Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons

High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+]i). We used fura-2 and magfura-2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+]i. We found that fura-2 was useful in measuring small increases in [Zn2+]i associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura-2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist-stimulated increases in [Zn2+]i. The coapplication of 300 microM Zn2+ and 100 microM glutamate/10 microM glycine resulted in a [Zn2+]i increase that was approximately 40-100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 microM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+]i. These findings demonstrate the feasibility of measuring changes in [Zn2+]i in neurons.     

Visualisation of extensive water ribbons and networks in a DNA minor-groove drug complex

The crystal structure is reported of a complex between an ethyl derivative of the minor-groove drug furamidine and the dodecanucleotide duplex d(CGCGAATTCGCG)2, which has been refined to 1.85 A resolution and an R factor of 16.6% for data collected at -173 degreesC. An exceptionally large number (220) of water molecules have been located. The majority of these occur in the first coordination shell of solvation. There are extensive networks of connected waters, both in the major and minor grooves. In particular, there are 21 water molecules associated with the minor-groove drug, via hydrogen bonds from the four charged nitrogen atoms. One cluster of four waters is situated in the groove itself; the majority are on the outer edge of the groove, and serve to bridge between the outward-directed drug nitrogen atoms and backbone phosphate oxygen atoms. These bridges are both intra- and inter-strand, with the net effect that the outer edge of the drug molecule is covered by ribbons of water molecules.     

Rolandic type cerebral palsy in children as a pattern of hypoxic-ischemic injury in the full-term neonate

Magnetic resonance images (MRIs) of the brains of 11 patients aged from 1 week to 12 years with a distinctive type of cerebral palsy were selected based on distribution of cerebral lesions, which were restricted to bilateral perirolandic cortical and subcortical regions, including frequent symmetric involvement of basal ganglia and ventrolateral nucleus of thalami. Retrospectively, the perinatal history and clinical features were reviewed to correlate clinical data with this distinctive pattern of brain injury. Clinically affected neonates had an encephalopathy associated with a severe perinatal asphyxial event. Older children with cerebral palsy survived a similar perinatal course and demonstrated spastic quadriparesis with bulbar or pseudobulbar involvement, lack of verbal speech and variable delays in cognitive development. The distribution of hypoxic-ischemic lesions involving bilateral perirolandic regions, basal ganglia, and thalami, appears to correlate with increased metabolic areas of primary myelination in full-term neonates, but not with arterial border zones nor a single cerebral artery distribution. Myelination is a critical process in maturing brain associated with marked increase in tissue respiration and thus greater susceptibility to oxygen deprivation. It is believed that the extent of hypoxic-ischemic brain injury is determined principally by brain maturity and regional metabolic rates at time of insult and this correlates with active myelination in full-term neonates. This study confirms previous data from neuropathologic literature and recent reports of neuroimaging studies of asphyxiated neonates. In addition, retrospective analysis of the clinical data enables recognition of a type of cerebral palsy that might be the hallmark of hypoxic-ischemic injury in term neonates.     

Lac/Tet dual-inducible system functions in mammalian cell lines

The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.     

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.     

Recognition of chitin and proteins in invertebrate cuticles using analytical pyrolysis/gas chromatography and pyrolysis/gas chromatography/mass spectrometry

Flash pyrolysis/gas chromatography (py/GC) and py/GC/mass spectrometry (MS) have been utilized to characterize the cuticles of invertebrates chemically. Pyrolysis products have been identified and assigned to specific cuticular components. Acetylpyridones, acetamidofuran, 3-acetamido-5-methylfuran and 3-acetamido-(2 and 4)-pyrones are proposed as characteristic pyrolysis markers for chitin. Pyrolysis products displaying ions of m/z 70, 154, 168, 194 are thought to derive from diketopiperazine structures and provide potential markers for proteins and peptides in which proline, alanine, valine, arginine and glycine are the dominant amino acids. These products, constituting specific pyrolysis markers for invertebrate cuticles, may reflect the amino acid composition of their constituent structural proteins. The source of the various pyrolysis products of proteins has been verified by pyrolysis of reference proteins, peptides and amino acid mixtures. The presence of additional pyrolysis products related directly to histidine and catechol moieties is consistent with the chemical structure and composition proposed for arthropod cuticles based on recent work utilizing solid state 13C and 15N nuclear magnetic resonance. This study constitutes the first comprehensive chemical characterization of the pyrolysis products of invertebrate cuticles and provides the basis for future investigations requiring qualitative screening for cross-linked chitin and proteins in modern and fossil cuticles and in materials, e.g. geopolymers, that may be derived from them.     

Comparative evaluation of first- and second-generation digene hybrid capture assays for detection of human papillomaviruses associated with high or intermediate risk for cervical cancer

In the present study we comparatively evaluated the first- and second-generation Digene Hybrid Capture assays for detection of human papillomaviruses (HPV) associated with high or intermediate risk for cervical cancer in cervical specimens. Concordant results were obtained with 468 of 483 (96.8%) specimens. All 15 specimens which gave repeatedly discordant results were positive by the second-generation test only, and 14 of them tested PCR positive. The enhanced sensitivity of the second-generation assay is mainly a result of the reformulation of hybridization reagents and, to a lesser extent, a result of the addition of new HPV probes.