Human T cells in cord blood: Abnormalities in Ia antigens induction by phytohemagglutinin and in autologous mixed lymphocyte reactions

About 25% of human T cells isolated from cord blood acquired Ia antigens following stimulation with phytohemagglutinin (PHA) for 72 hr. This percentage is markedly lower than that found in PHA-activated T-cell populations (PHA-T cells) isolated from peripheral blood of adults. The low expression of Ia antigens by human T cells from cord blood does not reflect abnormalities in the sensitivity to PHA stimulation and/or in the kinetics of induction of Ia antigens. PHA-T cells from cord blood display a low stimulatory activity in autologous mixed lymphocyte reactions (MLR). The defect does not reflect a nonspecific abnormality in the stimulatory activity of PHA-T cells from cord blood, since the latter do not differ from PHA-T cells from adults in their ability to stimulate allogeneic T cells from adults. Furthermore the defect does not reflect a nonspecific abnormality in the proliferative response of T cells from cord blood, since the latter display a normal proliferative response to PHA-T cells from adults. The defect in the proliferative response is not restricted to the autologous MLR with PHA-T cells, since it was found also in autologous MLR with non-T cells as stimulators. Correlation of the temporal evolution of the abnormalities of human T cells with the maturation of the immune system may contribute to our understanding of the role of Ia antigens in cell-cell interactions and of the biological significance of abnormalities of autologous MLR.     

Untersuchung der eiweißspindeln des kakteen-X-Virus mit fluoreszierenden Antikörpern

A?koliv na zá kladě mnoha pokus? se p? edpokládalo, ?e tzv. bÍlkovinná v?etena v buňkách tzn. buně?né inkluse X-viru kaktus? (Ca XV), jsou slo?ena z ?etních prodlou?ených ?ásti Ca XV, p?esto to dosud nebylo proká zá no. Proto jsme se pokusili pomocÍ fluoreskujÍcÍch protilátek doká ?at, ?e bilkovinná v?etena jsou skute?ně agregáty virových ?ástic. V těto práci jsme pouzili tzv. nep?Ímé metody. Nejprve jsme p? sobili na buňky obsahujÍci tato v?etena homologiokým antisé rem proti Ca XV, zÍskanym imunizacÍ králÍk? a teprve potom jsme buňky vlo?ili do roztoku fluoreskujicÍch protilátek proti králicimu γglobulinu. BÍlkovinná v?etena svitila potom ve fluorescen?nÍm mikroskopu silně ?lutozeleně (bylopou?ito fluoresceinisothiocyaná tu). Tato fluorescence ná m uká zala, ?e nastala pozitivnÍ reakce a ?e bÍlkovianá v?etena jsou slo?ena z virových ?ástic. ?etné kontrolnÍ pokusy potvrdily ná? základnÍ pokus.     

Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization.     

How fast is fast? Eco‐evolutionary dynamics and rates of change in populations and phenotypes

It is increasingly recognized that evolution may occur in ecological time. It is not clear, however, how fast evolution – or phenotypic change more generally – may be in comparison with the associated ecology, or whether systems with fast ecological dynamics generally have relatively fast rates of phenotypic change. We developed a new dataset on standardized rates of change in population size and phenotypic traits for a wide range of species and taxonomic groups. We show that rates of change in phenotypes are generally no more than 2/3, and on average about 1/4, the concurrent rates of change in population size. There was no relationship between rates of population change and rates of phenotypic change across systems. We also found that the variance of both phenotypic and ecological rates increased with the mean across studies following a power law with an exponent of two, while temporal variation in phenotypic rates was lower than in ecological rates. Our results are consistent with the view that ecology and evolution may occur at similar time scales, but clarify that only rarely do populations change as fast in traits as they do in abundance.     

Identification and characterization of DUSP27, a novel dual-specific protein phosphatase

A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4-14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50 kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism.