Acidification and hypoxia interactively affect metabolism in embryos, but not larvae, of the coastal forage fish Menidia menidia.

Ocean acidification is occurring in conjunction with warming and deoxygenation as a result of anthropogenic greenhouse gas emissions. Multistressor experiments are critically needed to better understand the sensitivity of marine organisms to these concurrent changes. Growth and survival responses to acidification have been documented for many marine species, but studies that explore underlying physiological mechanisms of carbon dioxide (CO2) sensitivity are less common. We investigated oxygen consumption rates as proxies for metabolic responses in embryos and newly hatched larvae of an estuarine forage fish (Atlantic silverside, Menidia menidia) to factorial combinations of CO2×temperature or CO2×oxygen. Metabolic rates of embryos and larvae significantly increased with temperature, but partial pressure of CO2 (PCO2 ) alone did not affect metabolic rates in any experiment. However, there was a significant interaction between PCO2  and partial pressure of oxygen (PO2 ) in embryos, because metabolic rates were unaffected by PO2  level at ambient PCO2 , but decreased with declining PO2  under elevated PCO2 For larvae, however, PCO2  and PO2  had no significant effect on metabolic rates. Our findings suggest high individual variability in metabolic responses to high PCO2 , perhaps owing to parental effects and time of spawning. We conclude that early life metabolism is largely resilient to elevated PCO2  in this species, but that acidification likely influences energetic responses and thus vulnerability to hypoxia.

Glutamine Imaging: A New Avenue for Glioma Management.

The glutamine pathway is emerging as an important marker of cancer prognosis and a target for new treatments. In gliomas, the most common type of brain tumors, metabolic reprogramming leads to abnormal consumption of glutamine as an energy source, and increased glutamine concentrations are associated with treatment resistance and proliferation. A key challenge in the development of glutamine-based biomarkers and therapies is the limited number of in vivo tools to noninvasively assess local glutamine metabolism and monitor its changes. In this review, we describe the importance of glutamine metabolism in gliomas and review the current landscape of translational and emerging imaging techniques to measure glutamine in the brain. These techniques include MRS, PET, SPECT, and preclinical methods such as fluorescence and mass spectrometry imaging. Finally, we discuss the roadblocks that must be overcome before incorporating glutamine into a personalized approach for glioma management.

Trait-mediated shifts and climate velocity decouple an endothermic marine predator and its ectothermic prey.

Climate change is redistributing biodiversity globally and distributional shifts have been found to follow local climate velocities. It is largely assumed that marine endotherms such as cetaceans might shift more slowly than ectotherms in response to warming and would primarily follow changes in prey, but distributional shifts in cetaceans are difficult to quantify. Here we use data from fisheries bycatch and strandings to examine changes in the distribution of long-finned pilot whales (Globicephala melas), and assess shifts in pilot whales and their prey relative to climate velocity in a rapidly warming region of the Northwest Atlantic. We found a poleward shift in pilot whale distribution that exceeded climate velocity and occurred at more than three times the rate of fish and invertebrate prey species. Fish and invertebrates shifted at rates equal to or slower than expected based on climate velocity, with more slowly shifting species moving to deeper waters. We suggest that traits such as mobility, diet specialization, and thermoregulatory strategy are central to understanding and anticipating range shifts. Our findings highlight the potential for trait-mediated climate shifts to decouple relationships between endothermic cetaceans and their ectothermic prey, which has important implications for marine food web dynamics and ecosystem stability.

Functional feeding responses of piscivorous fishes from the northeast US continental shelf.

The functional feeding response forms of piscivorous fishes used in multispecies and ecosystem modeling have been questioned because they were mostly conjectural or solely based on laboratory studies. Here, we investigate the functional feeding response of seven species of piscivorous fishes on four species of their prey from the northeast US continental shelf using field data that spans 30 years. Our study confirmed that Holling's types II and III functional responses are the most common functional responses for piscivorous fishes in this region. However, our analyses also revealed that differences exist between piscivorous fishes' functional responses, and, therefore, combining functional responses of piscivores is probably not appropriate in multispecies and ecosystem modeling. In the absence of specific predator-prey functional responses, we suggest that, for cruising, actively attacking predators, a type II functional response is slightly preferable; for a sedentary, ambush predator, a type III functional response is slightly preferable; at low prey densities for a generic fish predator, a type III functional response should be used; and at moderate to high prey densities, either should work sufficiently. Because we have shown that the functional response of a particular predator to individual prey species varies, these relationships must be further evaluated as we continue to develop and employ multispecies and ecosystem modeling.

Effects of coastal acidification on North Atlantic bivalves: interpreting laboratory responses in the context of in situ populations.

Experimental exposure of early life stage bivalves has documented negative effects of elevated pCO2 on survival and growth, but the population consequences of these effects are unknown. Following standard practices from population viability analysis and wildlife risk assessment, we substituted laboratory-derived stress-response relationships into baseline population models of Mercenaria mercenaria and Argopecten irradians. The models were constructed using inverse demographic analyses with time series of size-structured field data in NY, USA, whereas the stress-response relationships were developed using data from a series of previously published laboratory studies. We used stochastic projection methods and diffusion approximations of extinction probability to estimate cumulative risk of 50% population decline during ten-year population projections at 1, 1.5 and 2 times ambient pCO2 levels. Although the A. irradians population exhibited higher growth in the field data (12% per year) than the declining M. mercenaria population (-8% per year), cumulative risk was high for A. irradians in the first ten years due to high variance in the stochastic growth rate estimate (log λs = -0.02, σ2 = 0.24). This ten-year cumulative risk increased from 69% to 94% and >99% at 1.5 and 2 times ambient scenarios. For M. mercenaria (log λs = -0.09, σ2 = 0.01), ten-year risk was 81%, 96% and >99% at 1, 1.5 and 2 times ambient pCO2, respectively. These estimates of risk could be improved with detailed consideration of harvest effects, disease, restocking, compensatory responses, other ecological complexities, and the nature of interactions between these and other effects that are beyond the scope of available data. However, results clearly indicate that early life stage responses to plausible levels of pCO2 enrichment have the potential to cause significant increases in risk to these marine bivalve populations.

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

A thermostable bacterial cocaine esterase rapidly eliminates cocaine from brain in nonhuman primates.

A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a K(M) of 1.33+0.085 µM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [(11)C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity.

Sustainable production of orphan radionuclides at Wisconsin.

Over a hundred proton-induced reactions have been studied at the University of Wisconsin Medical Physics department since the installation of the first CTI RDS 112 in 1985. The focus has been to measure thick target yields at 11 MeV, in an effort to concentrate on the practical production of positron emitting radionuclides that have favorable decay characteristics, high yields and the potential for labeling pivotal biological tracers. This review covers our recent advances to scale-up the production of the heavy halogens and transition metals as feed-stock for non-conventional PET tracers that are currently attracting increased attention in oncology.

Alkylation interference identifies essential DNA contacts for sequence-specific binding of the eukaryotic transcription factor C/EBP.

Transcriptional regulators containing a "leucine zipper" and a flanking basic domain belong to a recently identified class of DNA-binding proteins. We have mapped the essential DNA contacts of one member of this group, the eukaryotic transcription factor C/EBP. Methylation and ethylation interference experiments detected major groove contacts over a full turn of the DNA helix on both an asymmetric and a symmetric C/EBP binding site. The contacts essential for C/EBP binding have two-fold symmetry yet differ significantly from the contacts of other dimeric DNA-binding proteins, including those bearing helix-turn-helix motifs and the type I restriction endonuclease EcoRI.

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein.

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.

Congestive cardiomyopathy and haemochromatosis--rapid progression possibly accelerated by excessive ingestion of ascorbic acid.

We describe rapidly fatal cardiomyopathy in a young man. He had for twelve months ingested large amounts of ascorbic acid and was admitted with severe heart failure having been symptomatic for two months. He died after eight days. Idiopathic haemochromatosis was diagnosed at autopsy. The clinical and laboratory features are discussed and the possible implications of ascorbic acid ingestion are explored.

Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein.

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.

Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus.

The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.

Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.

The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.