Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously “on,” tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells

We established stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form in which the active site cysteine is mutated to serine (PTPMEGCS). We found that both endogenous and recombinant enzyme were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts for only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, we found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix.

Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin

The synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium-dependent, neurotransmitter release and is likely to be the calcium sensor for exocytosis, because of its many calcium-dependent properties. Polyphosphoinositides are needed for exocytosis, but it has not been known why. We now provide a possible connection between these observations with the finding that the C2B domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate (PIns-4,5-P2), its isomer phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P3). Calcium ions switch the specificity of this binding from PIns-3,4,5-P3 (at calcium concentrations found in resting nerve terminals) to PIns-4,5-P2 (at concentration of calcium required for transmitter release). Inositol polyphosphates, known blockers of neurotransmitter release, inhibit the binding of both PIns-4,5-P2 and PIns-3,4,5-P3 to tagmin. Our findings imply that tagmin may operate as a bimodal calcium sensor, switching bound lipids during exocytosis. This connection to polyphosphoinositides, compounds whose levels are physiologically regulated, could be important for long-term memory and learning.

Calcium isotope fractionation between soft and mineralized tissues as a monitor of calcium use in vertebrates

Calcium from bone and shell is isotopically lighter than calcium of soft tissue from the same organism and isotopically lighter than source (dietary) calcium. When measured as the 44Ca/40Ca isotopic ratio, the total range of variation observed is 5.5‰, and as much as 4‰ variation is found in a single organism. The observed intraorganismal calcium isotopic variations and the isotopic differences between tissues and diet indicate that isotopic fractionation occurs mainly as a result of mineralization. Soft tissue calcium becomes heavier or lighter than source calcium during periods when there is net gain or loss of mineral mass, respectively. These results suggest that variations of natural calcium isotope ratios in tissues may be useful for assessing the calcium and mineral balance of organisms without introducing isotopic tracers.

Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10−3. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

Soft-tissue characters in higher primate phylogenetics

Recent research has cast doubt on the reliability of bones and teeth for reconstructing phylogenetic relationships among higher primate species and genera. Herein, we investigate whether this problem is confined to hard tissues by examining the utility of higher primate soft-tissue characters for reconstructing phylogenetic relationships at low taxonomic levels. We use cladistic methods to analyze 197 soft-tissue characters for the extant hominoids and then compare the resulting phylogenetic hypotheses with the group's consensus molecular phylogeny, which is widely considered to be accurate. We show that the soft-tissue characters yield robust phylogenetic hypotheses that are compatible with the molecular phylogeny. Given the strength of the evidence for molecular phylogeny, these results indicate that, unlike craniodental hard-tissue characters, soft tissues are reliable for reconstructing phylogenetic relationships among higher primate species and genera. Thus, in higher primates at least, some types of morphological data are more useful than others for phylogeny reconstruction.

The role of prefrontal cortex and posterior parietal cortex in task switching

Human ability to switch from one cognitive task to another involves both endogenous preparation without an external stimulus and exogenous adjustment in response to the external stimulus. In an event-related functional MRI study, participants performed pairs of two tasks that are either the same (task repetition) or different (task switch) from each other. On half of the trials, foreknowledge about task repetition or task switch was available. On the other half, it was not. Endogenous preparation seems to involve lateral prefrontal cortex (BA 46/45) and posterior parietal cortex (BA 40). During preparation, higher activation increases in inferior lateral prefrontal cortex and superior posterior parietal cortex were associated with foreknowledge than with no foreknowledge. Exogenous adjustment seems to involve superior prefrontal cortex (BA 8) and posterior parietal cortex (BA 39/40) in general. During a task switch with no foreknowledge, activations in these areas were relatively higher than during a task repetition with no foreknowledge. These results suggest that endogenous preparation and exogenous adjustment for a task switch may be independent processes involving different brain areas.

The neural basis of task-switching in working memory: Effects of performance and aging

We studied the performance of young and senior subjects on a well known working memory task, the Operation Span. This is a dual-task in which subjects perform a memory task while simultaneously verifying simple equations. Positron-emission tomography scans were taken during performance. Both young and senior subjects demonstrated a cost in accuracy and latency in the Operation Span compared with performing each component task alone (math verification or memory only). Senior subjects were disproportionately impaired relative to young subjects on the dual-task. When brain activation was examined for senior subjects, we found regions in prefrontal cortex that were active in the dual-task, but not in the component tasks. Similar results were obtained for young subjects who performed relatively poorly on the dual-task; however, for young subjects who performed relatively well in the dual-task, we found no prefrontal regions that were active only in the dual-task. Results are discussed as they relate to the executive component of task switching.

Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.

The hard-soft acid-base principle in enzymatic catalysis: dual reactivity of phosphoenolpyruvate.

In this paper, the chemical reactivity of C3 of phosphoenolpyruvate (PEP) has been analyzed in terms of density functional theory quantified through quantum chemistry calculations. PEP is involved in a number of important enzymatic reactions, in which its C3 atom behaves like a base. In three different enzymatic reactions analyzed here, C3 sometimes behaves like a soft base and sometimes behaves like a hard base in terms of the hard-soft acid-base principle. This dual nature of C3 of PEP was found to be related to the conformational change of the molecule. This leads to a testable hypothesis: that PEP adopts particular conformations in the enzyme-substrate complexes of different PEP-using enzymes, and that the enzymes control the reactivity through controlling the dihedral angle between the carboxylate and the C==C double bond of PEP.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.