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Objectives The aim of this study was to establish the accuracy of fetal gender assignment by sonography in the biometrical range of 18 to 29 mm of biparietal diameter (BPD). Methods Transvaginal and/or transabdominal sonography was used to detect the sagittal sign as a marker of fetal gender in 2593 fetuses with BPD between 18 and 29 mm. The results of sonographic examination were compared with the gender at birth or with karyotype obtained from amniotic fluid cells or chorionic villus sampling. Results Fetal gender assignment was feasible in 2374 of 2593 cases (91%). Of the 2188 fetuses with known fetal sex outcome, 1025 were males and 1157 were females, and 6 had genital anomalies. In fetuses without genital anomalies, an accuracy rate of 100% was achieved at a BPD of ≥24 mm. The results of the six cases with genital malformations were considered separately. Conclusion Sonography is a reliable method for the study of the morphological development of the external genitalia in fetuses ‘in vivo’; it is possible to assign fetal gender in 95 to 99% starting at a BPD of 20 mm and to achieve an accuracy rate of 99 to 100% from a BPD of 22 mm, but fetal sex assignment should not be undertaken below a BPD of 22 mm, and especially not in cases where fetal sexing affects pregnancy management. Copyright © 2004 John Wiley & Sons, Ltd.

Objectives We present a case of early prenatal diagnosis of recurrent 46,XY partial gonadal dysgenesis, by combining early genetic and sonographic evaluations. Methods The conceptus of a mother with a first child affected by 46,XY gonadal dysgenesis was sonographically evaluated at 21- and 23-mm BPD (12+2 and 12+6 LMP-based age) and the female genitalia were observed. Karyotype analyses was performed on amniotic fluid and it revealed a 46,XY complement without mosaicism. SRY was amplified by PCR for molecular analyses. Results We observed a discordance between female phenotype detected at 21 and 23 mm of biparietal diameter (12+2 and 12+6 LMP-based age) and male karyotype. In the child and the fetus, seminiferous cords were not recognisable, whereas rare Leydig cells and no germ cells could be identified. Internal and external genitalia were sexually ambiguous in the child and feminized in the fetus. Conclusion This is the first case of early prenatal diagnosis of recurrent 46,XY partial gonadal dysgenesis and it points to the importance of combining early analyses of genetic sex with sonography in the management of anomalies of sexual development, with particular regard to syndromes for which the risk of recurrence is little understood. Copyright © 2003 John Wiley & Sons, Ltd.

e23159 Background: ERCC1 (Excision Repair Cross-Complementation group 1) deficiency is the most frequent DNA repair defect in non-small cell lung cancers (NSCLC), making this enzyme an attractive therapeutic target for synthetic lethal (SL) approaches. Previous data support that DNA damage response is involved in regulation of metabolic homeostasis. We hypothesized that ERCC1 deficiency resulted in metabolic reprogramming of cancer cells that in turn generated specific vulnerabilities. Methods: High-throughput proteomic SILAC (stable isotope labeling by aminoacids in cell culture) and metabolomic (LC-MS, GC-MS, LC-QTOF) profiling were performed on a in-house generated NSCLC model of ERCC1 deficiency. Nicotinamide phosphoribosyltransferase (NAMPT) was selected as main hit and revalidated in low-throughput. Sensitivity to NAMPT inhibition was assessed in several NSCLC models. Potential SL interactions were investigated using functional DNA repair and metabolic assays, and transmission electronic microscopy (TEM). Results: We found marked metabolic rewiring in ERCC1-deficient populations, including decreased NAMPT and NAD+ levels and defects in the tricarboxylic acid cycle. These caused exquisite selective sensitivity to NAMPT inhibition in several in vitro and in vivoNSCLC models; these effects were reversed by the restoration of a functional ERCC1 isoform, establishing a causal link with the ERCC1 defective state. TEM, functional DNA repair and metabolic studies revealed structural and functional defects in the mitochondria of ERCC1-deficient cells, and allowed us to propose a model for this nuclear-mitochondrial SL link. A correlation between NAMPT expression and ERCC1 expression was observed in NSCLC patient samples. Conclusions: These findings open novel therapeutic opportunities that exploit a previously undescribed nuclear/mitochondrial SL link between ERCC1 deficiency and NAMPT inhibition in NSCLC. This highlights the potential for targeting DNA repair / metabolic crosstalk for cancer therapy, and support the evaluation of NAMPT inhibitors in ERCC1-deficient tumors.

Deletions of the distal short arm of chromosome 9 have been reported in association with gonadal dysgenesis and XY sex reversal. These findings suggest that this region harbors at least one gene that might be involved in sexual development. Recent studies narrowed the deletion interval to the subtelomeric region 9p24.3. This region contains three genes DMRT1, DMRT3, and DMRT2 characterized by a DM-domain that seems essential for sexual development in Nematoda and Arthropoda. We have investigated the possible role of this gene cluster in a fetus with ring chromosome 9 and male-to-female sex reversal. We could demonstrate by FISH analysis a very small deletion of the terminal short arm of chromosome 9 including the DM-domain gene cluster. This finding gives further evidence that hemiZygosity of this region can be the cause for male-to-female sex reversal.

Accumulating evidence suggests that haploinsufficiency of a dosage-sensitive gene(s) in human chromosome 9p24.3 is responsible for the failure of testicular development and feminisation in XY patients with monosomy for 9p. We have used molecular cytogenetic methods to characterise the sex-reversing 9p deletions in two XY females. Fluorescence in situ hybridisation (FISH) with YACs from the critical 9p region containing an evolutionarily conserved sex-determining gene, DMRT1, is a very fast and reliable assay for patient screening. Comparative YAC mapping on great ape and Old and New World monkey chromosomes demonstrated that the critical region was moved from an interstitial position on the ancestral primate chromosome to a very subtelomeric position in chimpanzee and humans by a pericentric inversion(s). Pathological 9p rearrangements may be the consequence of an evolutionary chromosome breakpoint in close proximity to the sex-reversal region.

Various three-dimensional configurations, such as polyester tissue and woven-nonwoven, hydrophilic polyester fabric, either collagen-coated or uncoated, were investigated as potential scaffold for hepatocyte culture, in view of their use in bioreactors for hybrid liver support systems. Attachment, morphology and ultrastructure of primary adult rat hepatocytes were evaluated, as well as urea production and ammonium detoxification during a 24h incubation period in serum-free tissue culture medium. As control, hepatocytes were also plated onto collagen-coated dextran microcarriers and on plastic petri dishes, either collagen-coated or uncoated. In all the three-dimensional cultures, hepatocytes appeared morphologically intact without any statistically significant difference in metabolic activity. Collagen-coating did not influence cell attachment to polyester substrates, whereas woven-nonwoven hydrophilic polyester fabric may offer some potential advantages as three-dimensional system for hepatocyte culture in hybrid liver support systems.

La medecine factuelle fait appel a des descriptions analytiques realisees selon des criteres scientifiques rigoureux. Idealement, on veut reperer chez le patient un signe pathognomonique quantifiable ou biomarqueur de la maladie et en suivre les variations sous traitement. Souvent, le biomarqueur necessite une analyse destructive du tissu vivant. Les techniques d’imagerie proposent un compromis entre la precision de l’information et l’innocuite de la methode. Idealement, l’image localise et quantifie un parametre precis sans modifier l’equilibre physiologique du sujet. L’interet de l’imagerie du petit animal est triple : – inventer, tester et optimiser des techniques d’imagerie transposables a l’homme ; – decrire l’histoire naturelle d’une pathologie par une approche non invasive, donc repetable au cours du temps ; – obtenir un biomarqueur non invasif de l’efficacite therapeutique. Comparee aux techniques in vitro, l’imagerie paye son caractere non invasif par un accroissement de la complexite et du cout des examens. En outre, une modalite d’imagerie ne donne acces qu’a quelques parametres biologiques a la fois, ce qui est souvent insuffisant. Il est donc important de valider, en amont de la clinique, les nouvelles approches multimodales d’imagerie chez les modeles animaux des pathologies humaines. Dans cet expose, nous presenterons a partir d’un exemple concret les recherches menees dans notre laboratoire en imagerie multimodale (IRM, imagerie optique et 18FDG-TEP) chez un modele murin de paragangliomes lies a une mutation du gene SDHB, et le transfert de l’une d’entre elles en clinique humaine.

Black and channel catfish (Ictalurus melas and Ictalurus punctatus) were gradually acclimated to 36°C. Ictalurus punctatus tolerated the higher temperature better than Ictalurus melas. An intravenous glucose tolerance test was performed on Ictalurus punctatus by intravenously injecting 25 mg/100 g body weight of d-glucose. Both in the control and in acclimated fish, the blood glucose peak was reached after 1 min, after 5 min it began to decrease and after 4 hr it was drastically reduced, reaching the starting level after 24 hr. There was no significant difference between the two groups of animals until the 24th hour, where the glucose level in blood was significantly higher (P < 0.05) in acclimated fish than in control animals. Immuno-histochemical and electron microscopy studies indicated the presence of insulin-positive beta cells with characteristic cytoplasmatic electron-dense granulations, without any apparently histomorphological difference between acclimated and control fish.

Inherited metabolic diseases are mostly due to enzyme deficiency in one of numerous metabolic pathways, leading to absence of a compound downstream from and the accumulation of a compound upstream from the deficient metabolite(s). Diseases of intoxication by proteins (aminoacidopathies, organic acidurias, urea cycle defects) and by sugars (galactosemia, fructosemia) usually do not give prenatal symptoms since mothers protect their fetuses from pathological metabolite accumulation. A well-known exception is hypoplasia of corpus callosum, as is sometimes observed in nonketotic hyperglycinemia and sulfite oxidase deficiency. Conversely, women with phenylketonuria "poison" their fetus if they are not treated (spontaneous abortions, intrauterine growth restriction [IUGR], cardiac malformations, and brain disease). Amino acid synthesis defects can lead to prenatal symptoms: microcephaly in serine deficiency (detectable by amino acid analysis in fetal cord blood), and brain malformations in glutamine synthetase deficiency. Impaired folate metabolism is involved in a large fraction of neurodevelopmental defects referred to as spina bifida, yet the underlying genetic component(s) are largely unknown. Energy metabolism diseases caused by defects in the synthesis or utilization of relevant metabolites lead to organ dysfunctions or malformations, but prenatal diagnosis is usually impossible unless genetic analysis can rely on a previously affected child in the family. A somewhat intermediate condition is defects of mitochondrial beta-oxidation of fatty acids, as they may sometimes be symptomatic prenatally (notably the HELLP syndrome or other presentations), and in this case, organic acid and acylcarnitine analysis in amniotic fluid can be informative in the absence of an index case. In contrast, complex molecule diseases commonly give prenatal symptoms that may permit the diagnosis even in the absence of index cases: hydrops fetalis and skeletal anomalies in lysosomal storage diseases, hydrops fetalis in congenital disorders of glycosylation (CDG) and transaldolase deficiency, brain malformations in O-glycosylation defects, brain malformations, kidney cysts and skeletal anomalies in peroxysomal diseases (Zellweger syndrome), syndactyly, genitalia malformations, and IUGR in Smith-Lemli-Opitz (SLO) syndrome. Although many metabolic disorders show biochemical abnormalities during fetal development that are informative for prenatal diagnosis, only a fraction of them are clinically/sonographically symptomatic before birth, thus allowing for prenatal diagnosis in the absence of an index case, i.e., serine deficiency, some fatty acid beta-oxidation defects, transaldolase deficiency, lysosomal diseases, CDG, Zellweger syndrome, and SLO syndrome.

The questions addressed in this study were: 1) whether insulin added to the incubation medium can down-regulate (125)I insulin binding to isolated hepatocytes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss); 2) whether quantitative assessment of insulin processing can be made on isolated fish liver cells; 3) how ambient temperatures can affect insulin binding, and down-regulation of insulin receptors.After isolation and a short (up to 4h) "metabolic recovery period", liver cells were used either directly in (125)I insulin binding assay or first preincubated for 18h at 4°C or for 3h at 15°C, with or without mammalian or salmon insulin in concentrations ranging from 1 to 1000 nM.Preincubation at 15°C, decreased binding capacity (number of binding sites per liver cell) in all five independent hepatocyte preparations treated with 1000 nM insulin and in four out of five preparations treated with 100 nM insulin. At 4°C insulin binding sites were down-regulated in less than 50% of all hepatocyte preparations and only in the presence of 1000 nM insulin.Differential quantitive assessment was made of a) intact free insulin; b) insulin degraded; c) intact insulin bound to the cell membrane; d) internalized but degraded insulin, and e) intact insulin internalized by liver cells. Hepatocytes preincubated with 100 - 1000 nM insulin at 15°C bound and internalized less (125)I insulin.We hypothesize that in vivo, at water temperatures of 15°C and higher, extreme physiological levels of plasma insulin may regulate the numbers of insulin receptors in the salmonid liver. In contrast, in fish inhabiting cold waters the regulation of insulin receptors by circulating plasma insulin seems to be of little physiological importance.

Induction of AmpC beta-lactamase in Enterobacter cloacae ATCC 13047 by D-methionine, glycine, or D-tryptophan was accompanied by alterations in peptidoglycan composition and structure; in the case of D-methionine, it was also accompanied by morphologic changes. A decrease in peptidoglycan tripeptides was seen. With glycine, there was an increase in the proportion of diaminopimelic-diaminopimelic cross-links. The possible implications of these changes for beta-lactamase induction are discussed.

Normative data for amniotic fluid (AF) levels of organic acids at different gestational ages are lacking. They can provide a useful framework to investigate the accuracy of prenatal diagnosis for organic acidemias.We report on the concentration of 21 organic acids in AF obtained by gas chromatography/mass spectrometry between the 12th and 34th weeks of gestation from 92 pregnancies that were not at risk for organic acidurias.We infer normal reference values that can be compared with 134 pregnancies at risk for several metabolic conditions, that is, propionic acidemia, methylmalonic acidemia (methylmalonyl-CoA mutase deficiency or defects in cobalamin metabolism), 4-hydroxybutyric acidemia, glutaric acidemia and pyroglutamic acidemia.Most of the metabolites tested did not show conspicuous variations across gestational ages in normal fetuses, with ranges that were consistently similar to available reference values from pooled samples in previous reports. With rare exceptions, knowledge of pathological versus normal values for relevant metabolites leads to clear-cut differentiation of affected versus unaffected fetuses. Nevertheless, it is strongly recommended that mutational analysis and/or additional biochemical approaches complement organic acid analysis for an adequate diagnostic workup.

A possible connection between septation/division and induction of cloned ampC beta-lactamase was investigated. When a ftsZ84(Ts) mutant of Escherichia coli carrying ampR-ampC from Citrobacter freundii was grown at the restrictive temperature (42 degrees C), induction of beta-lactamase by cefoxitin was inhibited by about 80%. Inhibition was virtually complete when a ftsZ84(Ts) mutant of different genetic background was tested. Although somewhat delayed, the induction of beta-lactamase in transformed ftsA(Ts) and ftsQ(Ts) mutants was similar to that observed in wild-type transformants. These results imply that FtsZ is involved in the process of beta-lactamase induction.

Two pools of liver cells, enriched either in periportal or in perivenous hepatocytes, were obtained from the catfish Ictalurus melas by the selective destruction of the liver by digitonin infusion. Following digitonin pulses directed either orthograde via the portal vein or retrograde via the hepatic vein, hepatocytes from undamaged perivenous and periportal zones, respectively, were isolated by the conventional collagenase perfusion procedure. Eluates from either periportal or perivenous zones were collected for a total of 9 min before, during, and immediately following the digitonin pulse. The eluates were assayed for protein, glycogen, glucose, and activities of lactate dehydrogenase and alanine aminotransferase, and their yield ("recovery") was estimated as a percentage of the total hepatic content. The aforementioned metabolic compounds and enzyme activities were measured also in periportal and perivenous liver cells. No significant differences were obtained between these cells. Both pools of hepatocytes were more than 90% viable and remained hormone responsive. Epinephrine (3 × 10−8 M) or salmon glucagon (3 × 10−8 M) elevated cAMP content, and stimulated glycogen phosphorylase a activity, glycogen depletion, and glucose release in periportal and perivenous liver cells. No differences were observed between periportal and perivenous cells in their metabolic responsiveness to either hormone.

The adenosine analogue 9-(Tetrahydro-2-furyl)adenine, SQ 22536, inhibited adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of crude membrane preparations from catfish (Ictalurus melas) and rat isolated hepatocytes in a non-competitive manner. The IC50s were reduced in the presence of NaF. SQ 22536 reduced the activity of adenylate cyclase also in the presence of increasing concentrations of GTP, as well as Mg++ and Mn++. In the presence of catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) SQ 22536 reduced their activating effect on adenylate cyclase in both catfish and rat membranes. SQ 22536 also inhibited the effect of glucagon (0.1 UM) on rat membrane cyclase activity.

Objectif Les mutations du gene SDHB sont un facteur de malignite et de mauvais pronostic des paragangliomes. L’elaboration de modeles animaux est necessaire pour l’evaluation preclinique de therapeutiques innovantes. Materiel et methodes Une lignee murine de cellules chromaffines immortalisees (imCC) Sdhb–/− a ete generee dans notre laboratoire et implantee en sous-cutane dans le « fatpad » de souris nudes NMRI (n = 15). Un groupe controle (n = 16) a ete realise par implantation d’imCC sauvages (WT). L’angiogenese tumorale a ete evaluee par imagerie optique in vivo grâce a un traceur fluorescent ciblant les integrines αvβ3, la microcirculation par IRM dynamique de contraste (IRM-DCE) et le metabolisme par spectroscopie par resonance magnetique (1H-MRS) et par 18FDG-PET. Resultats L’expression des integrines est plus marquee dans le groupe de tumeurs Sdhb–/− et est associee, en IRM-DCE, a un rehaussement tumoral nettement plus important que dans les tumeurs WT. Les tumeurs Sdhb–/− presentent de plus une accumulation de succinate mesurable par 1H-MRS. Enfin, la captation du glucose en 18FDG-PET est egalement plus marquee dans le groupe Sdhb–/−. L’evolution sous traitement antiangiogenique est en cours d’analyse. Discussion L’augmentation de la vascularisation et de la captation du glucose des tumeurs Sdhb–/− est evaluable in vivo par IRM et imagerie optique. Ces resultats permettent de mettre en place des etudes precliniques de reponse precoce au traitement. La detection de l’accumulation de succinate par MRS pourrait permettre par un diagnostic « moleculaire » preoperatoire afin d’identifier precocement les patients a risque.

Isolated hepatocytes in culture represent an idoneous system for the study of liver physiology and metabolism. Furthermore, they are also widely utilized in pharmacological and toxicological study, in evaluating xenobiotic substance effects on the liver. In this paper, we reviewed the enzymatic methods for liver cell isolation in some mammalian species, as well as the techniques for qualitative and quantitative evaluation of cell number, vitality, purity, morphology and function. Recently, there has been a renewed interest in hepatocyte transplantation and hepatocyte-based liver support systems. From a clinical point of view, isolated hepatocytes could be useful in temporarily substituting an acutely damaged liver, a liver affected by a chronic pathology, or to correct an inherited liver disease carrying a severe metabolic derangement. Early experimental results of allogeneic hepatocyte transplantation, as well as the first clinical trials of bioartificial liver support systems employing xenogeneic hepatocytes are promising and contribute to maintain that interest in liver cell isolation and purification methods.

Various three-dimensional configurations, such as polyester tissue and woven-nonwoven, hydrophilic polyester fabric, either collagen-coated or uncoated, were investigated as potential scaffold for hepatocyte culture, in view of their use in bioreactors for hybrid liver support systems. Attachment, morphology and ultrastructure of primary adult rat hepatocytes were evaluated, as well as urea production and ammonium detoxification during a 24h incubation period in serum-free tissue culture medium. As control, hepatocytes were also plated onto collagen-coated dextran microcarriers and on plastic petri dishes, either collagen-coated or uncoated. In all the three-dimensional cultures, hepatocytes appeared morphologically intact without any statistically significant difference in metabolic activity. Collagen-coating did not influence cell attachment to polyester substrates, whereas woven-nonwoven hydrophilic polyester fabric may offer some potential advantages as three-dimensional system for hepatocyte culture in hybrid liver support systems.

Isolated hepatocytes are a suitable system for the study of hepatic physiology and metabolism. They are also used for pharmacological and toxicological studies related to hepatic uptake, metabolism, excretion and toxicity of xenobiotics, as well as morphological and metabolic effects induced in the liver as a result of drug or toxic substance exposure. In this paper, the enzymatic methods for hepatocyte isolation in some mammalian species are reviewed, and methods for evaluating cell purification and assessment of cellular morphology and function are also examined. More recently, interest in hepatocyte transplantation has increased, and the clinical experimentation of hepatocyte-based liver support systems has attracted the attention of scientists and hepatologists. From a clinical perspective, using isolated hepatocytes could be useful both for supporting an acutely devastated liver, a chronically diseased liver, and for correcting genetic disorders resulting in metabolically deficient states. Reports of clinical usage of isolated allogenic hepatocytes in hepatocellular transplantation and of xenogenic liver cells in constructing bio-artificial liver support systems are promising, and are renewing interest in the development of methods for isolation and purification of hepatocytes.

Objectives: Membrane toxicity induced by hydrophobic bile salts may be important in liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases, but the nature of this effect is still unclear. We aimed at establishing a convenient in-vitro system for investigating the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate. Methods: About 100mg of freshly isolated rat hepatocytes were suspended on a resin column (Bio-Gel P4 fine) and perifused with different concentrations of bile salts. The effluent was collected at 5-min intervals and assayed for lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. Enzyme leakage induced by bile salts was compared with that induced by Triton X-100 (Union Carbide, Danbury, CT, USA) at different concentrations. After perifusion, hepatocytes were collected for electron microscopic observation. Results: Cytotoxicity of individual bile salts, assessed by enzyme release, was time and concentration dependent and corresponded to their hydrophilic-hydrophobic balance. Perifusion with hydrophilic bile salts, cholate and ursodeoxycholate, did not result in a significant enzyme release in concentrations up to 5mmol/l, whereas hydrophobic bile salts, chenodeoxycholate and deoxycholate, induced significant enzyme leakage even in low concentrations, 0.5 and 0.1 mmol/l, respectively. Addition of ursodeoxycholate significantly reduced the hepatotoxic effect of deoxycholate. This protective effect was evident within minutes. The ultrastructural appearance of hepatocytes exposed to hydrophobic bile salts was very similar to the nonspecific cellular lysis observed after exposition to Triton X-100, suggesting that they act mainly in a detergent-like fashion. Conclusion: Perifused rat hepatocytes seem a convenient in-vitro system for investigating the hepatotoxic properties of bile salts and hepatoprotective effect of ursodeoxycholate, offering the opportunity to investigate the effects of bile salts under dynamic conditions, mimicking the in-vivo situation, and allowing continuous enzyme release monitoring. Hydrophobic bile salts seem to act mainly in a detergent-like fashion; ursodeoxycholate-related hepatoprotection could be due not only to a dilution effect of toxic bile salts, but also to a direct cytoprotective effect.

Hepatocytes were isolated from catfish (letalurus melas) by conventional collagenase digestion. Sensitivities of liver cells isolated from the same fish to the glycogenolytic action of epinephrine, mammalian glucagon, catfish glucagon, catfish glucagon-like peptide, synthetic fragment 19-29 of anglerfish glucagon 1, fragment 19-29 of anglerfish glucagon II, and anglerfish glucagon II were compared in two different systems: perifusion in a Bio-Gel P4 column and flask incubation. Both experimental procedures were continued for a total of 100-120 min, while hormones were applied simultaneously to both preparations for 10 min. Effluent fractions from the columns and incubation media from the flasks were collected for glucose determination. The hormonal effects were clearly enhanced in perifused cells compared to those in cells incubated in flasks, the effect being especially evident at physiological concentrations of hormones. The hormonal effects in both systems were dose-dependent. Epinephrine and mammalian glucagon (10 nM), applied separately to the same column, produced two different peaks, glucagon causing more glucose production than epinephrine. In the presence of 0.4 mM glucose in the perifusion system, hormonal effects were diminished, implying that glucose accumulation during incubation of liver cells in flasks might affect hormonal effects. The results obtained in this study indicate that piscine hepatocytes suspended and perifused in a Bio-Gel column are more sensitive to physiological concentrations of glycogenolytic hormones and may represent a new tool for experimental studies of fish liver metabolism and its hormonal regulation.

This study aimed to characterize alpha-adrenergic receptors in catfish hepatocytes in which catecholamine-induced calcium transients have been observed. alpha-Adrenergic binding sites were studied in purified liver membranes using the specific alpha 1-receptor antagonist [3H]prazosin. At 22 degrees C specific binding of [3H]prazosin is saturable, reversible, and linear as a function of tissue concentration; the association reaches a maximum at 15 min, and the half-time for dissociation is about 4 min. Analysis of binding data suggests a single class of binding sites with a Kd of 1.6 nM and a Bmax of 182.1 fmol/mg protein. The relative potency of selective adrenoceptor ligands points to the presence in catfish liver of alpha-adrenergic receptors in addition to the beta-adrenergic receptor population previously characterized. Moreover, the binding data have been correlated to the glucose release from isolated hepatocytes suspended and perifused in a BioGel column, suggesting the involvement of alpha-adrenergic receptors in the glycogenolytic response to catecholamines in catfish hepatocytes.

Adult catfish, Ictalurus melas, hepatocytes respond to alpha-agonists by increasing intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner. Basal [Ca2+]i were approximately 78 nM in catfish hepatocytes; this value was increased by 213, 243, 131, and 238 nM in the presence of epinephrine (Epi; 10(-6) M), norepinephrine (NE; 10(-5) M), phenylephrine (PE; 10(-4) M), and isoproterenol (Iso; 10(-4) M), respectively. The binding constants were 3.4 x 10(-8), 2.6 x 10(-7), 9.8 x 10(-7), and 6.1 x 10(-5) M for Epi, NE, PE, and Iso, respectively, which is the same order of potency for these agonists reported for the alpha 1-adrenoceptor system of mammalian hepatocytes. The Epi-induced changes in [Ca2+]i were antagonized equally by phentolamine (PH; 10(-8) M) and yohimbine (10(-8) M) but poorly by propranolol (Prop; 10(-6) M), as indicated by their respective inhibitory constants. Epi (10(-7) M) induced a time-dependent increase in hepatocyte adenosine 3',5'-cyclic monophosphate concentration that was antagonized by Prop but not PH. There were, however, no clear agonist-induced changes in the activities of glycogen phosphorylase (total, a, or %a) under these conditions. These studies provide evidence that Epi and other adrenergic agonists increase [Ca2+]i by an alpha- or alpha 1-like adrenoceptor pathway in catfish hepatocytes, but the biochemical effector system responding to changes in [Ca2+]i has yet to be elucidated.

The induction of Class I β-lactamase (Bla) in the Enterobacteriaceae and other Gram negative organisms has been studied extensively but as yet no information is available as to the nature of the event(s) which triggers it nor of the specific molecule that might act as the inducer. It is the purpose of this paper to present evidence and to suggest a model which makes septation/division the controlling factor for the induction of Bla.

beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.

The simultaneous addition of epinephrine and salmon glucagon to catfish (Ictalurus melas) and trout (Salmo gairdneri) hepatocytes did not induce greater increases in glycogen phosphorylase a activity and in glucose release than those caused by epinephrine alone. The effects of epinephrine are greater than those of glucagon. Propranolol added to the hormonal pool blocked the epinephrine effects. In trout cells, epinephrine and glucagon-like peptide (GLP) had similar effects and when they were added simultaneously the stimulation of metabolic indices was higher compared to that obtained with either epinephrine or GLP. However, the effects were not additive. In the presence of epinephrine plus GLP the inhibitory effect of propranolol was not evident, due to the effect induced by GLP, on which propranolol was not effective. This may indicate that epinephrine masks the GLP effect. Results could mean that epinephrine and glucagon-family peptides act in catfish and trout hepatocytes through different receptors on the same pathway leading to glycogen phosphorylase a activation.

Chrysotile fibers promoted the fusion of human erythrocyte membranes and the hemolysis and fusion of fowl erythrocytes. Amosite and glass fibers did not show the same effect. These findings provide a model for the internalization of the fibers by animal cells. Images FIGURE 1. a FIGURE 1. b FIGURE 1. c FIGURE 2. a FIGURE 2. b FIGURE 3. a FIGURE 3. b FIGURE 3. c FIGURE 3. d

The effect of insulin was studied on blood glucose, and on the glycogen level of liver, muscles and heart in fed and in starved catfish (Ictalurus melas). Fish received intraperitoneally 60 iu/kg body weight of bovine insulin, or physiological saline and were sacrificed after 2, 4, 8, 24, 72 hr from injection. Insulin caused a decrease of blood glucose level, both in fed and in fasted animals, and the effect is more evident in fed animals. After insulin treatment, liver glycogen shows a decrease which is significant at the 8th and 24th hr in fasted and in fed animals respectively; after 72 hr the glycogen level in livers of fed and fasted animals is still very low. Insulin increases the glycogen level both in white and in dark muscle, both in fed and in fasted fish, although with different characteristics, but at the 72nd hr in all animals, the increases are significant. Hormone treatment does not change heart glycogen levels in fed catfish till the 24th hr, then there is a net decrease; in starved animals the decrease begins at the 2nd hr, but only at the 48th hr is it significant. The role of insulin was discussed in relation to the lowering of glycogen concentration in liver, in connection with the fact that many authors found different and even opposite effects of this hormone in various fish. It is possible that the glycogen depletion observed in liver after insulin injection is not due to a direct action of this hormone, but depends on the stimulated production of other specific glycogenolytic hormones, such as epinephrine and/or glucagon.

Different doses of glucagon and glucagon-like peptide (GLP) isolated from coho salmon, Oncorhynchus kisutch were tested in vivo and in vitro on juvenile coho and chinook (O. tshawytscha) salmon. Results obtained suggest an involvement of these peptides in the regulation of plasma glucose, plasma fatty acids, liver glycogen, and the hepatic enzymes: glycogen phosphorylase, pyruvate kinase, triacylglycerol lipase, and glucose-6-phosphate dehydrogenase. Metabolic effects were more enhanced in summer than either in spring or in autumn. GLP was less effective than glucagon in stimulating glycogenolysis in vivo. Salmon glucagon, especially in low concentrations, was generally more potent metabolically than mammalian (porcine/bovine) glucagon. The interaction between glucagon-family peptides and insulin seems to be different from the one described in mammals: glucagon and GLP either lowered plasma circulating levels of insulin or showed no effect. Only at the time of parr-smolt transformation did GLP slightly elevate plasma insulin levels in coho salmon.

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.

1. 1. The isolated and perfused catfish liver showed (a) a decrease in liver glycogen, (b) a continuous increase in glucose output, and (c) a decrease of lactate in the medium. 2. 2. Insulin did not influence liver glycogen decay during the first 2 hr; thereafter the hormone induced an increase of glycogen, particularly when glucose was added into perfusate. 3. 3. In insulin treated liver, the glucose output was lower than controls in the first hours of perfusion; thereafter a re-uptake of glucose occurred. 4. 4. After 2–3 hr of perfusion, the lactate present in the medium was increased by insulin towards the starting level. 5. 5. The long lasting effects of insulin on catfish in vivo were confirmed.

Glycogenolytic effects of salmon and mammalian glucagons, salmon glucagon-like peptide (GLP) and epinephrine were studied on liver cells isolated from catfish (Ictalurus melas). In spring and summer, salmo-glucagon (3×10(-10) to 3×10(-8) M) was more effective than its mammalian counterpart in the stimulation of glucose release and cAMP synthesis in hepatocytes. GLP was less potent as compared to both glucagons. γ-amylase activity was not affected by the treatment with either glucagon-family peptides or epinephrine.The comparison of the glycogenolytic effects of salmon glucagon to those of epinephrine reveals a greater potency of the latter hormone in the stimulation of cAMP synthesis, glycogen-phosphorylase activity and glucose release. Glycogen content in the liver cells was equally depleted after treatment with both of the two hormones.

1. 1. Catfish ( Ictalurus melas ) contain large amounts of glycogen in liver and other organs; these amounts are greater than those present in the corresponding tissues of mammals. 2. 2. All organs show seasonal variations in their glycogen content: in general, the values are lower in spring than in other seasons. 3. 3. Fasting induces variations in the glycogen content of organs according to the season and, for the liver, also according to sex.

Summary Bromosulfophthalein had been reported to inhibit the γ-glutamyl cycle. The γ-glutamyl cycle has been implicated in amino acid transport in mammalian cells. Bromosulfophthalein was therefore employed in an attempt to correlate function of the γ-glutamyl cycle with amino acid transport by two well-defined ascites cell amino acid transport systems. Inhibition of both transport systems occurred in the presence of 1 mM bromosulfophthalein. However, further studies suggested that this inhibition was associated with a functional and morphological alteration of the plasma membrane. The S37 cells had an increased non-specific permeability demonstrated by sulfate. Steady state retention of amino acids was decreased and 51Cr release was increased by bromosulfophthalein. Vital staining was also increased by bromosulfophthalein and electron micrographs revealed membrane solubilization in the presence of bromosulfophthalein.

Two doses of epinephrine were infused for 6 hr into isolated catfish liver previously perfused with a glucose-free medium or with a medium containing 10 mM glucose. The hormone induced (a) a continuous decrease in liver glycogen level, both in absence and in presence of glucose in the medium; low dose of epinephrine was without effect on the decay of glycogen; (b) a great release of glucose, both in absence and in presence of glucose in the medium; the low dose of epinephrine induced an effect similar to the maximal dose, only in experiments without glucose in the medium; (c) no effect on lactate uptake by liver; or (d) a prevention of the decline in liver glycogen phosphorylase activity observed during 1 hr incubation of liver slices. It has been concluded that epinephrine caused an increase of glucose in perfusion medium with different mechanisms according to the level of glucose and the dose of epinephrine. High doses of hormone cause the glycogenolysis by activation of glycogen phosphorylase, both in presence and in absence of glucose; low doses of epinephrine probably preferentially promote in liver the gluconeogenetic processes in glucose-free experiments.

Pathologic response of tissue to asbestos in vivo gives rise to fibromatoma, granuloma and mesothelioma. We are attempting to develop a model system in vitro using human cells in order to investigate the possible mechanisms responsible for these pathologies. Within the first 12 hr of exposure to chrysotile, the fibroblasts showed distinctive morphological changes. Cells appeared elongated with occasional vacuolated nuclei and granular cytoplasm. Cells showed no other obvious morphological changes by light microscopy and were serially passaged at confluence. The cells with vacuolated nuclei were successfully serially passaged. Binucleated cells were first observed 48 hr after passaging. As time in culture increased (3 days to 2 weeks) many cells lost their distinctive bipolar properties and developed "stress striations" and multiple vacuoles in the cytoplasm. Multinucleated giant cells (2-11 nuclei/cell) with lobate nucleoli became more numerous. With increasing passages, the confluent cell density decreased and cell size increased. Cells usually had condensed nucleoli and had lost all control of directional growth. Preliminary indications suggest that these in vitro morphological transformations are due--at least in part--to a lack of control over cytokinesis. Images FIGURE 1. a FIGURE 1. b FIGURE 1. c FIGURE 2. a FIGURE 2. b FIGURE 2. c FIGURE 3. a FIGURE 3. b FIGURE 3. c FIGURE 4. a FIGURE 4. b

The one-stage prothrombin time of human plasma was prolonged by the addition to the thromboplastic mixture of either colistin sulfate or polymyxin B. Colistimethate sodium, a derivative of colistin in which the amino groups of the cyclic polypeptide antibiotic are substituted, did not show this effect. The inhibition was greater if the antibiotics were incubated with thromboplastin in the absence of Ca++. In the Hicks–Pitney modification of the thromboplastin generation time test, the two polyamine antibiotics reduced the amount of thromboplastic activity generated. Generation, however, appeared to be initiated sooner. It is postulated that these actions by the antibiotics are the results of modifications in the zeta potentials of the thromboplastic micelles.