The Mdm2 gene of zebrafish (Danio rerio): preferential expression during development of neural and muscular tissues, and absence of tumor formation after overexpression of its cDNA during early embryogenesis

The Mdm2 protein is most probably the main negative cellular regulator of the p53 tumor-suppressor protein. It was found to be overexpressed in a great number of human tumors and is considered as a potential target for anti-tumor therapies. Mdm2 is an essential gene in mice, yet its role in normal development and tissue differentiation is unknown. In order to study the role of this important protein in an evolutionary perspective, we cloned an Mdm2 cDNA from the fish Danio rerio and analyzed its expression pattern as well as the phenotypic consequences of its overexpression. The main functional domains as well as the interaction between Mdm2 and p53 are conserved in zebrafish. Moreover, we show here that the gene is expressed specifically during early development in neural and muscular tissues. Surprisingly, microinjection of Mdm2 mRNA in two-cell-stage embryos led to inhibition of cellular convergence during gastrulation. The clones derived from Mdm2 microinjected blastomeres were significantly smaller than those derived from control microinjections, and, in contrast to what was observed in Xenopus, did not develop tumors. Our results suggest that Mdm2 expression may be important during the differentiation of neural and muscular tissues of zebrafish. They also point to important differences between phyla in the susceptibility to tumor formation.     

An association,in adult Japanese,between the occurrence of rogue cells among cultured lymphocytes (JC virus activity) and the frequency of "simple" chromosomal damage among the lymphocytes of persons exhibiting these rogue cells.

Data from a previous study of the cytogenetic effects, in cultured lymphocytes, of exposure to the atomic bomb in Hiroshima have been reanalyzed to determine the relationship between the occurrence of "rogue" cells in an individual and the frequency of "simple" chromosomal damage in the nonrogue cells of the same individual. Rogue cells are cells with complex chromosomal damage, currently believed to be a manifestation of the activity of a human polyoma virus termed "JC." Among a total of 1,835 persons examined, there were 45 exhibiting rogue cells. A total of 179,599 cells were scored for simple chromosomal damage. In both the exposed and the control populations, there was an absolute increase of approximately 1.5% in the frequency of simple chromosomal damage in the nonrogue cells of those exhibiting rogue cells, when compared with the frequencies observed in those not exhibiting rogue cells, which is a statistically significant difference. It is argued that this phenomenon, occurring not only in lymphocytes but possibly also in other cells/tissues, may play a contributory role in the origin of malignancies characterized by clonal chromosome abnormalities. Unexpectedly, among those exhibiting rogue cells, there was a disproportionately greater representation of persons who had received relatively high radiation exposures from the bomb. The reason for this is unclear, but it is tempting to relate the finding to some lingering effect of the exposure (or the circumstances surrounding the exposure) on immunocompetence.     

T Lymphocyte activation results in an increased expression of β-1,4-Galactosyltransferase: Phorbol ester induces a similar enhancement in the absence of mitosis

We previously showed that in vitro activated human T lymphocytes expressed increased amounts of -1,6-branched N-linked oligosaccharides (Lemaire S et al. (1994) J Biol Chem 269: 8069-74), which have been proposed to participate in the regulation of the immune process. In the present paper, we compared the activity and expression of -1,4-galactosyltransferase (GalT), one of the glycosyltransferases involved in the biosynthesis of these -1,6-branched N-linked oligosaccharides, before and after in vitro activation of T lymphocytes after a 40 h treatment with a mixture of phorbol 12-myristate 13-acetate and Phaseolus vulgaris lectin. After treatment, the enzymatic activity of the GalT was significantly increased and immunoblot experiments performed with a monoclonal antibody to human GalT showed an increased intensity of the GalT band at 49 kDa, attributable to an enhancement of GalT mRNA level, as shown by Northern blots. However, treatment of the same T-lymphocytes by phorbol ester alone, which is unable to induce mitosis, resulted in a comparable increase of the expression of GalT. Moreover, these phorbol ester-treated T lymphocytes, analysed by flow cytometry exhibited a two-fold increase in the expression of GalT. Finally, confocal fluorescence microscopy performed on all T lymphocytes (treated or not) showed that the flow cytometric signal of GalT originates from intracellular, Golgi-associated antigen only since no surface GalT was detected.     

A suggested role for mitochondria in Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.     

The thorough screening of the MUTYH gene in a large French cohort of sporadic colorectal cancers

The MUTYH gene encodes a key glycosylase of the base-excision repair system that is involved in maintaining genomic DNA stability against oxidative damage. Biallelic germline MUTYH mutations have been proved to greatly predispose to non-familial adenomatous polyposis (FAP) and non-hereditary non-polyposis colorectal cancer (HNPCC) familial recessive forms of colorectal cancer with multiple adenomas. To date, there is still much debate over the impact of monoallelic germline MUTYH mutations on colorectal carcinogenesis. To evaluate their role in the susceptibility to sporadic colon and rectum cancers, we screened 1024 French sporadic colorectal cancer cases and 1121 French healthy controls for Caucasian MUTYH-associated polyposis mutations, including already known mutations p.Gly382Asp and p.Tyr165Cys, and new mutation p.Val479Phe. We observed a nonstatistically significant association between these MUTYH mutations at a heterozygous state and an increase in colorectal cancer risk (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.70-2.27). As a result, we conclude that heterozygous MUTYH mutations do not play a major role in sporadic colorectal carcinogenesis although a modest effect on this process cannot be ruled out.     

3d interaction homology: The structurally known rotamers of tyrosine derive from a surprisingly limited set of information‐rich hydropathic interaction environments described by maps

Sidechain rotamer libraries are obtained through exhaustive statistical analysis of existing crystallographic structures of proteins and have been applied in multiple aspects of structural biology, for example, crystallography of relatively low‐resolution structures, in homology model building and in biomolecular NMR. Little is known, however, about the driving forces that lead to the preference or suitability of one rotamer over another. Construction of 3D hydropathic interaction maps for nearly 30,000 tyrosines reveals the environment around each, in terms of hydrophobic (π–π stacking, etc.) and polar (hydrogen bonding, etc.) interactions. After partitioning the tyrosines into backbone‐dependent (?, ψ) bins, a map similarity metric based on the correlation coefficient was applied to each map‐map pair to build matrices suitable for clustering with k‐means. The first bin (?200° ≤ ? < –155°; ?205° ≤ ψ < –160°), representing 631 tyrosines, reduced to 14 unique hydropathic environments, with most diversity arising from favorable hydrophobic interactions with many different residue partner types. Polar interactions for tyrosine include surprisingly ubiquitous hydrogen bonding with the phenolic OH and a handful of unique environments surrounding the tyrosine backbone. The memberships of all but one of the 14 environments are dominated (>50%) by a single χ₁/χ₂ rotamer. The last environment has weak or no interactions with the tyrosine ring and its χ₁/χ₂ rotamer is indeterminate, which is consistent with it being composed of mostly surface residues. Each tyrosine residue attempts to fulfill its hydropathic valence and thus, structural water molecules are seen in a variety of roles throughout protein structure. Proteins 2015; 83:1118–1136. © 2015 Wiley Periodicals, Inc.     

Conspecific and heterospecific attraction in assessments of functional connectivity

Functional connectivity is known to have an important, positive influence on species persistence. Measurements of functional connectivity traditionally focus on structural attributes of landscapes such as the distance and matrix type between habitat patches as well as on how species interact with those structural attributes. However, we propose that the social behavior of a species, through conspecific and heterospecific attraction, will also impact connectivity by changing how dispersers move with respect to each other and occupied patches. We analyzed functional connectivity patterns using circuit and graph theory for golden-headed lion tamarins (Leontopithecus chrysomelas) in Brazil under three scenarios. In the first scenario, we looked at connectivity without the effects of attraction under varying maximum dispersal distance and ecological movement cost thresholds. In the second scenario, we allowed dispersers to travel over more hostile matrix than they normally would to reach an occupied patch. In the final scenario, we allowed dispersers to move only to occupied patches. We found that, according to the first scenario, range-wide functional landscape connectivity for golden-headed lion tamarins is low at realistic maximum dispersal distance and movement cost thresholds. Incorporating the effects of conspecific or heterospecific attraction would increase functional connectivity, in the case of scenario two, or decrease functional connectivity, in the case of scenario three. Because conspecific/heterospecific attraction can have an impact on movement for some species, this factor should be incorporated in assessments of functional connectivity patterns for social species and others where patch occupancy is likely to influence the movements of dispersers.     

Applying an empirical hydropathic forcefield in refinement may improve low-resolution protein X-ray crystal structures

Background

The quality of X-ray crystallographic models for biomacromolecules refined from data obtained at high-resolution is assured by the data itself. However, at low-resolution, >3.0 Å, additional information is supplied by a forcefield coupled with an associated refinement protocol. These resulting structures are often of lower quality and thus unsuitable for downstream activities like structure-based drug discovery.

Methodology

An X-ray crystallography refinement protocol that enhances standard methodology by incorporating energy terms from the HINT (Hydropathic INTeractions) empirical forcefield is described. This protocol was tested by refining synthetic low-resolution structural data derived from 25 diverse high-resolution structures, and referencing the resulting models to these structures. The models were also evaluated with global structural quality metrics, e.g., Ramachandran score and MolProbity clashscore. Three additional structures, for which only low-resolution data are available, were also re-refined with this methodology.

Results

The enhanced refinement protocol is most beneficial for reflection data at resolutions of 3.0 Å or worse. At the low-resolution limit, ≥4.0 Å, the new protocol generated models with Cα positions that have RMSDs that are 0.18 Å more similar to the reference high-resolution structure, Ramachandran scores improved by 13%, and clashscores improved by 51%, all in comparison to models generated with the standard refinement protocol. The hydropathic forcefield terms are at least as effective as Coulombic electrostatic terms in maintaining polar interaction networks, and significantly more effective in maintaining hydrophobic networks, as synthetic resolution is decremented. Even at resolutions ≥4.0 Å, these latter networks are generally native-like, as measured with a hydropathic interactions scoring tool.