Instability of the human minisatellite CEB1 in rad27Delta and dna2-1 replication-deficient yeast cells

Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them. Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations. We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Delta cells. Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Delta cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit. Altogether, these results suggest that the improperly processed 5' flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events. This replication-dependent phenomenon differs from the recombination-induced instability in yeast meiotic cells.     

Comparison of the seed germination effects of synthetic analogs of strigol,gibberellic acid,cytokinins, and other plant growth regulators

Four synthetic multiring analogs of strigol, a naturally occurring sesquiterpene lactone that promotes germination of dormant seeds ofStriga (witchweed), were found to stimulate germination of dormantLactuca (lettuce) seeds. The effects on light-sensitive and light-insensitive lettuce seeds were concentration-dependent and exceeded those produced by equimolar (0.1 mM) solutions of gibberellic acid. Strigol and epistrigol promoted lettuce seed germination to a lesser degree than did the synthetic analogs. The strigol group compounds had minimal effect on the germination of monocot seeds. The results indicate that the synthetic strigol analogs have plant growth regulatory activity in dormant seeds of genera beyondStriga in which germination stimulation by strigol and the synthetic analogs was first demonstrated.Names of companies of commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Tome-1, a trigger of mitotic entry,is degraded during G1 via the APC

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.     

GIT1 mediates Src-dependent activation of phospholipase Cgamma by angiotensin II and epidermal growth factor

Critical events for vasoconstrictor and growth factor signal transduction include stimulation of phospholipase Cgamma (PLCgamma) and elevation of intracellular calcium. c-Src has been proposed as a common mediator for these signals activated by both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein-1 (GIT1) is a substrate for c-Src that undergoes tyrosine phosphorylation in response to angiotensin II (AngII) and EGF in vascular smooth muscle and 293 cells. GIT1 associates with PLCgamma via the PLCgamma Src homology 2 and 3 domains constitutively, and the interaction is unaltered by AngII and EGF. GIT1 interaction with PLCgamma is required for PLCgamma activation based on inhibition of tyrosine phosphorylation and calcium mobilization after GIT1 knockdown with antisense GIT1 oligonucleotides. GIT1 interacts with PLCgamma via a novel Spa homology domain (SHD) and a coiled-coil domain. Deletion mutation analysis showed that GIT1(SHD) is required for AngII- and EGF-mediated PLCgamma activation (measured by phosphorylation of Tyr783 and inositol 1,4,5-trisphosphate formation). We propose that GIT1 is a novel regulator of PLCgamma function that mediates PLCgamma activation by c-Src and integrates signal transduction by GPCRs and TKRs.     

Clinical relevance of serum angiogenic activity in patients with transitional cell carcinoma of the bladder

Angiogenesis is essential for tumor growth and progression and is mediated by positive and negative regulators of vessel growth. Since angiogenic mediators found in patient serum have been postulated to reflect the angiogenic potential of a malignant tumor, we investigated the angiogenic activity in the serum of patients with transitional cell carcinoma (TCC). The data were correlated to tumor characteristics and the clinical course of the patients. Eighty-one patients with transitional cell carcinoma and 53 control persons were included in the study. Preoperative serum samples were collected and both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were quantified by ELISA. Additionally, the serum evoked proliferative activity on human umbilical vein endothelial cells (HUVEC) was evaluated. Data were compared to the clinical course of the patients. Serum of tumor patients significantly enhanced the proliferative capacity of HUVEC, compared to cells grown in standard culture medium (p = 0.0032), but not when compared to serum from control persons. Serum from patients with superficial TCC and well differentiated tumors induced a significantly higher angiogenic response (ANG(hi)) than serum from patients with poorly differentiated and invasive carcinomas (ANG(lo); p = 0.037). VEGF level of ANG(hi) serum was 384.22 +/- 247.76 pg/ml (n = 37) which significantly differed from mean VEGF level detected in ANG(lo) serum (247.72 +/- 211.93 pg/ml, n = 42; p = 0.019). Similarly, mean bFGF levels were 9.58 +/- 5.91 pg/ml in ANG(hi) serum versus 5.74 +/- 3.52 pg/ml) in ANG(lo) serum (p = 0.0043). A negative correlation was established between VEGF/bFGF serum concentration and patient prognosis. The experiments demonstrate a positive correlation between VEGF and bFGF serum level and endothelial proliferation in vitro. The inverse relationship between angiogenic activity and tumor stage might disclose information about angiogenesis and tumor progression in TCC.     

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.     

Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR‐induced senescence markers could persist long‐term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16^INK4a expression, two hallmarks of cellular senescence and aging. BrdU‐labeling experiments revealed that IR‐induced damaged cells are preferentially eliminated, at least partially, in a tissue‐dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild‐type and Rag2^?/? γC^?/? mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long‐term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.     

Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants

Background  

Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS-associated toxoplasmosis.