Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sda blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc-GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GM1 and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV3NeuAcnLc4OseCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc4OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta (1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta (1-4)[NeuAc alpha (2-3)]Gal beta (1-3)Gal beta (1-4)GlcCer. 1H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta (1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.     

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.     

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.     

Seasonally of adult male japanese macaques (Macaca fuscata): Androgens and behavior in a confined troop

Seasonal variations in levels of serum testosterone, dihydrotestosterone (DHT), reproductive behavior, and social behavior were investigated in 12 adult males (5 to 20 + years of age) of the Oregon troop of Japanese macaques (Macaca fuscata). Blood samples were collected at 2- to 4-month intervals, and behaviors were monitored twice weekly over a 15-month period. Significant seasonal variations in levels of testosterone and DHT, and in frequencies of mount series, ejaculations, number of female partners, displays, courtship, and aggression were observed. Seasonal variations in reproductive and social behaviors did not correlate with seasonal variations in androgen levels because seasonal increases in these behaviors followed seasonal increases in the androgens with a 1- to 2-month delay. However, significant correlations between increased androgen levels and the onset of mating activity occur when mean monthly frequencies of mount series are shifted 1 to 2 months earlier to coincide with the rise in serum androgen levels. The frequency of adult male play and male-male mounting increased significantly when androgen levels were low. We suggest that photoperiod changes may function as a proximate cue in male Japanese macaques which induces a state of biological readiness for mating, and the behavioral consequences (i.e., mating) are then dependent upon the presence of receptive females.     

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.     

Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase

The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed.     

Heat shock proteins within the mammalian cell cycle: relationship to thermal sensitivity, thermal tolerance, and cell cycle progression

We have measured endogenous and induced rates of 70-kD, 89-kD, and 110-kD heat shock proteins in highly pure G1-, S-, or G2-M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence-activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two-dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S-phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70-kD polypeptide were measured in S and G2-M as compared to G1 following heat shock. Little increase in either the 89-kD or 110k-kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat-induced cell cycle delay or to the resumption of phase-specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Relationship between CO and transit times for dye and thermal indicators in central circulation

We investigated two factors that may influence the estimation of lung water by the thermal-dye double-indicator-dilution method: 1) changes in cardiac output (CO), and 2) thermal equilibration with cardiac tissue. In theory, the difference between mean transit times of thermal and dye indicators (delta MTT) is proportional to the extravascular volume of distribution of the thermal indicator (VODev) and inversely related to CO. The delta MTT also includes a time element DT due to the difference in response times of the measuring instruments such that delta MTT = VODev/CO + DT. In nine anesthetized dogs we recorded 286 aortic thermal and dye curves following left atrial (LA) and right atrial (RA) injections as CO was increased from 2.35 to 6.65 ml X s-1 X kg-1 by isoproterenol infusion, and a regression of delta MTT on CO-1 was performed. DT was measured in vitro for comparison with the y-intercept. In six of nine dogs the slope of the regression for LA injections was not different from zero, indicating that there is no measurable volume of distribution for thermal indicator in cardiac tissue. For RA injections the relationship between delta MTT and CO-1 was linear in all experiments, with an average correlation coefficient of 0.97 +/- 0.01 (SE), indicating that the VODev was constant over a threefold increase in CO. Although the in vitro measurement of DT agreed closely with the average of the y-intercepts of the regressions, small between-subject differences in DT can lead to apparent flow-related changes in extravascular thermal volume computed in the conventional fashion using the in vitro estimate of DT.