Glucocorticoid Effects on Cerebellar Development in a Chicken Embryo Model: Exploring Changes in PAX6 and Metalloproteinase-9 After Exposure to Dexamethasone.

The developing cerebellum is vulnerable to effects of glucocorticoids and cerebellar dysfunction is associated with neurodevelopmental disorders (e.g. autism). Transcription factor PAX6 and matrix metalloproteinase-9 (MMP-9) are critical for normal cerebellar development and are highly expressed in migrating neurones. Alterations in MMP-9 and PAX6 are associated with altered cerebellar development. In the present study, we characterised the growth rate and development of the cortical layers, and further investigated how the levels of PAX6 and MMP-9, as well as glucocorticoid receptor (GR) and proliferating cell nuclear antigen (PCNA), change in the cerebellum during the foetal period [embryonic day (E)12-21] in chicken, which corresponds to the human perinatal period. Dexamethasone (DEX) was administered in ovo at E13 and E16, aiming to investigate how prenatal exposure to glucocorticoids interferes with normal development. DEX reduced foetal and cerebellar weight at E17 in a dose-dependent manner linked to a reduced level of PCNA and, over time, down-regulation of GR. We report that promoter activity of PAX6 and MMP-9 increased as a result of GR-stimulation in vitro. Prenatal DEX increased the protein level of PAX6 in a transient manner. PAX6 is reduced in mature granule neurones, and this occurred earlier in embryos exposed to DEX than in non-exposed controls. DEX exposure also led to a slow-onset down-regulation of MMP-9. Taken together, these findings indicate that excess prenatal glucocorticoid stimulation disturbs normal development of the cerebellum through mechanisms associated with reduced proliferation and accelerated maturation where PAX6 and MMP-9 play important roles.

17α-Estradiol down-regulates glutathione synthesis in serum deprived PC-12 cells.

During the last decades it has been shown that estrogen may have neuroprotective functions in the CNS. However, we have previously reported that pretreatment with estradiol abolishes its protection of cultured cerebellar granule neurons from glutamate-induced cell death due to down-regulation of endogenous glutathione. 17α-Estradiol is considered a hormonally inactive isomer of 17ß-estradiol still containing its antioxidant potential. Here, we demonstrate that 17α-estradiol enhanced serum deprivation-induced cell death in the rat pheochromocytoma cell line PC-12, while antioxidants vitamins C and E in combination (vitamins C/E) tended to protect. We further examined mechanisms behind the glutathione lowering effect of 17α-estradiol in serum deprived PC-12 cells. Endogenous glutathione levels were reduced in the serum deprived cells. Serum deprivation-induced cell death seemed to depend partly on this reduction as supplemented N-acetylcysteine, a cysteine precursor with potential to restore glutathione levels, reduced cell death. 17α-Estradiol down-regulated glutathione, promoter activity of the rate-limiting enzyme in glutathione production, glutamate cysteine ligase (GCL), as well as c-Fos protein levels in serum deprived cells. The c-Fos transcription factor normally binds to the AP-1 response element in the GCL promoter resulting in increased production of glutathione as a stress response. Over-expression of AP-1 proteins partly restored the GCL promoter activity in serum deprived cells treated with 17α-estradiol. Nrf2, a transcription factor binding another response element in the GCL promoter was unaffected by 17α-estradiol. Conclusively, 17α-estradiol may have a long-term negative effect on the endogenous glutathione level through its ability to down-regulate the glutathione synthesis during serum deprivation.

Development and evaluation of an ultrasonic ground water seepage meter.

Submarine ground water discharge can influence significantly the near-shore transport and flux of chemicals into the oceans. Quantification of the sources and rates of such discharge requires a ground water seepage meter that provides continuous measurements at high resolution over an extended period of time. An ultrasonic flowmeter has been adapted for such measurements in the submarine environment. Connected to a steel collection funnel, the meter houses two piezoelectric transducers mounted at opposite ends of a cylindrical flow tube. By monitoring the perturbations of fluid flow on the propagation of sound waves inside the flow tube, the ultrasonic meter can measure both forward and reverse fluid flows in real time. Laboratory and field calibrations show that the ultrasonic meter can resolve ground water discharges on the order of 0.1 microm/sec, and it is sufficiently robust for deployment in the field for several days. Data from West Neck Bay, Shelter Island, New York, elucidate the temporal and spatial heterogeneity of submarine ground water discharge and its interplay with tidal loading. A negative correlation between the discharge and tidal elevation was generally observed. A methodology was also developed whereby data for the sound velocity as a function of temperature can be used to infer the salinity and source of the submarine discharge. Independent measurements of electrical conductance were performed to validate this methodology.

Effect of four laboratory decontamination procedures on the quantitative determination of cocaine and metabolites in hair by HPLC-MS.

The testing for drugs of abuse in hair is increasingly used to detect illicit substances. Laboratories have implemented various decontamination, or washing, procedures in order to eliminate concerns regarding potential contamination of the hair with drug from the environment. However, the effect of these decontamination procedures on drug incorporated into the hair shaft via systemic exposure is unknown. This study evaluated the effect of four simple laboratory wash procedures on the quantitative measurement of cocaine and its metabolites in hair from rats administered cocaine by intraperitoneal injection. Washes included (1) methanol only; (2) 0.1 M phosphate buffer, pH 6.0; (3) 0.1 M phosphate buffer, pH 8.0; and (4) isopropanol and phosphate buffer, pH 5.5. Cocaine and its major metabolites, benzoylecgonine, norcocaine, ecgonine methyl ester, and cocaethylene, were analyzed using high-performance liquid chromatography coupled to atmospheric pressure electrospray ionization mass spectrometry. All four washes resulted in significant differences from unwashed hair controls (p < or = 0.05) for some or all of the detectable analytes. Because different wash procedures lead to significant differences in the measured concentrations of analytes in hair known to contain drug, quantitative data must be interpreted cautiously based on the wash procedures employed.

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF.

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.

UV-light-dependent binding of a visual arrestin 1 isoform to photoreceptor membranes in a neuropteran (Ascalaphus) compound eye.

Arrestins are regulators of the active state of G-protein-coupled receptors. Towards elucidating the function of different arrestin subfamilies in sensory cells, we have isolated a novel arrestin 1, Am Arr1, from the UV photoreceptors of the neuropteran Ascalaphus macaronius. Am Arr1 forms a phylogenetic clade with antennal and visual Arr1 isoforms of invertebrates. Am Arr1 undergoes a light-dependent binding cycle to photoreceptor membranes, as reported earlier only for members of the arrestin 2 subfamily. This suggests a common control mechanism for the active state of invertebrate rhodopsins and G-protein-coupled receptors of antennal sensory cells. Furthermore, it implies that a strict correlation of distinct arrestin isoforms to distinct functions is not a general principle for invertebrate sensory cells.

5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism.

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products. Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised. Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium. There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase. Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting [3H]uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis. Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU. In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations. The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine. These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

Na(+)+K(+)-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of alpha-subunit mRNA and protein.

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other arthropods and 74-77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of alpha-subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na(+)+K(+)-ATPase alpha-subunit protein band of the expected size. The posterior gills contained a much higher level of alpha-subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na(+)+K(+)-ATPase alpha-subunit protein detected by alpha5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 per thousand salinity was not accompanied by notable differences in the relative proportions of alpha-subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na(+)+K(+)-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate.

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.

The Calliphora rpa mutant lacks the PDZ domain-assembled INAD signalling complex.

The visual transduction cascade of fly photoreceptors is a G protein-coupled phospholipase C-signalling pathway which is assembled into a supramolecular signalling complex by the PDZ (postsynaptic density protein-95, discs large, Z0-1) domain protein INAD (inactivation no afterpotential D). The norpA-encoded phospholipase Cbeta, the light-activated transient receptor potential (TRP) Ca2+ channel and an eye-specific protein kinase C are bound to INAD and together form the core of the signalling complex. In the present study we show that the Calliphora rpa mutant, which has previously been hypothesized to represent an equivalent of Drosophila norpA mutants, has normal amounts of norpA mRNA but fails to express inaD mRNA. Electrophysiological recordings from the eyes of the rpa mutant reveal that the electroretinogram is reduced (about 12% of wild type) but not completely absent, and that it exhibits markedly prolonged deactivation kinetics. Furthermore, rpa mutants display a slow, light-dependent degeneration of the photoreceptor cells. With respect to the INAD signalling complex, the rpa mutant is similar to the Drosophila inaD null mutant: not only INAD itself, but also the other core components of the INAD signalling complex, are reduced or absent in photoreceptor membranes of rpa flies. Residual TRP is localized throughout the plasma membrane of the photoreceptor cell, rather than being restricted to the microvillar photoreceptor membrane. [35S]methionine-labelling of newly synthesized retinal proteins reveals that TRP is synthesized in the rpa mutant at wild-type level, but is transported to or incorporated into the microvillar photoreceptor membrane at a much lower rate. We thus suggest, that the formation of the INAD signalling complex is required for specifically targeting its components to the photoreceptor membrane.

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

The PDZ assembled "transducisome" of microvillar photoreceptors: the TRP/TRPL problem.

Two types of ion channels, TRP and TRPL, are activated upon light-absorption in rhabdomeral photoreceptor membranes of fly compound eyes. Whereas TRP is associated with other signaling proteins into a multiprotein complex (transducisome), the molecular organization of TRPL is discussed controversely. We analysed the TRPL content of blowfly rhabdomeral membranes and investigated by co-immunoprecipitation studies whether or not TRPL is part of the transducisome. Compared to TRP there are at least ten times less TRPL molecules present in the rhabdomeral membrane. A small fraction of the total TRPL present co-immunoprecipitates with other proteins of the transducisome and vice versa. Our data suggest that a significant fraction of TRPL is not incorporated into the transducisome. This fraction may either form independent ion channels or bind to the transducisome transiently.

The visual G protein of fly photoreceptors interacts with the PDZ domain assembled INAD signaling complex via direct binding of activated Galpha(q) to phospholipase cbeta.

Visual transduction in the compound eye of flies is a well-established model system for the study of G protein-coupled transduction pathways. Pivotal components of this signaling pathway, including the principal light-activated Ca(2+) channel transient receptor potential, an eye-specific protein kinase C, and the norpA-encoded phospholipase Cbeta, are assembled into a supramolecular signaling complex by the modular PDZ domain protein INAD. We have used immunoprecipitation assays to study the interaction of the heterotrimeric visual G protein with this INAD signaling complex. Light-activated Galpha(q)- guanosine 5'-O-(thiotriphosphate) and AlF(4)(-)-activated Galpha(q), but not Gbetagamma, form a stable complex with the INAD signaling complex. This interaction requires the presence of norpA-encoded phospholipase Cbeta, indicating that phospholipase Cbeta is the target of activated Galpha(q). Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Galpha(q) forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cbeta.

A novel Ggamma isolated from Drosophila constitutes a visual G protein gamma subunit of the fly compound eye.

Visual transduction in the compound eye of flies is a well established model system for the study of G protein-coupled transduction pathways. To characterize key components of the phototransduction cascade we performed substractive hybridization screening. We cloned the cDNA coding for the visual Ggamma (Ggamma(e)) subunit from Drosophila which had so far eluded identification at the molecular level. Northern blot analysis revealed the presence of a major, 1.4-kilobase(kb) Ggamma(e) transcript and two minor transcripts of 1.8 and 6 kb in size. The major 1.4-kb mRNA is expressed preferentially in the eye. The spatial expression pattern determined for Ggamma(e) as well as co-immunoprecipitation experiments demonstrated that Ggamma(e) dimerizes with Gbeta(e) to form the heterodimeric Gbetagamma subunit which functions in visual transduction in the Drosophila compound eye. Ggamma(e) shares common characteristics with the visual Ggamma subunits of human rod and cone photoreceptors although different classes of Galpha subunits are employed in vertebrate and invertebrate phototransduction. By the molecular cloning and characterization of the visual gamma subunit of Drosophila one of the few missing links in the well studied Drosophila phototransduction cascade has been characterized to complete our knowledge about the Drosophila visual transduction pathway.

Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins.

Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. The Drosophila visual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the only Drosophila visual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1-R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in "blind" ninaE(17) mutant flies, which lack the gene encoding the visual pigment of the R1-R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1-R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured in Calliphora and Musca. Spectrophotometric analyses of Rh5 and Rh6 in vivo by microspectrophotometry, and of detergent-extracted pigments in vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (lambda(max) = 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (lambda(max) = 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized.

Long-term outcomes after carotid stent placement treatment of carotid artery dissection.

OBJECTIVE: To assess the long-term outcomes after stent placement for the treatment of carotid artery dissections. METHODS: Between 1992 and 1998, seven patients underwent stenting procedures for treatment of extracranial carotid artery dissections resulting from various causes, including trauma (n = 2), iatrogenesis (n = 2), spontaneous development (n = 2), and fibromuscular dysplasia (n = 1). Stenting procedures were performed for large, nonhealing, dissection-induced pseudoaneurysms (four cases) or severe preocclusive stenosis (three cases). A total of 11 stents were placed (Palmaz stents, n = 8; Wallstents, n = 3). Radiological follow-up examinations were performed after a mean period of 17.7 months (range, 1-67 mo), using conventional or computed tomographic angiography. Clinical follow-up data were obtained after a mean period of 42.9 months (range, 13-72 mo). RESULTS: All stent placements resulted in complete resolution of dissection-induced stenosis. For two of the four patients with aneurysms, the lesions occluded spontaneously at the time of the procedure. The third patient required coil embolization of the pseudoaneurysm. One patient exhibited progressive shrinkage of the aneurysm in serial follow-up examinations, with healing after 18 months. No clinical complications were associated with the procedures. One patient exhibited progression to asymptomatic occlusion 3 months after stenting. The remaining six patients exhibited no significant changes in luminal diameters. All patients remained in clinically stable condition, with no ischemic symptoms, during more than 3.5 years (mean period) of follow-up monitoring. CONCLUSION: This experience suggests that stents placed for treatment of extracranial carotid artery dissections remain patent and patients remain free of symptoms on a long-term basis. Additional studies will be required to determine the optimal types of stents and intervals for follow-up monitoring using imaging.

Modulation of myocardial function and [Ca2+] sensitivity by moderate hypothermia in guinea pig isolated hearts.

Cardiac hypothermia alters contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]i over a range of extracellular CaCl2 concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37 degrees C, 27 degrees C, and 17 degrees C. Transmural LV phasic [Ca2+] was measured using the Ca2+ indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+ released per beat (area Ca2+) were plotted as a function of 0.3-4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/dt) and relaxation (-dLVP/dt), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]i and slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]e was shifted upward at 17 degrees C and 27 degrees C, whereas that of phasic [Ca2+]) to [CaCl2]e was shifted upward at 17 degrees C but not at 27 degrees C. The relationships of phasic [Ca2+]i to developed LVP, +dLVP/dt, and LVP(area) were progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+ with little effect on systolic Ca2+ or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+ and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.