Morphology and life history of Scytosiphon canaliculatus comb. nov. (Scytosiphonales,Phaeophyceae) from Japan*

Morphology of field material and life history in culture were studied in Scytosiphon canaliculatus (Setchell et Gardner) comb. nov. from northern Japan. Erect gametophytes of S. canaliculatus are cylindrical, tubular, up to 7 mm wide and 40 cm long, and without regular constrictions. S. canaliculatus has pronounced anisogamy and ascocysts accompanied with plurilocular gametangia. The life history of S. canaliculatus showed an alternation between erect gametophytes and crustose prostrate sporophytes bearing unilocular sporangia. Since field sporophytes of S. canaliculatus were found to be identical with Hapterophycus canaliculatus Setchell et Gardner (Ralfsiaceae, Phaeophyceae), it is proposed to transfer H. canaliculatus to the genus Scytosiphon. In the field, gametophytes with plurilocular gametangia appeared in spring and disappeared in summer. Sporophytes with unilocular sporangia were collected in late autumn and winter. Unilocular sporangia were produced at 15°C in short-day culture conditions and unispores developed into erect gametophytes at 5–15°C. It is suggested that the seasonal Occurrence of gametophytes in the field is due to the seasonal formation of unilocular sporangia, which is regulated by temperature and photoperiod.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.     

Diverse Expression of β1,4-N-Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain

Abstract: Among various tissues of mouse, β1,4- N -acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

The relationship between amide proton chemical shifts and secondary structure in proteins

Summary The parameters for HN chemical shift calculations of proteins have been determined using data from high-resolution crystal structures of 15 proteins. Employing these chemical shift calculations for HN protons, the observed secondary structure chemical shift trends of HN protons, i.e., upfield shifts on helix formation and downfield shifts on -sheet formation, are discussed. Our calculations suggest that the main reason for the difference in NH chemical shifts in helices and sheets is not an effect from the directly hydrogen-bonded carbonyl, which gives rise to downfield shifts in both cases, but arises from an additional upfield shift predicted in helices and originating in residues i-2 and i-3. The calculations also explain the well-known relationship between amide proton shifts and hydrogen-bond lengths. In addition, the HN chemical shifts of the distorted amphipathic helices of the GCN4 leucine zipper are calculated and used to characterise the solution structure of the helices. By comparing the calculated and experimental shifts, it is shown that in general the agreement is good between residues 15 and 28. The most interesting observation is that in the N-terminal half of the zipper, although both calculated and experimental shifts show clear periodicity, they are no longer in phase. This suggests that for the N-terminal half, in the true average solution structure the period of the helix coil is longer by roughly one residue compared to the NMR structures.     

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain

Summary The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 Å. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Å from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.Abbreviations rmsd root-mean-square deviation - NOE nuclear Overhauser enhancement - 4D-SA simulated annealing in four-dimensional space - Myb-R2R3 repeats 2 and 3 of the DNA-binding domain of the c-Myb protein - DNA 16 Myb-specific binding DNA duplex with 16 base pairs - IHDD-C residues 3 to 59 of the C-chain of the engrailed homeodomain-DNA complex - DNA11 DNA duplex with base pairs 9 to 19 of the engrailed homeodomain-DNA complex     

In situ CO2 gas-exchange in fruits of a tropical tree,Durio zibethinus Murray

We examined the in situ CO₂ gas-exchange of fruits of a tropical tree, Durio zibethinus Murray, growing in an experimental field station of the Universiti Pertanian Malaysia. Day and night dark respiration rates were exponentially related to air temperature. The temperature dependent dark respiration rate showed a clockwise loop as time progressed from morning to night, and the rate was higher in the daytime than at night. The gross photosynthetic rate was estimated by summing the rates of daytime dark respiration and net photosynthesis. Photosynthetic CO₂ refixation, which is defined as the ratio of gross photosynthetic rate to dark respiration rate in the daytime, ranged between 15 and 45%. The photosynthetic CO₂ refixation increased rapidly as the temperature increased in the lower range of air temperature T _c (T _c <28.5 °C), while it decreased gradually as the temperature increased in the higher range (T _c 28.5 °C). Light dependence of photosynthetic CO₂ refixation was approximated by a hyperbolic formula, where light saturation was achieved at 100 mol m^–2 s^–1 and the asymptotic CO₂ refixation was determined to be 37.4%. The estimated gross photosynthesis and dark respiration per day were 1.15 and 4.90 g CO₂ fruit^–1, respectively. Thus the CO₂ refixation reduced the respiration loss per day by 23%. The effect of fruit size on night respiration rate satisfied a power function, where the exponent was larger than unity.