Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of selenite on glucocorticoid receptor

Rat liver glucocorticoid receptor was partially purified and characterized for its hormone binding using selenite. Selenite at very low concentrations irreversibly inhibited the hormone binding. The concentration for half maximal inhibition was approximately 2.8 microM. The inhibition was restored by dithiothreitol. The receptor-hormone complex became considerably insensitive to the selenite inhibition. The receptor inhibited by selenite was eluted at the same position as the native receptor from DEAE ion exchange and gel filtration columns. The results suggest that at least four sulfhydryl groups are located in the hormone binding domain of the receptor making a cluster.     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Pathological and microbiological studies on calf pneumonia occurring in mass rearing facilities

Pathological and microbiological studies were conducted on lesions in the lungs of 194 calves from mass rearing facilities. Macroscopically, the lesions were classified into six forms: nonlesion, atelectasis, mild pneumonia, moderate pneumonia, advanced pneumonia, and advanced pneumonia accompanied with abscess. Histopathological examination revealed bronchopneumonia in most of the calves. Lesions more advanced than moderate pneumonia were complicated with desquamation, severe exudation, and necrosis. Bacteriologically, Pasteurella sp. was isolated often in combination with Staphylococcus sp. from about a half of the atelectatic cases. With the development of pneumonic lesions, Pasteurella sp. was isolated at a high frequency in combination with Haemophilus sp., Streptococcus sp., and Corynebacterium sp. Prominent necrosis was more often seen in cases with Pasteurella haemolytica isolated than in cases with only Pasteurella multocida isolated. Mycoplasma sp. and Ureaplasma sp. were isolated from distinctly pneumonic lesions. Advanced pneumonic lesions were observed in many calves over 30 days of age. The importance of environmental and managerial improvement was also emphasized, since calf pneumonia tended to break out in facilities under unsatisfactory conditions in the present work.     

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.     

Synthesis and Evaluation of Pyrrole Polyamide- 2′-Deoxyguanosine 5′-Phosphate Hybrid

Pyrrole polyamide-2′-deoxyguanosine 5′-phosphate hybrid (Hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was found to exhibit dose-dependent inhibition of cell growth.     

Direct measurement of renal sympathetic nervous activity in high-fat diet-related hypertensive rats

The elevation of renal sympathetic nervous activity (SNA) is a possible cause of blood pressure (BP) elevation. Although a high-fat diet (FAT) often induces BP elevation in animals, the effect of FAT on renal SNA in animals is not consistent between studies. Thus, we compared the basal levels of efferent renal SNA and BP in FAT- or high-carbohydrate diet (CHO)-fed rats. Twenty-four male Sprague-Dawley rats were fed FAT (P/F/C=20/45/35% cal) or CHO (20/5/75) from 5 weeks of age. After 20-21 weeks of feeding, a 24-h urine sample was collected to measure sodium excretion. The next day, blood (0.2 ml) was withdrawn from a femoral artery, and basal efferent renal nerve discharges and mean arterial pressure (MAP) were recorded under anesthesia. Immediately after the experiment, abdominal (epididymal, perirenal and mesenteric) adipose tissues were dissected. Total abdominal fat weight was significantly greater in the FAT group than in the CHO group. The plasma level of leptin was significantly higher in the FAT group, but blood glucose and plasma insulin levels did not differ between the two groups. MAP and renal SNA were significantly higher in the FAT group. In addition, the ratio of urinary sodium excretion to dietary sodium intake was significantly lower in the FAT group than in the CHO group. The data suggest that the increased renal SNA may contribute to BP elevation in FAT-fed rats. The present study firstly demonstrated that renal SNA was elevated with FAT-related BP elevation.     

Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N⁴-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N⁴-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N¹-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k_cat/K_m value for N¹-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N¹-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k_cat/K_m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N¹-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N¹-aminopropylagmatine. Furthermore, spermine and N⁴-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.Polyamines are positively charged aliphatic compounds. Putrescine [4], spermidine [34], and spermine [343] are common polyamines observed in various living organisms, from viruses to humans (16). Polyamines, which play important roles in cell proliferation and cell differentiation (19, 34), are thought to contribute to adaptation against various stresses (9, 26). In thermophilic microorganisms, polyamines contribute to growth under high-temperature conditions. Indeed, in the thermophilic bacterium Thermus thermophilus, a mutant strain lacking the enzyme related to polyamine biosynthesis shows defective growth at high temperatures (23). Furthermore, thermophilic archaea and bacteria possess long-chain and branched-chain polyamines such as N⁴-aminopropylspermidine [3(3)4], N⁴-aminopropylspermine [3(3)43], and N⁴-bis(aminopropyl)spermidine [3(3)(3)4], in addition to common polyamines (11, 13, 14). N⁴-aminopropylspermine was detected in the cells of thermophiles, such as Saccharococcus thermophilus, thermophilic Bacillus and Geobacillus spp. (Bacillus caldolyticus, B. caldotenax, B. smithii, Geobacillus stearothermophilus, and G. thermocatenulatus), Caldicellulosiruptor spp. (C. kristjanssonii and C. owensensis) and Calditerricola spp. (C. satsumensis and C. yamamurae) (10, 12, 22), but it was not detected in archaea. These unique polyamines are thought to support the growth of thermophilic microorganisms under high-temperature conditions. An in vitro study indicated that long-chain and branched-chain polyamines effectively stabilized DNA and RNA, respectively (32).Polyamines are synthesized from amino acids such as arginine, ornithine, and methionine (26). In most eukaryotes, putrescine is synthesized directly from ornithine by ornithine decarboxylase (34). Plants and some bacteria possess additional or alternative putrescine biosynthesis pathways in which putrescine is synthesized from arginine via agmatine (18, 31, 35). In this pathway, agmatine is synthesized by arginine decarboxylase, and agmatine is converted to putrescine by agmatine ureohydrolase or a combination of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Longer polyamines are then produced by the addition of the aminopropyl group from decarboxylated S-adenosylmethionine. This pathway is shown on the left in Fig. ​Fig.11 (pathway I). On the other hand, the thermophilic bacterium T. thermophilus possesses a unique polyamine-biosynthetic pathway (23) in which spermidine is synthesized from agmatine via N¹-aminopropylagmatine by aminopropyl transferase followed by ureohydrolase, as shown on the right in Fig. ​Fig.11 (pathway II).Open in a separate windowFIG. 1.Predicted biosynthetic pathway of polyamines in T. kodakarensis. (A) Predicted biosynthetic pathway. Pyruvoyl-dependent arginine decarboxylase proenzyme (TK0149), arginine/agmatine ureohydrolases (TK0240/TK0474/TK0882), aminopropyl transferase (TK0147), and pyruvoyl-dependent S-adenosylmethionine decarboxylase proenzyme (TK1592) are shown based on the genome analysis. (B) Structures of unique polyamines.A sulfur-reducing hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, was isolated from Kodakara Island, Kagoshima, Japan (1, 21). This archaeon grows at temperatures between 60°C and 100°C but optimally at 85°C. Under low- or high-temperature-stressed conditions, T. kodakarensis produces cold- or heat-inducible chaperones to adapt to unfavorable growth environments (4, 5, 30). The lipid composition of the membrane also changes depending on the growth shift (20). In addition to acting as such tolerance factors, polyamines have been suggested to play an important role in maintaining nucleosomes in high-temperature environments (15). A complete genome analysis of T. kodakarensis has been performed, and the pathway of polyamine biosynthesis has been predicted (Fig. ​(Fig.1)1) (6, 7). It has been speculated that putrescine is synthesized from arginine via agmatine by arginine decarboxylase (PdaD_Tk) and agmatine ureohydrolase. Long- and/or branched-chain polyamines are then produced by the addition of the aminopropyl group derived from decarboxylated S-adenosylmethionine. Previously, we revealed that PdaD_Tk catalyzed the first step of polyamine biosynthesis and was essential for cell growth (6). The strain DAD, which lacks the gene pdaD_Tk, does not grow in medium without agmatine. Archaeal cells are known to use agmatine to synthesize agmatidine, which is an agmatine-conjugated cytidine found at the anticodon wobble position of archaeal tRNA^Ile (17). Agmatine is important for agmatidine synthesis as well as long-chain polyamine. In the present study, we focused on the subsequent steps in polyamine biosynthesis, especially from agmatine to spermidine. T. kodakarensis possesses three agmatine ureohydrolase homologues (TK0240, TK0474, and TK0882); however, it is unclear which one is dominantly functional in T. kodakarensis cells. In a closely related genus, Pyrococcus, TK0474 and TK0882 orthologues have been identified, but the TK0240 orthologue is missing in Pyrococcus genomes. In Pyrococcus horikoshii, PH0083, which is an orthologue of TK0882, was shown to possess agmatine ureohydrolase activity (8). TK0882, hence, appears to possess agmatine ureohydrolase activity as well. It is unclear whether other agmatine ureohydrolase homologues (TK0240 and TK0474) are involved in polyamine synthesis and cell growth in T. kodakarensis. In addition to agmatine ureohydrolase, aminopropyl transferase plays a crucial role in the synthesis of polyamines. TK0147 was annotated first as spermidine synthase and shares sequence identity with aminopropyl transferase (PF0127) from Pyrococcus furiosus (3). It is therefore expected to harbor the function of aminopropyl transferase for long-chain-polyamine synthesis. Recombinant PF0127 showed broad amine acceptor specificity for agmatine, 1,3-diaminopropane (3), putrescine, cadaverine (5), sym-nor-spermidine (33), and spermidine. While maximal catalytic activity was observed with cadaverine, agmatine was most often preferred on the basis of the k_cat/K_m value (3), suggesting that pathway II is a dominant route for polyamine synthesis in P. furiosus. In the present study, various disruptants lacking genes for polyamine biosynthesis were constructed in order to understand the physiological roles of these enzymes in T. kodakarensis. The cell growth profiles and cytoplasmic polyamines of the wild type and the disruptants were analyzed and compared. Recombinant enzymes were also purified and characterized. The obtained results are expected to provide useful information regarding the specific roles of polyamines in thermophiles.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells.

Oligodeoxynucleotides containing CpG motifs have been highlighted as potent Th1 activators. We previously reported that Ag and CpG, when conjugated together, synergistically promoted the Ag-specific Th1 development and inhibited the Th2-mediated airway eosinophilia. In this study, we examined the mechanisms underlying the synergism of the covalent conjugation. The CpG-OVA conjugate enhanced the Th1 activation and development. These characteristic features of the conjugate could not be ascribed to the polymerization of OVA, but mirrored the augmented binding of the CpG-tagged Ag to dendritic cells (DCs) in a CpG-guided manner, because phycobiliprotein, R-PE, conjugated to CpG stained a higher proportion of DCs with higher intensity than the mixture. R-PE fluorescence was emitted from cytoplasmic portions of the DCs, which simultaneously expressed costimulatory molecules and IL-12. The CpG-conjugated R-PE trafficking described above actually served as a potent Ag. These results indicate that CpG conjugated to Ag exhibit novel joint properties as promoters of Ag uptake and DC activators, thereby potentiating the ability of DCs to generate Th1 cells. The DNA-mediated promotion of Ag uptake would be advantageous for evoking host immune responses against invading microorganisms.