Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells.

Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.

Autoradiographic imaging of phosphoinositide turnover in the brain.

With [3H]cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product [3H]cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization.

Using a cDNA library prepared from circumvallate papillae of rat tongue, we have identified, cloned, and sequenced a novel K+ channel, designated cdrk. The cdrk channel appears to be a member of the Shab subfamily, most closely resembling drk1. Electrophysiologic analysis of expressed cdrk channels reveals delayed rectifier properties similar to those of drk1 channels. Localizations of cdrk mRNA in rat brain and peripheral tissues, assessed by in situ hybridization and Northern blot analysis, differ from any other reported K+ channels. In the brain cdrk mRNA is most concentrated in granule cells of the olfactory bulb and cerebellum. In peripheral tissues, mRNAs for cdrk and drk1 are reciprocally localized, indicating that the K+ channel properties contributed by mammalian Shab homologs may be important in a variety of excitable tissues.

Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase.

Nitric oxide is a free radical that has been recently recognized as a neural messenger molecule. Nitric oxide synthase has now been purified and molecularly cloned from brain. Using specific antibodies and oligonucleotide probes, we have localized brain nitric oxide synthase to discrete neuronal populations in the rat and primate brain. Nitric oxide synthase is exclusively neuronal, and its localization is absolutely coincident with NADPH diaphorase staining in both rat and primate.

Disruption of p53 in human cancer cells alters the responses to therapeutic agents.

We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

Molecular mechanisms of olfaction.

Recent studies provide initial insights into molecular mechanisms of olfaction. The identification of an odorant-sensitive adenylate cyclase which responds to most odorants, affords a second messenger system following odorant interactions with receptors. Cyclic nucleotide- and odorant-gated ion channels have been demonstrated in olfactory cilia, providing signalling systems in place of or in addition to protein phosphorylation. A unique odorant-binding protein localized to nasal mucosa binds odorants in proportion to their odoriferous potencies. Molecular cloning of the isolated protein reveals it to be a member of a family of proteins that serve as carriers for small lipophilic molecules such as retinol and cholesterol. The odorant-binding protein is localized to lateral nasal glands whose secretions are atomized into the tip of the nose where the binding protein presumably interacts with odorants in the inspired air.

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles.

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.

CDRK and DRK1 K+ channels have contrasting localizations in sensory systems.

Molecular cloning of mammalian potassium channels has revealed an extensively heterogeneous superfamily of potassium channels derived from four basic subfamilies, Shaker, Shaw, Shal and Shab, each with multiple members. The families were first identified in Drosophila, in which subfamily heterogeneity is derived by alternative splicing, while in mammals mainly distinct genes give rise to channel subtypes. Further diversity of mammalian potassium channels is demonstrated by the identification of some which do not belong to any of the four main subfamilies. Although potassium channels are differentiated into fast-inactivating and delayed rectifier types, differential functions of the many mammalian potassium channels are unclear. Moreover, potassium channels function as homotetramers, though in principle heterotetramers might have a physiological role as is the case with heteromers of neurotransmitter receptor subunits. Insight into differential functions of potassium channels may be provided by their regional and subcellular localizations. In the rat brain in situ hybridization and immunohistochemistry have revealed distinct regional localizations for various subfamilies. In one instance a particular subfamily predominated in cell bodies and another in axons. We demonstrated dramatically different localizations for two members of the Shab subfamily, circumvallate papilla delayed rectifier K+ channel (CDRK) and delayed rectifier potassium channel 1 (DRK1), which in major portions of their sequences display more than 90% amino acid identity. In a number of brain regions they occur in distinct neuronal cell types or subcellular compartments, with CDRK predominantly localized diffusely over soma and in fibers and DRK1 most evident in soma and dendritic process.

Structure-function relationships of antimicrobial peptides.

Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.

Structures of the platelet calcium- and integrin-binding protein and the alphaIIb-integrin cytoplasmic domain suggest a mechanism for calcium-regulated recognition; homology modelling and NMR studies.

Calcium- and integrin-binding protein (CIB) binds to the 20-residue alphaIIb cytoplasmic domain of platelet alphaIIbbeta3 integrin. Amino acid sequence similarities with calmodulin (CaM) and calcineurin B (CnB) allowed the construction of homology-based models of calcium-saturated CIB as well as apo-CIB. In addition, the solution structure of the alphaIIb cytoplasmic domain in 45% aqueous trifluoroethanol was solved by conventional two-dimensional NMR methods. The models indicate that the N-terminal domain of CIB possesses a number of positively charged residues in its binding site that could interact with the acidic carboxy-terminal LEEDDEEGE sequence of alphaIIb. The C-terminal domain of CIB seems well-suited to bind the sequence WKVGFFKR, which forms a well-structured alpha helix; this is analogous to calmodulin and calcineurin B, which also bind alpha helices. Similarities between the C-terminal domains of CIB and calmodulin suggest that binding of CIB to the cytoplasmic domain of alphaIIb may be affected by fluctuations in the intracellular calcium concentration.

Domain orientation in beta-cyclodextrin-loaded maltose binding protein: diffusion anisotropy measurements confirm the results of a dipolar coupling study.

Maltose binding protein (MBP) is a 370-residue two-domain molecule involved in bacterial chemotaxis and sugar uptake. Rotational diffusion tensors were calculated for a complex between MBP and beta-cyclodextrin using backbone 15N T1 and T1rho relaxation times and steady state 1H-15N NOE values. The tensors obtained for each of the two domains in the protein were subsequently used to determine the relative domain orientation in the molecule. The average domain orientation determined using this approach agrees well with results from dipolar coupling data, but differs significantly from the domain orientation deduced from X-ray studies of the complex.

Assessment of recent ozone short-term epidemiologic studies.

The U.S. Environmental Protection Agency (EPA) revised the National Ambient Air Quality Standards (NAAQS) for ozone in 1997 based largely on short-term ozone studies published up to 1995. The U.S. EPA's conclusions must now be updated because (1) the agency did not consider many new studies published since 1995 and (2) the agency did not critically review the studies published before 1995 (i.e., it accepted the stated conclusions). In this article, we examine many recently published short-term ozone studies including 17 hospital admissions studies, 10 mortality studies, and 6 summer-camp studies. Almost all of these studies reported a significant association between ambient levels of ozone and adverse health effects. However, on close examination, it is apparent that there are mixed findings from one study to another and even within the results of a single study. Moreover, questionable statistical analyses and failure to consider confounders make a number of the reported findings doubtful and even negative.

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.

Structure of the antimicrobial peptide tritrpticin bound to micelles: a distinct membrane-bound peptide fold.

Tritrpticin is a member of the cathelicidin family, a group of diverse antimicrobial peptides found in neutrophil granules. The three Trp and four Arg residues in the sequence VRRFPWWWPFLRR make this a Trp-rich cationic peptide. The structure of tritrpticin bound to membrane-mimetic sodium dodecyl sulfate micelles has been determined using conventional two-dimensional NMR methods. It forms two adjacent turns around the two Pro residues, a distinct fold for peptide-membrane interaction. The first turn involves residues 4-7, followed immediately by a second well-defined 3(10)-helical turn involving residues 8-11. The hydrophobic residues are clustered together and are clearly separated from the basic Arg residues, resulting in an amphipathic structure. Favorable interactions between the unusual amphipathic fold and the micelle surface are probably key to determining the peptide structure. NMR studies of the peptide in the micelle in the presence of the spin-label 5-doxylstearic acid determined that tritrpticin lies near the surface of the micelle, where its many aromatic side chains appear to be equally partitioned into the hydrophilic-hydrophobic interface. Additional fluorescence studies confirmed that the tryptophan residues are inserted into the micelle and are partially protected from the effects of the soluble fluorescence quencher acrylamide.

Effects of molecular oxygen on chick limb bud chondrogenesis.

Chondrogenesis is an important process in the development of the embryonic chick limb. If limb buds are dispersed just prior to the initiation of chondrogenic differentiation and their cells seeded densely in culture as three-dimensional "micromasses," some of the cells differentiate to form chondrogenic nodules. These nodules characteristically produce sulfated proteoglycans and type II collagen. Two conditions within the early avian limb core have been linked causatively to the initiation of chondrogenesis: a limitation in the availability of molecular oxygen and a low NAD content of the tissue. The O2 limitation is thought to be responsible for the low NAD level. We examined the effects of molecular oxygen on the NAD content of chick limb-bud cells in micromass culture, the formation of chondrocytic nodules, and the production of type II collagen and sulfated proteoglycans. The NAD content of the cells in the micromasses and the production of type II collagen did not vary greatly as a function of oxygen availability. The development of the nodules was modified, but not eliminated, by high oxygen partial pressure (0.95). It was eliminated by anoxia. Proteoglycan synthesis was decreased significantly by high oxygen tension and its sulfation was also decreased, more so in the wing-bud than the leg-bud cells. The results suggest that in culture, high oxygen tension is compatible with some, but not all, aspects of chondrogenic differentiation of cells from embryonic chick limbs.

Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily.

We have localized CDRK and DRK1, two novel K+ channels of the Shab subfamily by immunohistochemistry. The two channels are closely related in structure with about 90% amino acid identity in the N-terminal and middle portions and 60% identity in the C-terminal region. We observe striking differences in cellular localizations of the two channels. DRK1 tends to localize to cell bodies and proximal dendrites discretely, while CDRK is diffusely present in cell bodies and is also found on fibers in specific brain areas. In the cerebral cortex DRK1 is localized to pyramidal cells, whereas CDRK occurs in small cells, presumably interneurons. These localizations may reflect specialized delayed rectifier functions and targeting properties manifested differentially by K+ channel subfamily members.

PUMA induces the rapid apoptosis of colorectal cancer cells.

Through global profiling of genes that were expressed soon after p53 expression, we identified a novel gene termed PUMA (p53 upregulated modulator of apoptosis). The protein encoded by PUMA was found to be exclusively mitochondrial and to bind to Bcl-2 and Bcl-X(L) through a BH3 domain. Exogenous expression of PUMA resulted in an extremely rapid and profound apoptosis that occurred much earlier than that resulting from exogenous expression of p53. Based on its unique expression patterns, p53 dependence, and biochemical properties, PUMA may be a direct mediator of p53-associated apoptosis.