Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.     

Studies on transfer ribonucleic acids and related compounds. VIII(1). Further studies on aromatic phosphoramidates as a protecting group for phosphomonoesters

Stability of aromatic phosphoramidates was studied using 2',3'-O-dibenzoyluridine 5'-phosphoramidates and N,2',3'-O-tribenzoylcytidine 5'-phosphate. The effect of dicyclohexylcarbodiimide in this mixture was investigated. Decomposition of the anilidate was slower in the presence of DCC.Substituted anilidates of uridine 5'-phosphate were synthesized and the stability of these amidates in anhydrous pyridine was studied.2'-O-Benzoyluridine 3'-phosphoranilidate and the corresponding beta-naphthylidate were compared in their stabilities in anhydrous pyridine, 50% aqueous pyridine and 80% acetic acid. 2'-O-Benzoyluridine 3'-phosphoro-beta-naphthylidate was used for synthesis of dinucleotides.     

Genetic diversity and divergence of the endangered freshwater goby Rhinogobius sp. BB in Okinawa Island

The genetic diversity and population structure of Rhinogobius sp. BB, the fluvial landlocked endangered goby, were investigated and were compared with those of the closely related amphidromous Rhinogobius sp. MO at 11 microsatellite loci. Specimens of Rhinogobius sp. BB were collected from the Genka and Takae-A rivers, and those of Rhinogobius sp. MO were collected from the Genka and Suginda rivers in Okinawa Island. At 11 microsatellite loci, the two populations of Rhinogobius sp. BB showed lower variation than the two Rhinogobius sp. MO populations: the average number of alleles was 3.6 and 2.0 vs. 8.6 and 7.6, respectively; and the observed heterozygosity was 0.263 and 0.281 vs. 0.440 and 0.545, respectively. Pairwise F_st tests showed significant differences (P < 0.001) among the populations: F_st was 0.525 between the two Rhinogobius sp. BB populations, 0.079 between the two Rhinogobius sp. MO populations, and varied from 0.456 to 0.462 for comparisons among Rhinogobius sp. BB and MO. Nei’s genetic distance between the two Rhinogobius sp. BB populations is extremely large (0.604) compared with that between the two Rhinogobius sp. MO populations (0.126). The two populations of Rhinogobius sp. BB are genetically divergent, and they have extremely low genetic diversity. Therefore, the conservation of Rhinogobius sp. BB in Okinawa Island requires the assessment of each river’s population.     

National surveillance of <Emphasis Type="Italic">Salmonella enterica</Emphasis> in food-producing animals in Japan

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 β-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.     

Aminoglycoside binding sites in the cochlea as revealed by neomycin-gold labelling

Neomycin/bovine serum albumin/gold was used as a probe to detect the binding sites of aminoglycosides on the thin sections of the cochlea embedded in Spurr. The binding sites were mainly located on the stereocilia, the cuticular plate of hair cells, the head plates of Deiters' cells, fibrous structures in pillar cells, in the spiral limbus and tectorial membrane and basilar membrane, plasma membranes, mitochondria and the chromatin of various kinds of cells. Triphosphoinositide, acidic glycosaminoglycans, and RNA were considered to be responsible for the binding activity.     

Identification of β-galactofuranosyl residues and their rapid internal motion in the Penicilliumochro-chloron cell wall probed by 13C NMR

Polysaccharides, which come into resonances in the ¹³C NMR spectrum of $\text{Penicillium}\text{ochro-chloron}$ intact mycelium and give anomeric carbon signals at 107.5 and 108.3 ppm, are associated with the cell wall. By ¹³C NMR and gas liquid chromatography analysis, it is shown that the polysaccharides are two types of β-galactofuranosyl residues, one of which has (1→2)-β-galactofuranosyl linkages. Both β-galactofuranosyl residues, which are minor cell wall components, experience rapid internal motion in the cell wall.     

Morphology and evolution of the ectethmoid-mandibular articulation in the meliphagidae (Aves)

The ectethmoid-mandibular articulation in Melithreptus and Manorina (Meliphagidae: Aves) consists of the dorsal mandibular process fitting into and abutting against the ventral ectethmoid fossa; it forms a brace for the mandible. This articulation in Melithreptus is a typical diarthrosis with long folded capsular walls. The mandible, thus, has two separate articulations, each with a different axis of rotation. No other genus of Meliphagidae (except Ptiloprora) or any other avian family possesses a similar feature. The jaw and tongue musculature of Melithreptus are described. The two muscles opening the jaws are well developed, while those closing the jaws are small. The tongue muscles show no special developments. A large maxillary gland, presumably muscus secreting, covers the ventral surface of the jaw muscles. Its duct opens into the oral cavity just behind the tip of the upper jaw. The frilled tip of the tongue rests against the duct opening. The ectethmoid-mandibular articulation braces the adducted mandible against dorsoposteriorly directed forces. The mandible can be held closed without a compression force exerted by the mandible on the quadrate, permitting the bird to raise its upper jaw with greater ease and less loss of force. The tongue can be protruded through the slight gap between the jaws, moving against the duct opening and thus be coated with mucus. Presumably, these birds capture insects with their sticky tongue. Hence, the ectethmoid-mandibular articulation is an adaptation for this feeding method; it evolved independently in three genera of the Meliphagidae. The ectethmoid-mandibular articulation demonstrates that a bone can have two articulations with different axes of rotation, that the two articular halves can separate widely, and that articular cartilages can be flat and remain in contact over a large area. Its function suggests that the basitemporal articulation of the mandible found in many other birds has a similar function. And it demonstrates that in the evolution of the mammalian dentary-squamosal articulation, the new hinge did not have to lie on the same rotational axis as the existing quadrate-articular hinge.     

Analysis of Nuclear Accumulation of Influenza NP Antigen in von Magnus Virus-Infected Cells

When 1–5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5⁺ virus by four successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cells NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble form and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells is not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.