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.     

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     

Survival and Repopulation of Irradiated 'Pre-Cfu-C' In Mice

ABSTRACT Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c. burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). the CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely ‘pre-CFU-c’, in the maturation sequence of granulopoiesis, and this maturation of the ‘pre-CFU-c’ to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

A specific concanavalin A-mediated binding of bovine serum α-mannosidase to cultured human skin fibroblasts

A specific elevation of cell-associated α-mannosidase was observed in human skin fibroblasts cultured with concanavalin A for 12–72 hours. There was a latency of several hours before the increase of the enzyme activity occurred. When the cells were washed with α-methylmannoside, α-mannosidase activity was not increased. Other lysosomal enzymes including β-mannosidase showed a slight decrease in activity. It was concluded that the elevation of this enzyme activity was the result of a specific binding to the cell surface mediated by concanavalin A.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.