Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.

Identification of a site in glutamate receptor subunits that controls calcium permeability.

The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus been identified that regulates the permeation properties of these glutamate receptors.

Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Alcohol abuse--a persistent preventable risk for congenital anomalies.

OBJECTIVE: To examine whether alcohol abuse (ALC) continued to be a health hazard to pregnant women in the 1990s. STUDY DESIGN: Analysis of a perinatal data base comprising 170,258 women with singleton pregnancies. Univariate cross table analysis and logistic regression were conducted to examine the association between alcohol abuse and congenital malformations coded according to the International Classification of Diseases (ICD). RESULTS: 14,727/170,258 mothers (8.6%) admitted to ALC during pregnancy and 36,705/170,258 (21.6%) to smoking. Anomaly rates for ALC (365/14,092, 4.3%) vs. Non-ALC (6187/149,344, 4.0%) differed significantly (p<0.001). The rates of specific anomalies varied between <0.1% and 1.1%. Odds ratios for 16 ICD 9 anomaly categories were >1 in 14 instances overall (Sign test, p=0.004), in 12 instances in women <30 years (p=0.08), and in 13 instances in women over 30 years (p=0.02). Congenital anomalies of the "respiratory system" (ICD9 748), of "genital organs" (ICD9 752.1), of the "integument" (ICD9 757), and "other anomalies of limbs/other musculoskeletal anomalies" (ICD 755/756) were statistically significantly associated with ALC, especially in women>30 years. CONCLUSION: ALC in pregnancy continued to be an important factor independently associated with an increased incidence of a broader range of congenital anomalies than previously recognized. Risk for anatomic anomalies was increased in offspring of ALC women over age 30, consistent with previous reports of increased risk of neurobehavioral abnormality in offspring of women over 30.

Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

The development of glutathione S-transferase and glutathione peroxidase activities in human lung.

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.

Differential expression of alpha and pi isoenzymes of glutathione S-transferase in developing human kidney.

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies to determine the developmental expression of these isoforms in human kidney. Before 35 weeks of gestation, both isoenzymes were expressed by the collecting tubules and developing nephrons. After this time, expression of the alpha set was restricted to the proximal tubule and that of the pi set to the distal and collecting tubules and the loop of Henle.

Studies on the relationship between glutathione S-transferase phenotype and bile acid binding by human liver cytosol.

The possibility that the GST1 phenotype of human liver cytosol is a determinant of bile salt binding has been investigated by using equilibrium dialysis and gel-exclusion chromatography. Binding of bile salts was non-saturable and whereas the glutathione S-transferases did not appear to be major bile salt binders, other binding components with molecular weights of 35 000 and 11 000 were identified in both fetal and adult cytosols.

The alpha and pi isoenzymes of glutathione S-transferase in human fetal lung: in utero ontogeny compared with differentiation in lung organ culture.

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies of developing human lung. In utero expression of the pi set was down-regulated in distal airway cells and the first appearance of pi-negative cells coincided with phenotypic differentiation. In contrast, in the early phase of fetal lung organ culture pi isoenzyme was detected in all differentiated epithelial cells and only as culture progressed did focal negativity develop. The alpha set showed no developmental changes in utero or in organ culture.

Human glutathione S-transferases: radioimmunoassay studies on the expression of alpha-, mu- and pi-class isoenzymes in developing lung and kidney.

The developmental expression of the alpha-, mu- and pi-class glutathione S-transferases has been defined in human lung and kidney using radioimmunoassay, immunohistochemistry and column chromatography. Expression of alpha-class enzymes increased significantly after about 40 weeks gestation in kidney but not lung, while expression of mu isoenzymes was continuous throughout development in both tissues. Expression of the pi isoenzyme fell during in utero ontogeny in lung, the pattern of down-regulation being similar to that previously observed in liver. There was no change in the expression of this isoenzyme in kidney. Comparison of the expression of the glutathione S-transferases in developing lung, kidney and liver shows some common patterns of expression suggesting these genes are under similar regulatory control.

Membrane specific carbonic anhydrase (CAIV) expression in human tissues.

Membrane-bound carbonic anhydrase IV (CAIV) expression has been evaluated in a range of fetal and adult human tissues and in cell culture. All tissues tested showed expression of CAIV, assessed by Western blotting, with a single immunodetected band at 55 kDa. The levels varied in fetal lung and liver during development and in various zones of the fetal brain. CAIV was clearly expressed in lung, pancreatic tumour and skin cell cultures.

Studies on the expression of Cu,Zn superoxide dismutase in human tissues during development.

The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.

The development expression of alpha-, mu- and pi-class glutathione S-transferases in human liver.

The developmental expression of the alpha, mu and pi class glutathione S-transferases has been defined in human liver using radioimmunoassay and immunohistochemistry. Expression of alpha and mu class isoenzymes increased significantly at birth, while that of the pi isoenzyme declined during the first trimester. Mu-class isoenzymes (GST1 1, GST1 2, GST1 2-1) were expressed in hepatocytes but not in other liver cell types.

Studies on the developmental expression of glutathione S-transferase isoenzymes in human heart and diaphragm.

The developmental expression of the basic, near-neutral and acidic isoenzymes of glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) has been studied in heart and diaphragm. Neither these enzymes nor the putative muscle-specific GST4 isoenzyme demonstrated any developmental trends in expression. In vitro hybridisation and SDS-discontinuous polyacrylamide gel electrophoresis were used to show that the GST4 isoenzyme is a homodimer composed of monomers that have a slightly larger molecular weight than the near-neutral isoenzyme. The sensitivity of GST4 to inhibitors also appeared similar to that of the GST1 2 isoenzyme. Immunodiffusion and immunoblotting techniques were used to show that the acidic enzyme in muscle is immunologically identical to that in other tissues.

Thioredoxin reductase and cytoplasmic glutathione peroxidase activity in human foetal and neonatal liver.

Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.

Dehydroepiandrosterone sulfotransferase in the developing human fetus: quantitative biochemical and immunological characterization of the hepatic, renal, and adrenal enzymes.

The sulfation of the adrenal steroid dehydroepiandrosterone (DHEA) is a critical step in the provision of substrates for estrogen biosynthesis by the placenta during pregnancy. This enzyme reaction is catalyzed by a cytosolic sulfotransferase (ST) found in many key body tissues, and we have examined the ontogeny and localization of expression of this important enzyme in three tissues: the liver, adrenal, and kidney. Hepatic DHEA ST expression increased with advancing gestational age before reaching near-adult levels in the early postnatal period, suggesting an increased requirement for this enzyme in the liver as development progresses, whereas in the adrenal and kidney there was no obvious ontogenic pattern. The enzyme was expressed at a 5-fold higher level in the adrenal than in the liver and some 40-fold higher than in the kidney. Comparison of enzyme activity measurements and quantitation of the expression of DHEA ST by immunodot blot analysis with an anti-DHEA ST antibody preparation demonstrated the fragility of the enzyme activity and suggested that immunoquantitation was a superior method for assessment of levels of expression of this enzyme in widely different tissue sources. Examination of the localization of DHEA ST in these tissues by immunohistochemistry showed that in liver, DHEA ST was expressed in embryonic hepatocytes and continued to be expressed in these cells into adulthood, when there was some concentration of immunostaining around central veins. In the fetus, the adrenal enzyme was expressed in the fetal zone, whereas in adult tissue, staining was localized principally to the zona reticularis. Renal DHEA ST was present in the proximal and distal tubules, loops of Henle, collecting ducts, and their progenitors, but was at no time expressed in the vascular glomerulus. In light of the broad substrate specificity of this enzyme toward other steroids, in particular bile acids and cholesterol, the information presented forms a strong basis for further studies into the role of DHEA ST in modulating the activity of a number of biologically active and potentially toxic steroids in the developing human.

Differential expression of sulfotransferase enzymes involved in thyroid hormone metabolism during human placental development.

Thyroid hormone is essential for normal human development, and disruption of thyroid hormone homeostasis at critical developmental stages can result in severe and often long-term effects on crucial organs such as the brain and lungs. Numerous factors control the bioavailability of receptor active thyroid hormone T(3). Sulfation, catalyzed by sulfotransferase enzymes (SULTs), is an important pathway of thyroid hormone metabolism by which T(4) is irreversibly converted to inactive reverse T(3) rather than active T(3). The human fetus and neonate have high levels of circulating sulfated iodothyronines, although the source of these is not clear. The placenta forms the link between the fetus and its mother and is involved in transfer of thyroid hormone early in pregnancy, although its capacity for sulfation is unknown. We therefore examined expression of the SULTs involved in iodothyronine metabolism during human placental development. SULT activity was measured in human placental cotyledon and membranes (amnion, chorion, and decidua basalis) from 13-42 wk of gestation, and Western blot analysis was employed to verify enzyme activity data. Phenol and catecholamine sulfotransferases were expressed at the highest levels and were generally higher in the villous than membranous tissues. SULT1A1 activity showed significant correlation with sulfation of 3,3'-T(2), suggesting that this enzyme is primarily responsible for placental T(2) sulfation. Estrogen sulfotransferase was present at extremely low levels during early pregnancy, although in mid- and late gestation increased expression in the (predominantly maternal-derived) decidual component of the placenta was observed. Hydroxysteroid sulfotransferase, T(3), reverse T(3), and T(4) SULT activities were also low in all tissues examined, and expression of SULTs 1B1 and 1C2 were essentially undetectable by Western blot analysis. The results highlight a tissue-specific regulation of SULT expression during placental development, demonstrate very low sulfation of iodothyronines suggesting that the placenta is not a major source of circulating sulfated iodothyronines in the fetus.

The effect of mifepristone (RU486) on the immunohistochemical distribution of prostaglandin E and its metabolite in decidual and chorionic tissue in early pregnancy.

The widespread tissue distribution of prostaglandin dehydrogenase (PGDH), the main enzyme for the metabolism and deactivation of prostaglandin (PG), suggests that local effective levels of PG are controlled by catabolism. Previous reports have suggested that after the administration of mifepristone (RU486) in vivo, the levels of PGDH in uterine tissues fall, such results support earlier suggestions that PGDH is under progesterone control in reproductive tissues. In this study we have used immunohistochemistry to assess tissue concentrations of PGE and its main metabolite 13,14-dihydro-15-keto-PGE in chorionic villi and decidua from women treated with RU486 12, 24, and 36 h previously. In control villous tissue, PGE and 13,14-dihydro-15-keto-PGE2 (PGEM) are prominent in the syncytiotrophoblastic layer, whereas PGE stains only weakly in cytotrophoblasts, due to the presence of PGDH in this region; treatment with RU486 in vivo causes little change in distribution or intensity in villi. However, in decidua, staining for PGE2 was intense in the glands after RU486 and localized in the supranuclear region of the cells. Small blood vessels that were PGE negative and PGEM positive in the controls were PGE positive and PGEM negative in treated tissue. These findings show that PG stimulation by antiprogestin is by means of a direct effect on PGDH, and secondly, that the prominent rise in PGE in blood vessels may be a major mode of action of this steroid in causing abortion, as PGs may synergize with leukocyte chemotactic agents to stimulate neutrophil ingress and tissue destruction.

The human adrenal microsomal glucose-6-phosphatase system.

Microsomal glucose-6-phosphatase (EC 3.1.3.9) is a multicomponent enzyme system traditionally thought only to be present in gluconeogenic tissues. The enzyme is associated with transport systems, for its substrate glucose-6-phosphate, and its products phosphate and glucose. It has been shown, using immunohistochemical methods and monospecific antibodies, that the component proteins of the enzyme system are present in human embryonic and fetal adrenal gland and are predominantly located in the fetal zone with lesser reactivities in the definitive zone. In addition, specific glucose-6-phosphatase activity was shown, and the rates of entry of glucose-6-phosphate, phosphate, and glucose into microsomes isolated from human fetal adrenals were measured. Although the complete enzyme system is present, the ratio of the component activities and comparison with human fetal and adult liver indicate that the regulation of the adrenal and liver glucose-6-phosphatase systems is different. In the human postnatal adrenal, immunoreactivies to the protein components decrease dramatically and are confined predominantly to the zona reticularis, suggesting a specialized role for adrenal glucose-6-phosphatase in fetal life.