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.     

Is serotonin a chemical transmitter in the frog taste organ?

By artificially perfusing the frog tongue with serotonin (5HT) and its antagonists, the possibility of 5HT as a chemical transmitter from taste cells to nerve terminals in frog taste organ was examined. Although serotonin creatinine sulfate, when perfused through the lingual artery, produced impulse discharges in the glossopharyngeal nerve, creatinine sulfate elicited a similar response. Neural responses to taste stimuli were depressed by perfusion with 5HT. Among many antiserotonergic drugs perfused through the lingual artery, LSD was the only one which modified responses to taste stimuli. LSD suppressed taste responses to NaCl, CaCl₂ and water, while LSD at a high concentration (10^?5 g/ml) enhanced responses to guinine and HCl. When PCPA (DL-p-chlorophenylalanine) was injected intraperitoneally in conbination with reserpine, the agent did not significantly change taste responses. The above results possibly suggest that 5HT would not be a chemical mediator from taste cells to nerve terminals.     

Relative potency in acute and chronic suppressive effects of prednisolone and betamethasone on the hypothalamic-pituitary-adrenal axis in man

The relative potency in the hypothalamic-pituitary-adrenal (HPA) suppression of both prednisolone and betamethasone was examined in an acute study with normal volunteers and in a chronic study with glucocorticoid-treated patients. Circadian rhythm of plasma cortisol was studied after a single dose administration of 5 to 30 mg prednisolone or 0.5 to 3.0 mg betamethasone at 8:00 hr. Morning-rise of plasma cortisol occurred on the morning after the administration of 30 mg or less prednisolone but no morning rise was noted after the administration of 1.0 mg or more betamethasone. Plasma ACTH was slightly elevated on the morning after 30 mg prednisolone administration but showed low levels throughout the night after 3.0 mg betamethasone administration. Plasma cortisol responsiveness to ACTH was examined in patients before and during therapy with either prednisolone or betamethasone. The basal cortisol level was not suppressed and the responsiveness to ACTH remained nearly normal during long-term 5 mg prednisolone therapy, but these were completely suppressed during long-term 5 mg betamethasone therapy. The responsiveness to ACTH was nearly normal in patients receiving alternate-day therapy with prednisolone in such large doses as 50 or 60 mg every other day, but was completely suppressed in patients receiving 1.0 mg betamethasone every other day. The relative potency of betamethasone in acute and chronic suppressive effects on the HPA system seems to be much stronger than that of prednisolone in equivalent doses with comparable anti-inflammatory effects. It is also suggested that the alternate-day therapy with such long-acting steroids as betamethasone are useless in preventing HPA suppression.     

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.     

The polymerization of α-aminopropionitrile

The mixture of -aminopropionitrile and ,-iminodipropionitrile polymerized to solidify almost at the temperature near 0°C during 8 years. The conversions based on decreasing of those reactants were 61 and 98% at 4 and 8 years, respectively. The fractionation of 4 and 8 years product using Sephadex G 10 yielded their predominant amounts in the oligomer and polymer section, respectively. The oligomer section product of 8 years product was analyzed by means of ion exchange chromatography and its trimethylsilyl derivative was also analyzed by means of gas chromatography combined with mass spectrometry. These results identified dialanine and trialanine and their amides and nitriles.