevents parenteral nutrition-induced gut hypoplasia. Therefore, we determined whether GLP-2 would prevent loss of gut integrity after major burn trauma. Osmotic minipumps were implanted into the peritoneum of 22 adult, male, Sprague-Dawley rats to infuse saline (10 microl/hr; n = 14) or GLP-2 (1 microg/hr; n = 8). On the next day 8 saline-infused and 8 GLP-2-infused rats were subjected to a 25 sec duration 30% BSA open flame burn, with the remaining rats serving as sham-burn controls. Five days after burn, all rats were killed. Gut protein was assessed, and immunosuppression was estimated by the mitogenic response of cultured splenocytes to phytohemagglutinin, pokeweed, and concanavalin A. Bacterial translocation was determined by culturing the mesenteric lymph nodes. Although protein content was significantly decreased in the ileum of burned rats treated with saline, the burned rats treated with GLP-2 exhibited significant increases in protein levels in duodenum, jejunum. and ileum. Colon protein was not affected by GLP-2 infusion. Saline-treated burned rats also exhibited immunosuppression, as suggested by significantly decreased responses to each of the mitogens. Infusion of GLP-2 normalized the response by the burned rats to each of the mitogens. Lymph nodes taken from sham rats exhibited no colony forming units, whereas in both of the burn groups, 50% of the cultures were positive. However, more aggressive colonization may have occurred in the saline-infused burned rats as compared with the GLP-2-infused burned rats (81 +/- 63 vs 3 +/- 2 colony forming units). These results suggest that GLP-2 may stimulate gut mucosa and reduce immunosuppression in burned rats. However, there does not seem to be a statistically significant positive effect of GLP-2 on bacterial translocation. Thus, improving small intestine mucosa may increase immunity while being ineffective against bacterial translocation.

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells.

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

Crystallization and preliminary X-ray analysis of CTLA-4 (CD152) membrane-external domain.

CTLA-4 (CD152) is involved in T-lymphocyte co-stimulatory pathways modulating both humoral and cellular immune response. The membrane-external domain has been prepared and crystallized. The unit-cell parameters are a = b = 43, c = 143 A with the symmetry of space group P3(1)21 or its enantiomer and the crystals diffract to 2. 7 A resolution at synchrotron beamlines.

CL1-GFP: an androgen independent metastatic tumor model for prostate cancer.

PURPOSE: The mechanisms responsible for tumor progression to androgen independence in prostate cancer (CaP) remain unknown. To characterize these changes and provide a basis for rational therapeutic strategies for advanced CaP, an in vivo model from a highly aggressive androgen independent CaP cell line with distinct cellular and molecular properties was developed. MATERIALS AND METHODS: An aggressive androgen-independent cell line designated CL1 was derived from a slow-growing, and androgen-dependent, parental LNCaP cell line through in-vitro androgen-deprivation and selection. CL1 was stably transfected with a green fluorescence protein gene (CL1-GFP) and orthotopically injected into SCID mice. The pathologic behavior, histology, and molecular determinants of CL1 tumor and metastases were determined and characterized by standard light and fluorescent microscopy, and quantitative RT-PCR analysis. RESULTS: CL1 is an anaplastic prostate cancer cell line which demonstrates extensive local invasion and metastases to various organs that can be visualized via GFP expression. When compared with parental LNCaP cells, RT-PCR analysis of the tumor revealed an over-expression of EGFR, b-FGF, VEGF, TGF-beta, IL-8, IL-6, and bcl-2 and a down regulated expression of the p53, E-cadherin and PTEN. In contrast to LNCaP cells, CL1 tumors express lower levels of androgen receptor and barely detectable PSA mRNA. CONCLUSIONS: CL1-GFP represents an aggressive androgen-independent CaP tumor model derived through androgen deprivation whose pathologic development and molecular properties in animals resembles the clinical characteristics of hormone refractory prostate cancer (HRPC). Metastatic sites of CL1-GFP can be visualized with fluorescence microscopy offering a unique therapeutic model for the evaluation of drug sensitivity and other therapeutic modalities.

[D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake.

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.

Maintaining gut integrity during parenteral nutrition of tumor-bearing rats: effects of glucagon-like peptide 2.

Maintaining tumor-bearing rats on total parenteral nutrition (TPN) for eight days significantly reduced mass, protein, and DNA in small intestine and colon. Coinfusion of glucagon-like peptide 2 (GLP-2) significantly increased each of these variables in the duodenum, jejunum, and ileum, but not in the colon. Histological analysis of tissue revealed normal mucosa thickness and villus height in the small intestine of GLP-2-treated rats, whereas non-treated rats maintained on TPN exhibited villus shortening and thinning of the mucosa. Compared with TPN alone, no significant effects of GLP-2 were noted on tumor growth, liver weight, or heart weight. Coinfusion of GLP-2 with TPN had no significant effect on TPN-associated immunosuppression, as measured by mitogen-induced proliferation of cultured splenocytes. Although translocation of bacteria to the mesenteric lymph nodes appeared to be reduced in GLP-2-treated rats, the difference between groups was not statistically significant. These results suggest that hormonal alterations may be more important than an absence of luminal nutrition in TPN-associated mucosa changes in tumor-bearing rats. Additionally, maintenance of gut integrity during TPN does not appear to be a sufficient condition for the avoidance of the negative sequelae associated with this route of supplemental nutrition.

Coordinate and divergent regulation of corticotropin-releasing factor (CRF) and CRF-binding protein expression in an immortalized amygdalar neuronal cell line.

CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.

Regulation of corticotropin-releasing factor messenger RNA by nicotine in an immortalized amygdalar cell line.

Preliminary data suggest that amygdalar corticotropin-releasing factor (CRF) is regulated by nicotinic agonists. We sought to confirm and extend these observations by determining the effects of various concentrations of nicotine on CRF messenger RNA expression in the AR-5 immortalized amygdalar cell line. Nicotine produced concentration- and time-dependent increases in CRF mRNA. This cell line thus confirms that nicotinic agonists stimulate amygdalar CRF and appears to be a useful model for studying molecular factors important in this interaction.

Sarcoidosis presenting as multilobular limbal corneal nodules.

PURPOSE: To review reported external ocular manifestations of sarcoidosis and to present bilateral, multilobular, nodular, limbal, corneal nodules as being a unique manifestation of the disease. PATIENTS AND METHODS: A 16-year-old Saudi girl presented with bilateral, multilobular, solid, limbal nodules, with a vascular supply from the conjunctival vessel, and associated membraneous conjunctivitis and healed trachoma. The Schirmer's test revealed less than 2 mm in both eyes with tear meniscus less than 2 mm. Biopsy of an associated palpebral conjunctival nodule was performed, in addition to a gallium scan, chest X-ray, and a serum angiotensin-converting enzyme (SACE) level. RESULTS: The culture showed beta-hemolytic streptococci. Gallium scan showed intake by both lacrimal glands. Her chest X-ray results were normal, as was the SACE level. Biopsy of the excised conjunctival nodule disclosed a noncaseating granulomatous reaction with epithelioid and giant cells, and chronic inflammatory cell infiltrate confirming a diagnosis of sarcoidosis. CONCLUSION: A multilobular, nodular, perilimbal mass as a unique manifestation of sarcoidosis is presented. A streptococcal membraneous conjunctivitis and healed trachoma superimposed. A review of sarcoidosis of the external eye is included.

NPY upregulates genes containing cyclic AMP response element in human neuroblastoma cell lines bearing Y1 and Y2 receptors: involvement of CREB.

Four NPY receptor subtypes have been cloned, and shown to be coupled to both Ca2+ and cAMP. However, very little is known about the downstream elements mediating NPY actions. It has recently been demonstrated in our laboratory that intrahypothalamic (i.h.t.) administration of NPY induces hypothalamic CaM kinase activity, cyclic AMP response element binding protein (CREB) phosphorylation and cyclic AMP response element (CRE) binding activity in rat hypothalamic nuclear proteins. In the present study, we have investigated whether these changes in CRE binding transcriptional factors activated by NPY results in gene regulation using a human neuroblastoma cell line (SK-N-BE2). This cell line which expresses the Y2 subtype of NPY receptors was transfected with a fusion gene containing 1.305 kb of human CRF 5' flanking region with a perfect palindromic CRE site linked to firefly luciferase gene. NPY treatment increased CaM kinase II activity, CREB phosphorylation and CRE binding in these cells. In transfected cells, luciferase activity was also increased by NPY (1.8-4-fold) within 4 h of treatment. Moreover, forskolin (7-30-fold), which stimulates cAMP production, and thapsigargin (6-8-fold), which mobilizes intracellular calcium, also increased luciferase activity within 4 h of treatment. PMA (phorbol-12-myristate-13-acetate), an activator of protein kinase-C, induced luciferase activity by 1.8-fold. NPY augmented forskolin-stimulated luciferase activity from 11- to 15-fold, but had no significant effect on thapsigargin-induced luciferase activity. These findings suggest that activation of protein kinase A (PKA) or CaM kinase leads to the induction of fusion gene. NPY treatment upregulated fusion gene expression through Ca2+ pathway in SK-N-BE2 cell line. Pretreatment with CREB antisense, but not the sense oligodeoxynucleotides, inhibited forskolin-, thapsigargin- and NPY-stimulated luciferase activity. However, CREB sense or antisense oligodeoxynucleotide treatment had no effect on PMA-stimulated luciferase activity. Furthermore, NPY induced CRE binding activity and the expression of CRE containing Y1 receptor gene in SK-N-MC cell line. These findings suggest that NPY can upregulate CRE containing reporter gene including Y1 receptor gene and NPY-induced reporter gene regulation in SK-N-BE2 cells is mediated by intracellular Ca2+ and CREB protein.

NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats.

Previous investigations suggest that neuropeptide Y (NPY) feeding mechanisms and corticotropin releasing factor (CRF) are altered in anorectic tumor-bearing (TB) rats. To better determine the relationship of NPY and CRF synthesis to cancer anorexia we measured mRNA for these peptides in medial and lateral hypothalamus of TB and control rats. NPY and CRF mRNA were reliably detected by Northern blot analysis only in medial hypothalamus, where NPY message was elevated significantly in TB rats. CRF mRNA tended to be reduced in both pair-fed (PF) and TB rats, but did not reach statistical significance. Concentrations of NPY or CRF were not altered significantly in either the lateral or medial hypothalamus of TB or PF rats. These results suggest that the transcription of NPY is elevated in PF rats and is increased further in anorectic TB rats. The lack of significant increases in levels of peptides may be related to dilution, due to measuring a relatively large block of hypothalamic tissue. Alternatively, translation of the signal for NPY production may be inhibited, or degradation of peptide levels may be increased.

Reciprocal changes in hypothalamic receptor binding and circulating leptin in anorectic tumor-bearing rats.

Although reduced biological activity of the obese gene product, leptin, has been associated with obesity, little information is available concerning leptin alterations during anorexia. Therefore, we measured circulating leptin concentrations and hypothalamic leptin binding in anorectic tumor-bearing and pair-fed control rats. Plasma concentrations of leptin decreased in tumor-bearing rats early in the course of tumor growth, and fell to nearly non-detectable levels during severe anorexia. The pair-fed control rats that ate the same amount of food as did the anorectic tumor-bearing rats exhibited a 50% decrease in plasma leptin concentration. Concentrations of free fatty acids were elevated in both tumor-bearing and pair-fed groups, while circulating levels of triglycerides were increased only in anorectic tumor-bearing rats. Leptin receptor density was doubled in the hypothalamus of tumor bearing rats, while binding affinity was decreased by 50%. These results suggest that peripheral leptin production is down-regulated, perhaps due to increased lipolysis in tumor-bearing rats. It appears that hypothalamic leptin systems up-regulate receptor numbers in response to decreased blood leptin level, however, the decrease in binding affinity may compensate for these alterations. Therefore, the influence of leptin on hypothalamic neuropeptide Y feeding systems may be minimal in anorectic tumor-bearing rats.

Neuropeptide Y treatment and food deprivation increase cyclic AMP response element-binding in rat hypothalamus.

Intrahypothalamic (IHT) administration of neuropeptide Y (NPY) induces a robust feeding response in rats. We have shown previously that NPY-induced feeding is mediated by a pertussis-toxin-sensitive G protein in rats. NPY receptors are coupled to cAMP and Ca2+. Because these second messengers are known to activate cAMP response element binding proteins, (CREB), cAMP response element modulators, or activating transcription factor 1, we investigated the involvement of these transcription factors in NPY-induced feeding in rats. Compared with control injections of cerebrospinal fluid (1 microl), IHT administration of NPY increased cAMP response element (CRE) binding to rat hypothalamic nuclear extracts in a time-dependent manner, as detected by an electrophoretic mobility shift assay. In contrast, IHT administration of the anorectic neuropeptide, pituitary adenylate cyclase activating polypeptide, strongly inhibited the CRE binding. Food deprivation for 48 hr also increased CRE binding, whereas 8 hr of refeeding normalized CRE activity. Preincubation of the hypothalamic nuclear extracts of NPY-treated and unfed rats with antibody specific to CREB blocked CRE binding, whereas preincubation with phosphoCREB antibody retarded the migration of CRE-protein complex, indicating that phosphoCREB is involved in this process. Consistently, immunohistochemical studies with food-deprived rats showed an intense phosphoCREB signal in the paraventricular nuclei and ventromedial hypothalamus in comparison to rats fed ad libitum. Hypothalamic calcium/calmodulin-dependent protein kinase II activity was also increased by IHT-NPY. These results suggest that calcium/calmodulin-dependent protein kinase II induced phosphorylation of CREB may be involved in regulating feeding behavior induced by NPY.