Ovarian Cancer Presenting as Cryptogenic Stroke from Patent Foramen Ovale.

A woman, aged 52 years, experienced severe headache, confusion, nausea, dizziness, and diplopia for three days. Magnetic resonance imaging of the brain showed multiple acute and subacute infarcts suggestive of embolic events. Dermatological examination was notable for splinter hemorrhages and macular patches on the fingernails and feet, respectively. Further diagnostic imaging of the chest and abdomen revealed pulmonary emboli and an ovarian mass with omental deposits and splenic infarcts. Fine-needle aspiration cytology and surgery confirmed a diagnosis of high-grade serous adenocarcinoma of the ovary with clear cell features. Extensive evaluation for malignancy should be considered on a case-by-case basis for patients with thromboembolic disease and an initial negative diagnostic evaluation for stroke. Consideration of patent foramen ovale closure is reasonable in patients with malignancy who are at risk for recurrent strokes.

Distal Tibia and Foot Involvement in a Patient With Waldenstrom's Macroglobulinemia.

Bone lesions are a rare presentation in Waldenstrom's macroglobulinemia patients. Although lytic bone lesions and generalized osteoporosis have been described variably in literature on Waldenstrom's macroglobulinemia patients, distal long bone and foot involvement has not been described to our knowledge. We report a patient with Waldenstrom's macroglobulinemia with IgM monoclonal spike, plasmacytic infiltration of bone marrow, and symptoms of foot pain, and found to have distal tibia and foot involvement. The symptoms of bone lesions in our patient were significantly improved with radiation treatment. The possibility of distal involvement of long bones in a clinically relevant presentation should be kept in mind in these patients.

Symptomatic factor XIII deficiency with normal urea solubility test.

BACKGROUND: Factor XIII deficiency is a rarely encountered bleeding disorder traditionally identified by clot dissolution in 5 molar urea (urea solubility test). METHODS: We report a patient with delayed post-surgical bleeding characteristic of factor XIII deficiency with a normal urea solubility test. RESULTS: Factor XIII deficiency was identified by an automated assay that measured factor XIII antigen. The patient was successfully treated with cryoprecipitate. CONCLUSIONS: Patients with excessive bleeding with normal screening tests should be tested for factor XIII using a sensitive assay procedure. The urea solubility assay for factor XIII should be discontinued due to its lack of sensitivity.

ADAMTS13 deficiency and thrombotic thrombocytopenic purpura associated with trimethoprim-sulfamethoxazole.

Thrombotic thrombocytopenic purpura (TTP) is a hematological disease characterized by microangiopathic hemolytic anemia and thrombocytopenia. Although the link between ADAMTS13 deficiency and idiopathic TTP has been well-established, the role of trimethoprim-sulfamethoxazole (TMP-SMX) in the pathogenesis of TTP is not yet well elucidated. To the best of our knowledge, there have been only two previous reports linking this medication with the development of TTP. We present the case of a healthy woman, age 26 years, who developed TTP during TMP-SMX therapy for urinary tract infection. She was found to have ADAMTS13 deficiency with anti-ADAMTS13 antibodies. Her condition responded to discontinuation of the TMP-SMX, plasmapheresis, and rituximab therapy. We speculate that the acquired ADAMTS13 deficiency might have been triggered by the TMP-SMX therapy.

Assay and inhibition of diacylglycerol lipase activity.

A series of N-formyl-α-amino acid esters of ß-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.

Binding between a distal C-terminus fragment of cannabinoid receptor 1 and arrestin-2.

Internalization of G-protein-coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB1(5P)(454-473)). Complexes formed between the peptide and human arrestin-2 (wt-arr2(1-418)) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2(1-382)) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1(5P)(454-473) adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh(7P)(330-348)), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1(5P)(454-473) peptide binds to tr-arr2(1-382) with higher affinity than to the full-length wt-arr2(1-418). As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred NOE data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB1(5P)(454-473) peptide.

Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Sound propagation in water containing large tethered spherical encapsulated gas bubbles with resonance frequencies in the 50 Hz to 100 Hz range.

The efficacy of large tethered encapsulated gas bubbles for the mitigation of low frequency underwater noise was investigated with an acoustic resonator technique. Tethered latex balloons were used as the bubbles, which had radii of approximately 5 cm. Phase speeds were inferred from the resonances of a water and balloon-filled waveguide approximately 1.8 m in length. The Commander and Prosperetti effective-medium model [J. Acoust. Soc. Am. 85, 732-746 (1989)] quantitatively described the observed dispersion from well below to just below the individual bubble resonance frequency, and it qualitatively predicted the frequency range of high attenuation for void fractions between 2% and 5% for collections of stationary balloons within the waveguide. A finite-element model was used to investigate the sensitivity of the waveguide resonance frequencies, and hence the inferred phase speeds, to changes in individual bubble size and position. The results indicate that large tethered encapsulated bubbles could be used mitigate low frequency underwater noise and that the Commander and Prosperetti model would be useful in the design of such a system.

Interaction of a fragment of the cannabinoid CB1 receptor C-terminus with arrestin-2.

Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419-439 of the CB1 C-terminus upon binding to arrestin-2. This segment is pivotal to the desensitization of CB1. Using high-resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared.

Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

Phase III study of matrix metalloproteinase inhibitor prinomastat in non-small-cell lung cancer.

PURPOSE: Matrix metalloproteinases (MMPs) degrade extracellular proteins and facilitate tumor growth, invasion, metastasis, and angiogenesis. This trial was undertaken to determine the effect of prinomastat, an inhibitor of selected MMPs, on the survival of patients with advanced non-small-cell lung cancer (NSCLC), when given in combination with gemcitabine-cisplatin chemotherapy. PATIENTS AND METHODS: Chemotherapy-naive patients were randomly assigned to receive prinomastat 15 mg or placebo twice daily orally continuously, in combination with gemcitabine 1,250 mg/m2 days 1 and 8 plus cisplatin 75 mg/m2 day 1, every 21 days for up to six cycles. The planned sample size was 420 patients. RESULTS: Study results at an interim analysis and lack of efficacy in another phase III trial prompted early closure of this study. There were 362 patients randomized (181 on prinomastat and 181 on placebo). One hundred thirty-four patients had stage IIIB disease with T4 primary tumor, 193 had stage IV disease, and 34 had recurrent disease (one enrolled patient was ineligible with stage IIIA disease). Overall response rates for the two treatment arms were similar (27% for prinomastat v 26% for placebo; P = .81). There was no difference in overall survival or time to progression; for prinomastat versus placebo patients, the median overall survival times were 11.5 versus 10.8 months (P = .82), 1-year survival rates were 43% v 38% (P = .45), and progression-free survival times were 6.1 v 5.5 months (P = .11), respectively. The toxicities of prinomastat were arthralgia, stiffness, and joint swelling. Treatment interruption was required in 38% of prinomastat patients and 12% of placebo patients. CONCLUSION: Prinomastat does not improve the outcome of chemotherapy in advanced NSCLC.

Hydrogen-bonded His93 as a sensitive probe for identifying inhibitors of the endocannabinoid transport protein FABP7.

The human brain FABP (FABP7) has been shown to be an intracellular carrier protein that can significantly potentiate the uptake of the endocannabinoid anandamide. For this reason, there is a great interest in the discovery and development of FABP7 inhibitors for treating stress, pain, inflammation, and drug abuse. We found that in the (1) H-NMR spectrum of the protein, a well-separated downfield resonance arising from the hydrogen-bonded His93 side chain is very sensitive to ligand binding. Using this characteristic spectral marker together with another well-resolved upfield resonance from the side chain of Val84, we have identified that an adipocyte FABP (FABP4) inhibitor BMS309403 also binds tightly to FABP7. Our data demonstrated that this unique His93 downfield resonance can be used as a sensitive probe for rapidly and unambiguously identifying novel high-affinity FABP7 ligands. The findings should help accelerate the discovery of potential drug leads for the modulation of endocannabinoid transport.

Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels.

Cyclic nucleotide gated cation channels (CNGCs) are a large (20 genes in Arabidopsis thaliana) family of plant ligand gated (i.e. cyclic nucleotides activate currents) ion channels, however, little is known about their functional properties. One reason for this is the recalcitrance of plant CNGC expression in heterologous systems amenable to patch clamp studies. Here, we show results demonstrating the efficacy of using growth of a K+ uptake-deficient yeast (trk1,2) as a functional assay of CNGCs as inwardly-conducting cell membrane cation (K+) transporters. Prior work demonstrated that trk1,2 is hypersensitive to the antibiotic hygromycin (hyg) and that expression of an inwardly conducting K+ transporter suppresses hyg hypersensitivity. We find that increasing [hyg] in solid YPD medium inhibits trk1,2 growth around a filter disk saturated with 3 M K+. Northern analysis indicated that message is transcribed in trk1,2 transformed with the CNGC coding sequences. Confocal imaging of yeast expressing CNGC-fluorescent fusion proteins indicated channel targeting to the cell membrane. Trk1,2 expressing several plant CNGCs grown in the presence of hyg demonstrated (a) greater growth than trk1,2 transformed with empty plasmid, and (b) enhanced growth when cAMP was added to the medium. Alternatively, cAMP inhibited growth of yeast transformed with either the empty plasmid, or the plant K+ channel KAT1; this channel is not a CNGC. Growth of trk1,2 was dependent on filter disk [K+]; suggesting that complementation of hyg hypersensitivity due to presence of a functional plant CNGC was dependent on K+ movement into the cytosol. We conclude that plant CNGC functional characterization can be facilitated by this assay system.

hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation.

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novel hCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines.

Ligand-binding architecture of human CB2 cannabinoid receptor: evidence for receptor subtype-specific binding motif and modeling GPCR activation.

The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, "ligand-assisted protein structure" (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.

Gene ancestry of the cannabinoid receptor family.

Genome sequencing projects, and their available resources, have revealed two distinct genes encoding cannabinoid receptors, CB(1) and CB(2). Biochemical evidence in support of a third cannabinoid receptor includes signal transduction events and vasodilation in the vasculature of cannabinoid receptor knockout mice after exposure to the endogenous cannabinoid, anandamide. In addition, a nonpsychoactive ingredient in marijuana, abnormal cannabidiol, which does not activate the two characterized cannabinoid receptor homologues, has been shown to induce vasodilation in the endothelium. Our work distinguishes the biochemical differences by way of a phylogenetic analysis of cannabinoid receptors. Recently a putative orthologue to CB(1) and CB(2) has been identified in the urochordate, Ciona intestinalis, indicating the presence of cannabinoid receptors previous to the evolution of vertebrates. Moreover, the Ciona sequence shares equal identity to both cannabinoid paralogous sequences and no other GPCR sequence identified in an exhaustive database search is as similar. We propose that, although an alternate cannabinergic-activating pathway may be present, it does not include a GPCR (or other receptor type) phylogenetically related to the CB(1)/CB(2)Ciona lineage.

The interrater reliability of a functional capacity evaluation: the physical work performance evaluation.

The purpose was to evaluate the interrater reliability of the Dynamic Strength, Position Tolerance, and Mobility tasks of the Physical Work Performance Evaluation (PWPE), a Functional Capacity Evaluation often used with workers disabled due to back pain. For each worker's evaluation, two raters were preselected among five trained raters. One of the raters administered the PWPE while the other functioned as a silent rater. A convenience sample of 40 workers disabled due to back pain and referred to an occupational rehabilitation center was used. In general, the reliability was "substantial" (0.61 < or = kappa < or = 0.80) to "almost perfect" (0.81 < or = kappa < or = 1.00) for most of the 21 tasks and three sections of the PWPE evaluated with the exception of three tasks in the Mobility section (ladder climbing (kappa = 0.47), repetitive trunk rotation--standing (kappa = 0.54), and repetitive trunk rotation--sitting (kappa = 0.37)) task and the Mobility section itself (kappa = 0.54). Several reasons could explain the lower agreement on the observation of the physical signs associated with these tasks. Since these tasks involve rotation movements or complex neuromuscular integration, it seemed difficult for the raters to define what are the normal physical signs and when physical signs of maximal functional capacity are present. The criteria for establishing the presence of the physical signs in the PWPE should be improved.

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel.

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels.

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.