Metabolic-associated fatty liver disease and hepatocellular carcinoma: a prospective study of characteristics and response to therapy.

BACKGROUND AND AIM: The rising incidence of hepatocellular carcinoma (HCC) in Australia is related to increasing rates of metabolic-associated fatty liver disease (MAFLD). This study aimed to prospectively characterize the metabolic profile, lifestyle, biometric features, and response to treatment of HCC patients in an Australian population. METHOD: Multicenter prospective cohort analysis of newly diagnosed HCC patients at six multidisciplinary team meetings over a 2-year period. RESULTS: Three hundred and thirteen (313) newly diagnosed HCC patients with MAFLD (n = 77), MAFLD plus other liver disease (n = 57) (the "mixed" group), and non-MAFLD (n = 179) were included in the study. Alcohol-associated liver disease (ALD) (43%) and MAFLD (43%) were the most common underlying liver diseases. MAFLD-HCC patients were older (73 years vs 67 years vs 63 years), more likely to be female (40% vs 14% vs 20%), less likely to have cirrhosis (69% vs 88% vs 85%), showed higher ECOG, and were less likely to be identified by screening (29% vs 53% vs 45%). Metabolic syndrome was more prevalent in the MAFLD and mixed groups. The severity of underlying liver disease and HCC characteristics were the same across groups. While the MAFLD population self-reported more sedentary lifestyles, reported dietary patterns were no different across the groups. Dyslipidemia was associated with tumor size, and those taking statins had a lower recurrence rate. CONCLUSION: Equal to ALD, MAFLD is now the most common underlying liver disease seen in HCC patients in Australia. Future HCC prevention screening and treatment strategies need to take this important group of patients into consideration.

Expression of genes involved in lipogenesis is not increased in patients with HCV genotype 3 in human liver.

Hepatitis C virus (HCV) infection is frequently associated with hepatic steatosis, particularly in patients with HCV genotype-3 (HCVGT3). It has variously been hypothesized, largely from in-vitro studies, to be the result of increased synthesis, decreased metabolism and export of triglycerides. We measured by real-time PCR the expression of genes involved in lipid metabolism [acetyl-Coenzyme A carboxylase alpha, apolipoprotein B (APOB), diacylglycerol O-acyltransferase 2, fatty acid-binding protein 1, fatty acid synthase, microsomal triglyceride transfer protein (MTTP), peroxisome proliferator-activated receptor alpha (PPARA), peroxisome proliferator-activated receptor gamma (PPARG), protein kinase AMP-activated alpha 1 catalytic subunit (PRKAA1) and sterol regulatory element-binding transcription factor 1 (SREBF1)] in liver biopsies from patients infected with HCV genotype-1 (HCVGT1), HCVGT3 and Hepatitis B (HBV) using ß-glucuronidase (GUSB) and splicing factor arginine/serine-rich 4 (SFRS4) as housekeeping genes. Patients infected with HCVGT3 were younger than those infected with HCVGT1 (36.3 ± 2.5 vs 45.6 ± 1.5, P < 0.05, Mann-Whitney) and were more likely to have steatosis (69.2%vs 11.8%). No significant difference was found in the expression of genes involved in lipogenesis or transport in patients infected with HBV or HCV of either genotype. Contrary to expectation, given the greater degree of steatosis in HCVGT3-infected liver, expression of enzymes involved in lipogenesis was not elevated in HCVGT3 compared with HCVGT1 or HBV-infected liver. Significantly less mRNA for SREBF1 was found in HCVGT3-infected liver tissue compared with HCVGT1-infected liver (1.00 ± 0.06 vs 0.70 ± 0.15 P < 0.05). These results suggest that steatosis in patients infected with HCVGT3 is not the result of a sustained SREBF1 driven increase in expression of genes involved in lipogenesis. In addition, a significant genotype-independent correlation was found between the expression of APOB, MTTP, PRKAA1 and PPARA, indicating that these networks are functional in HCV-infected liver.

Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion.

Adhesion of keratinocytes in a wound outgrowth to laminin 5 in the basement membrane via integrins alpha6beta4 and alpha3beta1 is distinct from adhesion to dermal collagen via alpha2beta1 or to fibronectin via alpha5beta1. Leading cells in the outgrowth are distinguished from following keratinocytes by deposition of laminin 5, failure to communicate via gap junctions and sensitivity to toxin B, an inhibitor of RhoGTPase. Laminin 5 deposited by leading keratinocytes onto dermal collagen dominates over dermal ligands and changes the cell signals required for adhesion from collagen-dependent to laminin-5-dependent. Thus, deposition of laminin 5 can instruct keratinocytes to switch from an activated phenotype to a quiescent and integrated epithelial phenotype.

Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody-drug conjugate SGN-75.

BACKGROUND: CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody-drug conjugate, SGN-75. RESULTS: We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo. CONCLUSION: Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

Separation of physical loading from photosynthesis/respiration processes in rivers by mass balance.

Diurnal variations in physical and chemical concentrations, including nutrients, are observed in river ecosystems. Understanding these cycles and separating the effects of physical loading (from point and non-point sources) and biogeochemical processes are necessary for water management and the TMDL process. A chloride mass balance approach is used to separate the relative influences of physical loading and biogeochemical processes in the Bow River through Calgary, Canada, which has a significant influence on the river water chemistry. Sampling campaigns were conducted in December 2005, when minimal photosynthesis and respiration occur, and in July 2006, when river discharge is high and some photosynthesis and respiration activity is present. Samples in each campaign were collected at point source input and output along the river reach through the city every hour for a 24-hour period, allowing for time of travel. The two wastewater treatment facilities within the city contributed the majority of physical mass loading to the river, with temporal variations in effluent discharge, chloride, and nutrient concentrations. Wastewater effluent chloride to nutrient (as well as other parameter relationships) concentrations also varied diurnally. An hourly chloride mass balance was achieved, within 0.5% (average, S.D.=4.4) for December and 7.7% (average, S.D.=4.2) for July, between estimated cumulative sum values from all inputs and measured values at the river output downgradient of the city, allowing for the investigation of other parameter conservativeness. Some slight diurnal variations associated with photosynthesis and respiration were observed even with limited productivity in the river. Nitrate mass fluxes appeared to be most strongly influenced by photosynthesis and respiration processes, with phosphate being less influenced. Ammonia mass fluxes appeared to be most strongly influenced by wastewater effluent loading. Physical loading can mask or enhance biogeochemical diurnal fluctuations, creating errors in river process interpretations. Chloride was a useful tracer in the mass balance to distinguish between and assist in separating physical loading and biogeochemical processes in the river.

Junctional epidermolysis bullosis: defects in expression of epiligrin/nicein/kalinin and integrin beta 4 that inhibit hemidesmosome formation.

Junctional epidermolysis bullosis (JEB) is a heterogeneous inherited blistering disorder of human epithelial basement membranes (BMs). Characteristically, the epidermis detaches from the BM between the basal cells and the lamina lucida due to reduced numbers of hemidesmosomes (HDs). Attempts to identify a candidate gene for JEB led to the characterization of nicein, a protein complex in normal BMs that is absent from BMs of patients with JEB gravis. In independent research, two related BM glycoproteins, epiligrin and kalinin, were identified as functional adhesion components of HDs. Epiligrin was characterized as a BM ligand for basal cell adhesion via integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4 in HDs. Kalinin was characterized as an adhesive ligand and a component of anchoring filaments. Recent antibody and sequence studies on epiligrin/nicein/kalinin have identified limited homologies with laminin. Ongoing studies in multiple laboratories seek to identify mutations in one or more of the three subunits of epiligrin that are causal in JEB gravis. Consistent with the genetic heterogeneity of JEB, we have identified a patient with a variant form of JEB that is associated with pyloric atresia. This patient has negligible HDs, normal epiligrin, but reduced expression of integrin beta 4. A defect in the beta 4 expression may define a subset of JEB cases that also present with pyloric atresia. These results testify to the dual requirements for epiligrin in the BM and integrin beta 4 in the plasma membrane in regulating function of HDs in epithelium.

The functions of laminins: lessons from in vivo studies.

This series of three short reviews is an attempt to summarize our current knowledge of the in vivo tests of hypotheses of laminin functions. The structures of the laminins have been thoroughly reviewed recently (P. Ekblom and R. Timpl, in press), and I will not attempt to repeat this information here. Instead, I will focus on the recent evidence gathered from gene knock out experiments in mice and from naturally occurring human and mouse gene mutations. The most obvious lesson from the above studies--other than demonstrating the importance of laminins in general--is that the structural diversity of the laminin family members makes highly specialized functions possible. While all laminins may share many functional properties, the individual chains are involved in interactions which cannot be substituted for by other laminins or by other basement membrane components. While this concept is not new, it is very satisfying to see its validity so dramatically confirmed. It is therefore predictable that additional gene ablation experiments using other known and yet undescribed laminin genes will be equally interesting and informative. To me, one of the most striking lessons from these studies is how strongly the induced mouse mutations mimic human disease. With all the concerns with genetic background differences and species specific effects, manipulation of the laminin genes appears to be a particularly good first approach to identifying the causes of human disease. There is an abundant literature accumulated from biochemical and, more recently, molecular structural analyses, and from in vitro systems, suggesting a role of laminins contributing directly to the stability of the basement membrane. There is an equally vast literature supporting an indirect role in mediating cellular behavior, through interactions with various receptors. It is interesting that the in vivo studies summarized above support both activities. In the case of laminin 5 mutations, the phenotypic consequence appears to be due primarily to the loss of an important structural link between the epithelial cytokeratins and the dermal anchoring fibrils. The ultrastructure of the epithelium appears normal, as does the architecture of the papillary dermis. Only the anchoring complex itself is aberrant. The absence of laminin 5 appears not to compromise the development or viability of the epidermis. The basement membrane appears normal-other than the anchoring complex itself. The pathology observed in the newborn is believed to be due to the frictional trauma of birth, with the expectation that the function of the fetal skin is normal in utero. The Herlitz epidermolysis bullosa phenotype is obvious immediately at birth, and it does not progress postnatally beyond the extent to which the affected individual experiences additional frictional trauma or secondary consequences such as infection or fluid loss. Since laminin 5 is only one of a series of structural links within the anchoring complex, one would predict that a loss of any of these links would result in the same phenotype. Current evidence supports this view, as the absence of integrin alpha 6 beta 4 (Vidal et al., 1995; Dowling et al., 1996; Georges-Labouesse et al., 1996; van der Neut et al., 1996) or of collagen VII (A. M. Christiano and J. Uitto, in press) also results in dramatic neonatal dermal-epidermal fragility. The differences in phenotype, such as the pyloric atresia in the case of loss of integrin alpha 6 beta 4, are presumably due to additional functions of the integrin in other tissues or in other developmental processes. Therefore, the laminin 5 mutations may be unique, in that the in vivo studies suggest that the primary role of the molecule is in the elaboration and stability of the anchoring complex, but not in the basement membrane itself. Of course, since the in vivo phenotype reflects only losses that cannot be compensated, this interpretation may be much too narrow. (ABSTRACT TRUNCATED)

Anatomical localisation of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridisation histochemistry: novel identification in olfactory regions.

Atrial natriuretic peptide (ANP) is one of three structurally homologous natriuretic peptides present in heart and brain, which is thought to be involved in the regulation of water and salt intake, blood pressure, and hormone secretion. In the present study, the distribution of preproatrial natriuretic peptide (ppANP) mRNA in the central nervous system of the rat was examined by in situ hybridisation histochemistry by using [35S]-labelled oligonucleotides. Cells expressing ppANP mRNA were apparent in several major neuronal systems, being present in hypothalamic, limbic, pontine and forebrain olfactory regions. Relatively high densities of ppANP mRNA-positive neurones were found in the anterior medial preoptic hypothalamic nucleus, medial habenular nucleus, and in Barrington's nucleus in the pons. Moderate numbers of ppANP mRNA-positive cells were present in a number of amygdaloid nuclei, including the posterolateral and anterior cortical nuclei, in the zona incerta, and the pedunculopontine tegmental nucleus. Other areas, including the ventromedial hypothalamic nucleus and the laterodorsal tegmental nucleus, displayed only low densities of ppANP mRNA-positive neurones. A number of structures in which ppANP mRNA (or ANP-like immunoreactivity) has not previously been reported were found to contain moderate to high numbers of ppANP mRNA-positive neurones including several nuclei associated with the olfactory system, such as the anterior olfactory nucleus and neurones of the tenia tecta and ventrolateral orbital cortex. Although ppANP mRNA in CA1 pyramidal cells of the hippocampus has been described, we also detected labelling in CA2 and ventral CA3 regions of the hippocampus. Conversely, nuclei such as the bed nucleus of the stria terminalis and the nucleus of the solitary tract, which are reported to possess ANP-like immunoreactivity, were found not to contain ppANP mRNA. Overall, these results demonstrate the presence of ANP gene expression in discrete neuronal populations of the rat central nervous system and provide additional evidence to support a putative role for this peptide in regulating and integrating hypothalamic, olfactory, limbic, and neuroendocrine systems.

Localization of preprogalanin messenger RNA in rat brain: identification of transcripts in a subpopulation of cerebellar Purkinje cells.

Galanin, a 29 amino acid peptide, is widely distributed throughout both the peripheral and central nervous systems and is thought to be involved in multiple physiological functions including smooth muscle relaxation, stimulation of feeding, blood pressure regulation, control of hormone secretion and modulation of nociception. Galanin has been shown to co-exist with several neurotransmitters throughout the neuroaxis and in some cases to modify their presynaptic and postsynaptic actions. In the present study, the anatomical distribution of preprogalanin messenger RNA in rat brain was examined by in situ hybridization histochemistry using specific 35S-labelled oligonucleotide probes. Neurons expressing preprogalanin messenger RNA were found throughout the brain and were particularly abundant in the hypothalamus. High densities of preprogalanin messenger RNA-positive neurons were found in the anteroventral preoptic, supraoptic, paraventricular and dorsomedial nuclei of the hypothalamus, in the locus coeruleus and in the nucleus of the solitary tract. Moderate densities of preprogalanin messenger RNA-positive cells were apparent in the periventricular and arcuate nuclei of the hypothalamus, in the dorsal raphe and dorsal cochlear nuclei. Low densities of preprogalanin messenger RNA-expressing neurons were observed in the piriform cortex, medial septum and the retrochiasmatic area. These findings are consistent with results of previous in situ localization studies of preprogalanin messenger RNA and also with studies reporting the distribution of galanin-like immunoreactivity in rat brain. A novel finding, however, was the detection of preprogalanin messenger RNA in Purkinje cells in the caudal cerebellar vermis (lobules 6 to 10) and the flocculus and paraflocculus of the lateral hemispheres of the cerebellum. Galanin is presumably co-localized in these cells with GABA, which is normally present in Purkinje cells and possibly with tyrosine hydroxylase, which has recently been detected in a similar subpopulation of cerebellar Purkinje cells in both rat and mouse. Thus, the present study reveals a previously unreported site of galanin gene expression in the cerebellum which represents a novel, putative site of action for galanin to add to its already varied physiological roles.

Galanin messenger RNA during postnatal development of the rat brain: expression patterns in Purkinje cells differentiate anterior and posterior lobes of cerebellum.

Following our initial mapping of preprogalanin messenger RNA in adult brain and its presence in a subpopulation of cerebellar Purkinje neurons [Ryan M. C. and Gundlach A. C. (1996) Neuroscience 70, 709-728], the present study examined the ontogenic expression of preprogalanin messenger RNA in the postnatal rat brain focussing on the Purkinje cells of the cerebellar cortex. Using in situ hybridization histochemistry, preprogalanin messenger RNA was detected in the developing forebrain and hindbrain from postnatal day 4 to day 60 (adult). On postnatal day 4 very light hybridization signal (labelling) was observed in cells of a number of nuclei including the central amygdaloid nucleus, the medial preoptic area, paraventricular nucleus and dorsomedial hypothalamic nucleus of the forebrain while lightly-labelled cells were detected in neurons of the nucleus of the solitary tract and locus coeruleus of the hindbrain. Hybridization signal was not apparent in other nuclei until later, with positively-labelled neurons first apparent in the dorsal cochlear nucleus at postnatal day 21. The abundance of preprogalanin messenger RNA-positive neurons and the intensity of the hybridization signal increased, in most regions, until postnatal day 28 when labelling resembled that of the mature rat. Preprogalanin messenger RNA was first detected in the cerebellum on postnatal day 10 only in Purkinje cells of lobule 10 of the posterior vermis and increased in distribution throughout Purkinje cell layers of the entire cerebellar cortex by postnatal day 13. The intensity of hybridization signal in Purkinje cells varied between lobules, with Purkinje cells in lobule 10 displaying a moderate to heavy degree of labelling, while lobules 6-9 and the more posterior lobules of the hemisphere including crus 2 of the ansiform lobule, the paramedian lobule and the copula pyramis, displayed only light labelling. The intensity of labelling in the anterior vermis and the remaining lobules of the hemisphere including crus 1 of the ansiform lobule, the simple lobule, the paraflocculus and the flocculus, was homogeneously weak. By postnatal day 21, Purkinje cell labelling reached maximum intensity in all lobules. Regional differences were still apparent, however, with labelling in the posterior vermis and hemisphere ranging from moderate to heavy, with only light to moderate labelling detected in the anterior vermis. The intensity of labelling in the posterior vermis and most lobules of the hemisphere was similar from postnatal day 21 to adulthood, while, in the anterior vermis, crus 1 of the ansiform lobule and the simple lobule, the intensity of hybridization decreased slightly by postnatal day 28 and was completely absent in Purkinje cells of the adult rat. Differential expression of preprogalanin messenger RNA in Purkinje cells of the developing rat cerebellum and transient expression in certain lobules suggests that galanin gene products may have a role in both the developing and mature rat brain and that galanin gene expression may represent a useful marker for differentiating the anterior and posterior cerebellar lobes.

HPA axis response to a psychological stressor in generalised social phobia.

Social phobia may be associated with a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis. In this study we determined HPA axis responsivity to a psychological stressor in patients with social phobia and compared them to healthy controls. Fifteen patients with DSM IV social phobia with a mean score of 77.7 on the Liebowitz Social Anxiety Scale and 15 age and sex matched controls underwent the stressor consisting of mental arithmetic and a short term memory test performed in front of an audience. Plasma levels of cortisol and corticotropin were measured at various intervals throughout the test. Although baseline measures of cortisol did not differ between patients (319.8+/-34.6 nmol/l) and controls (279.5+/-42.7 nmol/l)(t=0.7, df=28, P<0.5) nor did baseline corticotropin values (8.6+/-2.1 pg/ml vs 13.7+/-2.0 pg/ml respectively) (t=-1.8, df=28, P<0.08) this stressor resulted in a significantly greater delta max cortisol response (the difference between baseline values and the maximum increase during the stressor) in patients (167.1+/-23.7 nmol/l) than in controls (106.7+/-16 nmol/l) (t=2.1, df=28, P<0.04). There was no significant difference in delta max corticotropin between groups (patients 8.8+/-2.1 pg/ml vs controls 9.1+/-1.9 pg/ml) (t=-0.08, df=28, P<0.9). This preliminary study indicates that patients with social phobia appear to have a hyper-responsive adrenocortical response to psychological stress.

Differential regulation of angiotensinogen and natriuretic peptide mRNAs in rat brain by osmotic stimulation: focus on anterior hypothalamus and supraoptic nucleus.

Central angiotensin II and natriuretic peptide systems have been shown to be involved in the central regulation of blood fluid homeostasis with alterations in central peptide and/or receptor levels observed following changes in osmotic status. The present study investigated the effects of sodium loading on mRNA encoding the angiotensin II precursor, angiotensinogen (AOGEN), and the natriuretic peptides, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) in rat brain using quantitative in situ hybridization histochemistry of [35S]- and [33P]-labeled oligonucleotide probes. Following 7 and 14 days of 2% sodium chloride in drinking water a significant increase was detected in preproAOGEN (ppAOGEN) mRNA in presumed astrocytes in regions of the anterior hypothalamus, including the periventricular nucleus, the medial preoptic area and medial preoptic nucleus, while a decrease was observed in astrocytes in the supraoptic nucleus. Other forebrain regions examined including the subfornical organ, bed nucleus of the stria terminalis and the arcuate nucleus showed no significant alteration in the level of ppAOGEN mRNA. Sodium loading did not appreciably alter ppANP or ppCNP mRNA levels in neurons of the anteromedial preoptic or arcuate nuclei or hippocampus at the times studied. PpANP mRNA levels were also unaltered in Barrington's nucleus following sodium loading, while preprocorticotropin-releasing hormone mRNA was significantly decreased. These results indicate that AOGEN mRNA transcription/stability in vivo is modulated by alterations in osmotic balance, consistent with previous reports of a central role for AII in cardiovascular and body fluid homeostasis. In contrast, despite reports of modulation of hypothalamic ANP-immunoreactivity following changes in osmotic status, it would appear that osmotic stimulation over periods of 7-14 days does not markedly alter the transcription or stability of hypothalamic natriuretic peptide mRNAs in vivo.

Angiotensinogen and natriuretic peptide mRNAs in rat brain: localization and differential regulation by adrenal steroids in hypothalamus.

Adrenal steroids have been shown to modulate angiotensin II and natriuretic peptide systems--peptide synthesis and metabolism--in vitro. In the present study the effects of adrenal steroids on mRNA encoding the angiotensin II precursor, angiotensinogen (AOGEN), and the natriuretic peptides, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) in the rat hypothalamus were investigated using quantitative in situ hybridization histochemistry of [35S]- and [33P]-labeled oligonucleotide probes. Adrenalectomy produced an apparent overall decrease in preproAOGEN (ppAOGEN) mRNA in presumed astrocytes in the anterior hypothalamus with significant decreases (ANOVA) measured in the medial preoptic area, the ventral region of the medical preoptic area, the paraventricular, suprachiasmatic, supraoptic, and periventricular nuclei. ppAOGEN mRNA levels were restored by both glucocorticoid (dexamethasone; 2 micrograms/ml in drinking water) and mineralocorticoid (aldosterone; 50 micrograms/kg, SC) replacement. Treatment of intact animals with dexamethasone (2 micrograms/ml in drinking water for 5 days) and aldosterone (100 micrograms/kg, SC, daily for 10 days) produced a significant increase in ppAOGEN mRNA in those hypothalamic regions affected by adrenalectomy. ppANP and ppCNP mRNA-positive neurons were successfully detected using [35S]- and [33P]-labeled probes, respectively, and were abundant in the anterior hypothalamus, particularly in the anteromedial preoptic nucleus of the medial preoptic area. In contrast to the effects on ppAOGEN mRNA, however, alterations in adrenal steroid levels did not significantly change ppANP or ppCNP mRNA levels in neurons of the anteromedial preoptic nucleus or in the arcuate nucleus. These results indicate that adrenal steroids modulate AOGEN gene transcription in vivo, consistent with previous reports of increased levels of ppAOGEN mRNA in a number of brain regions in response to acute dexamethasone treatment and reports of decreased AOGEN immunoreactivity in brain regions of adrenalectomized rats. In contrast, despite reports of modulation of hypothalamic ANP immunoreactivity following adrenalectomy and dexamethasone treatment, it would appear that adrenal steroids do not alter the transcription or stability of hypothalamic natriuretic peptides mRNA in vivo.

Heterocyclic amino alcohols related to ifenprodil as sigma receptor ligands: binding and conformational analyses.

The interaction of a novel series of heterocyclic amino alcohols with the sigma receptor site was assessed using radioligand binding and computerized molecular modelling. All heterocyclic amino alcohols, like the structurally related ifenprodil, fully inhibited the specific binding of [3H]R(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H]3-PPP) to rat cerebral cortical membranes. All compounds recognised two populations of binding sites labelled by [3H]3-PPP and the proportion of sites in the high affinity state was 60-80% of the total sites. Some of the heterocyclic amino alcohols also displayed similar affinity for alpha 1-adrenoceptors labelled by [3H]prazosin, where the pattern of inhibition appears to be stereospecific, unlike that seen with the binding of [3H]3-PPP. The amino alcohols had negligible affinity for sites labelled by the N-methyl-D-aspartate channel ligand, [3H]-(N-1-[thienyl]cyclohexyl)piperidine. Quantitative conformational analyses indicated that the heterocyclic amino alcohols and ifenprodil fitted well to a sigma receptor site model; low energy conformers could be superimposed like other potent sigma receptor ligands with confidence to the sigma receptor model. Our results define a new class of sigma receptor ligands and extend the understanding of the molecular requirements for drugs active at the sigma receptor.

Structure-activity relationships of competitive NMDA receptor antagonists.

The interaction of structurally constrained competitive NMDA receptor antagonists, (+/-)-cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), (2-amino-4,5-(1,2-cyclohexyl))-7-phosphonoheptanoic acid (NPC 12626), (+/-)-6-phosphonomethyl-de-cahydroisoquinoline-3-carboxylic acid (LY 274614), (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (SDZ EAB-515) and (S)-alpha-amino-5-phosphonomethyl[1,1':4',1"-terphenyl]-3-propa noi c acid (SDZ 215-439), with their receptor was assessed using radioligand binding, protection against neurotoxicity in cortical neuronal cultures and computerised molecular modelling. All compounds inhibited the specific binding of [3H]CGS 19755 and/or [3H]CGP 39653 (inhibition constants 40-2000 nM), and protected neuronal cultures from NMDA-mediated injury (IC50 values 1.3-5.6 microM). Quantitative conformational analyses indicated that the molecules fitted well to a NMDA receptor model. Our results draw attention to a deep hydrophobic pocket, defined by the bi- and terphenyl containing antagonists (SDZ EAB-515, SDZ 215-439), which may influence potency and selectivity.

Ontogenic expression of natriuretic peptide mRNAs in postnatal rat brain: implications for development?

The central natriuretic peptide system is composed of at least three structurally homologous and uniquely distributed peptides and receptors which are thought to be involved in the central regulation of cardiovascular and autonomic function and more recently been shown to affect cellular growth and proliferation, processes pertinent to mammalian development. As such, following our initial mapping of preproatrial natriuretic peptide (ppANP) mRNA in adult brain [M.C. Ryan, A.L. Gundlach, Anatomical localization of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridization histochemistry: in olfactory regions, J. Comp. Neurol., 356 (1995) 168-182], it was of interest to determine the ontogenic expression of natriuretic peptide mRNAs in the developing rat brain. Using in situ hybridization histochemistry of specific [35S]- or [33P]-labeled oligonucleotides, ppANP and preproC-type natriuretic peptide (ppCNP) mRNAs were detected in the developing rat brain from postnatal day 4 to day 60 (adult). PpANP mRNA was observed in many hindbrain, but only some forebrain, regions at postnatal day 4. Regional differences in the temporal expression of ppANP mRNA were apparent with ppANP mRNA detected in the medial preoptic area, mammillary nuclei and medial habenular nucleus at postnatal day 4 and in other areas including the arcuate and dorsomedial hypothalamic nuclei and in olfactory and limbic regions at postnatal day 10. A number of regions also exhibited transient expression of ppANP mRNA such as the bed nucleus of the stria terminalis and the medial cerebellar nucleus. In contrast, ppCNP mRNA was detected at relatively high levels in several regions on postnatal day 4 including olfactory nuclei, the hippocampus and particularly the pontine nucleus. The level of expression appeared to increase markedly in most regions including forebrain olfactory and hippocampal areas and in brainstem regions including the pontine nucleus, the parvocellular and lateral reticular and spinal trigeminal nuclei by postnatal days 10 and 13, but decreased from this peak to equivalent to adult levels by postnatal day 28. The differential and transient expression of the natriuretic peptides during postnatal development, together with previous reports of the ontogenic regulation of natriuretic peptide receptor expression and binding patterns, further suggests their involvement in developmental processes in the rat CNS and provides information relevant to the likely functional development of natriuretic peptide-utilizing pathways.

Bacterial water quality in the personal water bottles of elementary students.

BACKGROUND: Samples of drinking water were collected directly from the personal water bottles of students at an elementary school in Calgary, Alberta. METHODS: Total and fecal coliforms and heterotrophic bacteria were enumerated using membrane filtration and agar plate count methods respectively. RESULTS: The Canadian Drinking Water Quality Guidelines (CWQG) criterion was exceeded for total coliform in 13.3% of 75 samples. Fecal coliform and total heterotrophic criteria were exceeded in 8.9% (of 68 samples) and 64.4% (of 76 samples) respectively. FINDINGS: The use of personal water bottles for students in elementary classrooms is not recommended.

Contrasting nitrate adsorption in Andisols of two coffee plantations in Costa Rica.

Fertilizer use in coffee plantations is a suspected cause of rising ground water nitrate concentrations in the ground water-dependent Central Valley of Costa Rica. Nitrate adsorption was evaluated beneath two coffee (Coffea arabica L.) plantations in the Central Valley. Previous work at one site had identified unsaturated zone nitrate retardation relative to a tritium tracer. Differences in nitrate adsorption were assessed in cores to 4 m depth in Andisols at this and one other plantation using differences in KCl- and water-extractable nitrate as an index. Significant adsorption was confirmed at the site of the previous tracer test, but not at the second site. Anion exchange capacity, X-ray diffraction data, extractable Al and Si, and soil pH in NaF corroborated that differences in adsorption characteristics were related to subtle differences in clay mineralogy. Soils at the site with significant nitrate adsorption showed an Al-rich allophane clay content compared with a more weathered, Si-rich allophane and halloysite clay mineral content at the site with negligible adsorption. At the site with significant nitrate adsorption, nitrate occupied less than 10% of the total anion adsorption capacity, suggesting that adsorption may provide long-term potential for mitigation or delay of nitrate leaching. Evaluation of nitrate sorption potential of soil at local and landscape scales would be useful in development of nitrogen management practices to reduce nitrate leaching to ground water.