Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Simulation Model of the Coupling between Nitrification and Denitrification in a Freshwater Sediment

A model was constructed to simulate the results of experiments which investigated nitrification and denitrification in the freshwater sediment of Lake Vilhelmsborg, Denmark (K. Jensen, N. P. Sloth, N. Risgaard-Petersen, S. Rysgaard, and N. P. Revsbech, Appl. Environ. Microbiol. 60:2094-2100, 1994). The model output faithfully represented the profiles of O₂ and NO₃^- and rates of nitrification, denitrification, and O₂ consumption as the O₂ concentration in the overlying water was increased from 10 to 600 μM. The model also accurately predicted the response, to increasing O₂ concentrations, of the integrated (micromoles per square meter per hour) rates of nitrification and denitrification. The simulated rates of denitrification of NO₃^- diffusing from the overlying water (D_w) and of NO₃^- generated by nitrification within the sediment (D_n) corresponded to the experimental rates as the O₂ concentration in the overlying water was altered. The predicted D_w and D_n rates, as NO₃^- concentration in the overlying water was changed, closely resembled those determined experimentally. The model was composed of 41 layers 0.1 mm thick, of which 3 represented the diffusive boundary layer in the water. Large first-order rate constants for nitrification and denitrification were required to completely oxidize all NH₄⁺ diffusing from the lower sediment layers and to remove much of the NO₃^- produced. In addition to the flux of NH₄⁺ from below, the model required a flux of an electron donor, possibly methane. Close coupling between nitrification and denitrification, achieved by allowing denitrification to tolerate some O₂ (~10 μM), was necessary to reproduce the real data. Spatial separation of the two processes (no toleration by denitrification of O₂) resulted in too high NO₃^- concentrations and too low rates of denitrification.     

Differential surface characteristics of M cells from mouse intestinal Peyer''s and caecal patches

Summary The mouse caecal patch is located near the blind end of the caecum, and consists of a group of lymphoid follicles. In common with the Peyer's patches, the follicle-associated epithelium overlying these follicles is largely composed of enterocytes, goblet cells and membranous epithelial (M) cells. Each of these types of cell was readily identified by electron microscopy, although caecal patch enterocytes and M cells were morphologically distinct from those of the Peyer's patches. Staining for alkaline phosphatase activity demonstrated that the majority of caecal follicle-associated epithelial cells were alkaline phosphatase-negative, positive cells consisting of a mixture of enterocytes and M cells. In contrast, it has previously been found that Peyer's patch enterocytes are positive for alkaline phosphatase while the M cells are relatively lacking in alkaline phosphatase activity. Lectin histochemistry revealed that surface glycoconjugate expression differs between the caecal and Peyer's patch follicle-associated epithelial cells; in particular, the characteristic staining of Peyer's patch M cells by Ulex europaeus agglutinin 1 was absent on the caecal patch follicle-associated epithelium. These altered surface characteristics indicate that the development of the caecal patch follicle-associated epithelial cells is influenced by the local environment, and these altered properties may be indicative of modified functional roles for the cells at this site.     

Genic diversity in Red River pupfish Cyprinodon rubrofluviatilis (Atheriniformes: Cyprinodontidae) and its implications for the conservation genetics of the species

Starch gel electrophoresis of proteins was used to study geographic variation at 26 gene loci in the Red River pupfish ( Cyprinodon rubrofluviatílís ), a species restricted to west Texas and Oklahoma. Marked differences were detected between populations in the Red and Brazos river drainages, with fixed or nearly fixed differences occurring at five gene loci. In addition, mean heterozygosity was uniformly high for the Red River form ( = 0·076–0·101) while samples of the Brazos River form were genetically depauperate ( =0·00–0·017). Introduced populations in the South Canadian and Colorado river drainages appear to have been derived from the Red River drainage. The presence of alleles diagnostic of the Red and Brazos river forms supports the suggestion from previous work that they may represent cryptic species. Regardless of taxonomy, however, the presence of two genetically distinct forms must be taken into consideration by those concerned with maintenance of biotic diversity.     

A (1→3,1→4)-β-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)-β-glucans

Monoclonal antibodies were raised against a (1→3,1→4)-β-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)-β-glucan, displaying no binding activity against a (1→3)-β-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)-β-glucan, purified by size exclusion chromatography and characterized by ¹H-NMR and anion exchange chromatography. These (1→3,1→4)-β-oligoglucosides, together with (1→3)-β- and (1→4)-β-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 10⁴ M⁻¹ for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)-β-glucans. The assay operates in the range 1–10 ng ml⁻¹ and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl-pachyman ((1→3)-β-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1→3)-β-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10^–7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10^–7). No colonies were seen on control plates (frequency < 0.96 × 10^–9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.     

Characterization of pea chloroplastic carbonic anhydrase. Expression inEscherichia coli and site-directed mutagenesis

A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed inE. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.