Cloning and sequencing of the rabbit gene encoding T-cell costimulatory molecules

The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D49841 (RCD28), D49844 (RCTLA-4), D49842 (RCD80), and D49843 (RCD86)     

Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface

The mechanism underlying molecular recognition of lectins waselucidated by a novel solid phase binding assay system basedon surface plasmon resonance. When the apparent affinities ofinteractions between chitooligosaccharides and wheat germ agglutininwere compared between lectin-immobilized and oligosaccharide-immobilizedassay systems, the affinity constants (Ka) calculated for theformer system were in good agreement with the previously reportedvalues measured in solution. On the other hand, in the lattersystem, the calculated Ka could be more than 10,000 times higherthan the values in solution at lower lee tin concentrations.To elucidate the reason for this, we systematically investigatedthe effects of the oligosaccha-ride immobilized density andthe lectin valence on the apparent affinity in the oligosaccharide-immobilizedassay system. Both the apparent association (k_ass) and dissociationrate constants (k_diss) showed a tendency to decrease as theoligosaccharide density increased. This effect was most remarkablefor the interaction possessing an extremely fast intrinsic K_ass.Oligomerization of lectin enhanced the avidity due to a significantreduction in k_diss. These phenomena could be explained by consideringthe nonhomogeneous conditions under which binding occurred.The reaction in a nonhomogeneous state is limited by the masstransport effect, and the effect of rebinding becomes so largethat it cannot be disregarded. These findings are the firstto demonstrate the importance of the mass transport effect inmodulating the affinity of lectin for oligosaccharides on asolid phase surface. avidity clustering effect lectin mass transport surface plasmon resonance     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Interaction of cross-sectional area, driving pressure, and airflow of passive velopharynx

Isono, Shiroh, Thom R. Feroah, Eric A. Hajduk, Rollin Brant,William A. Whitelaw, and John E. Remmers. Interaction ofcross-sectional area, driving pressure, and airflow of passive velopharynx. J. Appl. Physiol. 83(3):851-859, 1997.Previous studies have shown that, when thepharyngeal muscles are relaxed, the velopharynx is a highly compliantsegment of the pharynx. Thus, under these circumstances,cross-sectional area of the velopharynx (A_VP), drivingpressure across the velopharynx (P), and inspiratory airflow(I) willbe mutually interdependent variables. The purpose of the presentinvestigation was to describe the interrelation among these threevariables during inspiration. We studied 15 sleeping patients withobstructive sleep apnea/hypopnea when the pharyngeal muscles wererendered hypotonic by applying continuous positive airway pressure tothe nasal airway.A_VP, determined by endoscopic imaging, was significantly greater at onset ofI limitationthan at minimum oropharyngeal pressure(P < 0.01). Snoring was neverobserved duringIlimitation. In a subgroup of six patients, values for P,I, andA_VP were obtainedat 0.1-s intervals at various levels of mask pressure. For these sixpatients, the mathematical expressionI = 0.657(A_VP/A_max) · P^0.332,where A_max ismaximal A_VP,described the relationship among the three variables(R² = 0.962) forflow-limited and non-flow-limited inspirations. The impedance of thepassive velopharynx, defined asP^0.33/,was inversely related toA_VP and increaseddramatically when A_VP was <0.3cm². In summary, we observed aprogressive decrease inA_VP during flow-limited inspiration in patients with obstructive sleep apnea. Thisconstriction of the velopharynx contributes to an increase invelopharyngeal impedance that, in turn, counterbalances the increase inP during flow limitation.

     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

The pollination ecology ofBletilla striata (Orchidaceae)

Anthecological observations of an orchidBletilla striata were carried out in Kobe, Japan. The purplish pink flowers were visited by 26 species of insects belonging to Hymenoptera, Diptera and Lepidoptera, although the flower did not offer food rewards of any kind. Candidates for pollination were seven species of aculeate Hymenoptera with a body size that well fitted the space in between the column and labellum. The most effective candidate was the male of the longhorn beeTetralonia nipponensis, judging from its abundance and legitimate intrafloral behavior. The female ofT. nipponensis and the other six species also contributed to promote out-crossing, but to a lesser extent than the maleT. nipponensis, since they occasionally exhibited illegitimate intrafloral behaviors for pollination and also, their flower visitation rate was lower.     

Ribosomal protein modification in Escherichia coli. I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity.

A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 (rpsE). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ, it has been found that the wild-type allele is dominant over the mutant one. Ts⁺ revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed.