A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.     

Intraspecific brood-mixing of the cichlid fishPerissodus microlepis in Lake Tanganyika

Synopsis The frequency and origin of intraspecific brood-mixing in the biparental cichlid fishPerissodus microlepis were investigated by the cohort analysis of schooling young and the underwater observation of guarding parents. The cohort analysis showed that brood-mixing started from the early guarding state when the young were smaller than 10 mm standard length and nearly all schools of young larger than 16 mm contained alien young from up to 6 broods. Brood farming-out of this fish, which was originally proposed to be a way adopted only by a deserted parent, was performed also by paired parents. We suggest that brood-mixing inP. microlepis is attributed mainly to brood farming-out by paired and deserted parents.     

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     

Synthesis of desmosterol and epidesmosterol from hyodeoxycholic acid

A new synthesis of desmosterol was described using hyodeoxycholic acid (3,6-dihydroxy-5β-cholanic acid) as a starting material. Epidesmosterol (3-hydroxycholesta-5,24-diene) was also synthesized for the first time from the same starting material.     

Hereditary respiration deficiency in Saccharomycodes ludwigii

Saccharomycodes ludwigii, supposed to be petite-negative, gave rise to respiration-deficient mutants when acriflavine and ultraviolet irradiation, respectively, were applied to this yeast, strain IFO 1194. The frequency of such mutants was very low as compared with that in Saccharomyces cerevisiae and other petite-positive yeasts. Cytochrome composition was characterized by spectrophotometry at the temperature of liquid nitrogen. The respiratory mutants examined contained cytochrome c unaltered in quality and quantity. Cytochrome b was often present only in small amounts though never absent, while cytochrome a+a₃ was either present or absent. The respiratory mutants could form zygotes after conjugation with a wild-type culture of opposite mating type ( vs. a). The hybridization and segregation analysis of spore tetrads showed the inheritance of respiratory mutant character to be either Mendelian or non-Mendelian and similar to that of pet (nuclear) and rho^- (cytoplasmic) mutants, respectively, in Saccharomyces cerevisiae.     

Serum T3 level in the patients with hyperthyroidism after therapy.

Serum T3 level in various thyroid diseases was determined in unextracted serum with the Dainabot kit for T3 RIA. The serum T3 level in 33 normal subjects was 0.8-1.6 ng/ml. It was 5.7 +/- 3.5 ng/ml (mean +/- S.D.) in 36 hyperthyroid patients, and undetectable to 0.8 ng/ml in 21 hypothyroid patients. Generally the serum T4 and serum T3 decreased in parallel after radioiodine therapy for hyperthyroidism. However, in some cases the serum T3 level remained high in spite of normalized serum T4 after radioiodine therapy. This state indicated "T3-toxicosis", and hyperthyroidism was apt to recur. When thyroid function was observed for 2 years following radioiodine treatment, the ratio of serum T3 (T3 level before treatment/T3 level after treatment) decreased more significantly as compared with the ratio of serum T4 in euthyroid cases. Serum T3 provides a more sensitive index of thyroid function than serum T4 in euthyroid states after radioiodine or anti-thyroid drug therapy. The present data indicate that the serum T3 level and the T4/T3 ratio are valuable aids in the estimation of prognosis of hyperthyroid patients after various treatments.     

Metabolism of 1alpha-hydroxyvitamin D3 to 1alpha, 25-dihydroxyvitamin D3 in perfused rat liver.

The metabolism of 1α-hydroxyvitamin D₃ (1α-OH-D₃) was studied in rat liver perfused with [³H]-1α-OH-D₃. [³H]-1α-OH-D₃ was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D₃ [1α,25-(OH)₂-D₃] by co-chromatography with synthetic 1α,25-(OH)₂-D₃ as well as by gas chromatography-mass spectrometry. [³H]-1α,25-(OH)₂-D₃ appeared in the perfusate as early as 20 min after addition of [³H]-1α-OH-D₃, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D₃ is metabolized to 1α,25-(OH)₂-D₃, which exerts biological effects on bone and intestine.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.