Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5''-flanking region.

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.     

The real objective of Mendel's paper: A response to Monaghan and Corcos

Mendel's work in hybridization is ipso facto a study in inheritance. He is explicit in his interest to formulate universal generalizations, and at least in the case of the independent segregation of traits, he formulated his conclusions in the form of a law. Mendel did not discern, however, the inheritance of traits from that of the potential for traits. Choosing to study discrete non-overlapping traits, this did not hamper his efforts.     

Cloning and Mapping of a Novel Nodulation Region from Bradyrhizobium japonicum by Genetic Complementation of a Deletion Mutant

The phenotypes of a set of Bradyrhizobium japonicum 110 mutants with large deletions in the region of symbiotic gene cluster I were tested. The majority of the mutants showed a delayed nodulation on soybean and, by mixed-infection experiments, were found to be strongly reduced in their competitiveness. Phenotypic comparison of mutants with different deletion endpoints allowed a preliminary localization of two genomic regions, called nod-1 and nod-2, which were required for normal nodulation on soybean. Loss of nod-1 was found to result in a Nod⁻ phenotype on cowpea, mung bean, and siratro. A recombinant cosmid was identified which fully restored nodulation ability of a mutant lacking nod-1. Using Tn5-containing derivatives and subclones of this cosmid for complementation, we delimited the nod-1 region to a DNA segment of 3.1 to 3.5 kilobase pairs.     

Capacity of different cell types to stimulate cytotoxic T lymphocyte precursor cells in the presence of interleukin 2

Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by 1H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

The Taung endocast: a reply to Holloway

Indices of rostrality (ir, ir') are developed to assess the extent to which the medial end of the lunate sulcus (L) is rostrally positioned in photographs and figures of lateral views of primate brains and endocasts, and indices are determined for chimpanzees, SK 1585 and the Taung endocast. Ir quantifies the extent of rostrality as it has traditionally been viewed (in A-P projections) while ir' takes dorsal curvature into account. The ir of the feature that I have identified as the lunate sulcus of Taung is within one standard deviation of the mean ir for Pan and its ir' is within 1.5 standard deviations from the mean ir' for Pan. Both findings are compatible with my earlier statement that the medial end of the lunate sulcus of the Taung endocast is in a pongid-like position. Use of stereoplotting to transfer the position of L from chimpanzee endocasts and brains to australopithecine endocasts is critically assessed: Holloway stereoplotted five chimpanzee brains and then transferred their mean coordinates that describe the lunate sulcus to the Taung endocast. If stereoplotting successfully transfers the extent to which L is rostrally located, one would expect the mean L of Pan and its transferred counterpart in Taung to have identical index values of rostrality. However, the ir of the lunate sulcus that Holloway located on Taung is over two standard deviations lower than the mean ir for the five chimpanzees he stereoplotted to determine its angular coordinates, and Holloway's ir' for Taung is one standard deviation lower than the five chimpanzees' mean ir'. These discrepancies are shown to be due to shape differences, and it is concluded that stereoplotting should not be used to transfer sulci between differently shaped endocasts without correcting for these differences. I also reply to Holloway's criticisms of my use of L/H indices, palpation, techniques for sampling endocasts, and illustration of the Taung endocast. It is shown that there is room on the Taung specimen for the lateral end of L, and the pongid-like sulcal pattern of Taung is reaffirmed. Thus, we do not yet know when human-like sulcal patterns first appeared in the hominid fossil record.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.