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Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

The atypical chemokine receptor C-X-C chemokine receptor type 7 (CXCR7) is an attractive therapeutic target for a variety of cardiac and immunological diseases. As a strategy to mitigate known risks associated with the development of higher molecular weight, basic compounds, a series of pyrrolidinyl-azolopyrazines were identified as promising small-molecule CXCR7 modulators. Using a highly enabled parallel medicinal chemistry strategy, structure-activity relationship studies geared towards a reduction in lipophilicity and incorporation of saturated heterocycles led to the identification of representative tool compound 20. Notably, compound 20 maintained good potency against CXCR7 with a suitable balance of physicochemical properties to support in vivo pharmacokinetic studies.

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (, Graphical abstract Image 1, Highlights • Bacillus thuringiensis (Bt) Cry5B is a new pan-hookworm cure. • Bt Cry5B is highly active against Ancylostoma caninum hookworm infections in dogs. • Bt Cry5B is highly active against Necator americanus. • Cry5B acts independent of the host immune system and directly on hookworms. • Cry5B retains full bioactivity after repeated dosing.

Summary RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment “paraspeckles,” are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3′ UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation., Graphical Abstract, Highlights • TDP-43 maintains pluripotency by regulating expression of pluripotency factors • TDP-43 represses formation of paraspeckles in ESCs by regulating Neat1 • The paraspeckle-inducing isoform of Neat1 promotes differentiation of ESCs and embryos • Cross-regulation between TDP-43 and Neat1 enhances pluripotency-differentiation axis, Modic et al. uncover opposing roles of TDP-43 and paraspeckles in pluripotency and differentiation that are further enhanced by their cross-regulation. TDP-43 represses paraspeckles through processing of the scaffolding lncRNA Neat1, whereas paraspeckles partially sequester TDP-43. This reciprocal relationship promotes coordinated changes in alternative polyadenylation essential for efficient exit from pluripotency.

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified 'switch' kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to locate primarily not only in the perinuclear region of Rat-1 cells but also at the cell margins. We propose that the sequestration of PDE4D9 in a specific complex together with AMPK, ERK, MK2 and the H2O2-activatable 'switch' kinase allows for its selective multi-site phosphorylation, activation and regulation in mitosis.

The present article summarizes Metabolites in Safety Testing (MIST) studies on a glucokinase activator, N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), which is under development for the treatment of type 2 diametes mellitus. Metabolic profiling in rat, dog, and human hepatocytes revealed that PF-04937319 is metabolized via oxidative (major) and hydrolytic pathways (minor). N-Demethylation to metabolite M1 [N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide] was the major metabolic fate of PF-04937319 in human (but not rat or dog) hepatocytes, and was catalyzed by CYP3A and CYP2C isoforms. Qualitative examination of circulating metabolites in humans at the 100- and 300-mg doses from a 14-day multiple dose study revealed unchanged parent drug and M1 as principal components. Because M1 accounted for 65% of the drug-related material at steady state, an authentic standard was synthesized and used for comparison of steady-state exposures in humans and the 3-month safety studies in rats and dogs at the no-observed-adverse-effect level. Although circulating levels of M1 were very low in beagle dogs and female rats, adequate coverage was obtained in terms of total maximal plasma concentration (∼7.7× and 1.8×) and area under the plasma concentration-time curve (AUC; 3.6× and 0.8× AUC) relative to the 100- and 300-mg doses, respectively, in male rats. Examination of primary pharmacology revealed M1 was less potent as a glucokinase activator than the parent drug (compound PF-04937319: EC50 = 0.17 μM; M1: EC50 = 4.69 μM). Furthermore, M1 did not inhibit major human P450 enzymes (IC50 > 30 μM), and was negative in the Salmonella Ames assay, with minimal off-target pharmacology, based on CEREP broad ligand profiling. Insights gained from this analysis should lead to a more efficient and focused development plan for fulfilling MIST requirements with PF-04937319.

The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.

A novel series of glucagon receptor antagonists has been discovered. These pyrazole ethers and aminopyrazoles have lower molecular weight and increased polarity such that the molecules fall into better drug-like property space. This work has culminated in compounds 44 and 50 that were shown to have good pharmacokinetic attributes in dog, in contrast to rats, in which clearance was high; and compound 49, which demonstrated a dose-dependent reduction in glucose excursion in a rat glucagon challenge experiment.

Canine heartworm is endemic in many parts of the world, and veterinarians rely on rapid in-clinic antigen tests to screen for this infection. Recently, an in-clinic, instrument-based rotor employing a colloidal gold agglutination immunoassay was launched in the marketplace (VetScan VS2(®) Canine Heartworm (HW) Antigen Test Kit; Abaxis, Inc.). Because of the widespread use of heartworm prevention and possible false negative test results in dogs with low heartworm burdens, the performance of the VetScan VS2(®) HW test and a commercially available in-clinic, membrane-based ELISA test (SNAP(®) Heartworm RT Test; IDEXX Laboratories) was compared using samples from dogs with low heartworm burdens and/or low levels of circulating antigen. Ninety serum samples were evaluated using the two methods. Testing was performed according to the manufacturer's product insert by personnel blinded to sample status. The samples were derived from two populations: dogs with necropsy-confirmed heartworm status (40 with 1-4 female worms, 30 with no worms), and field dogs (20) confirmed positive for antigen by microtiter plate ELISA (PetChek(®) Heartworm PF Antigen Test; IDEXX Laboratories). All 40 dogs with heartworms on necropsy were also confirmed to have circulating antigen by the PetChek HW ELISA. In necropsy-negative dogs (n=30), neither the VetScan VS2 HW nor SNAP HW tests detected heartworm antigen. Of the samples testing positive for antigen by PetChek HW (n=60), the VetScan VS2 HW and SNAP HW tests detected antigen in 15 and 56 samples, respectively. Percent agreement (plus 95% confidence interval) for each test relative to the PetChek HW qualitative result was 50% (40-60%) for VetScan VS2 HW and 96% (89-98%) for SNAP HW. Relative to the presence or absence of female worms at necropsy, agreement was 61% (50-72%) for VetScan VS2 HW and 99% (92-99.6%) for SNAP HW tests. It is clinically important that dogs with low heartworm burdens and/or low levels of circulating heartworm antigen be correctly identified by veterinarians in order to ensure prompt treatment, and the VetScan(®) VS2 HW test does not appear to be as accurate as the SNAP HW or PetChek HW tests when performed on this subset of patients.

Heartworms can cause serious cardiopulmonary disease in their canid hosts. Canine heartworm has become widespread in many parts of the world, and its range continues to expand. Wildlife reservoirs play a role in perpetuation and transmission of this parasite to dogs. Human heartworm infection is incidental and is typically not associated with severe clinical disease; however, because no serological test is readily available, patients must undergo invasive procedures to differentiate heartworm from other more serious diseases. Human cases have been reported mainly in areas of high canine prevalence, highlighting the importance of heartworm testing and chemoprophylaxis in all dogs to reduce transmission. Future efforts should focus on the development of a non-invasive diagnostic test for people, and on epidemiological surveys for both animals and people.

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.

The endocrine hormone glucagon stimulates hepatic glucose output via its action at the glucagon receptor (GCGr) in the liver. In the diabetic state, dysregulation of glucagon secretion contributes to abnormally elevated hepatic glucose output. The inhibition of glucagon-induced hepatic glucose output via antagonism of the GCGr using small-molecule ligands is a promising mechanism for improving glycemic control in the diabetic state. Clinical data evaluating the therapeutic potential of small-molecule GCGr antagonists is currently emerging. Recently disclosed clinical data demonstrates the potential efficacy and possible therapeutic limitations of small-molecule GCGr antagonists. Recent pre-clinical work on the development of GCGr antagonists is also summarized.

The central melanocortin system and particularly the melanocortin-4 receptor (MC4R) subtype, plays an important role in the regulation of body weight. The discovery of orally active MC4R agonists suitable for evaluation in human clinical trials as weight loss agents has attracted considerable interest over the past decade, but has proved challenging, in part because of cardiovascular and behavioral side effects. Currently, the only MC4R agonist in clinical trials is a peptide identified as RM-493. To avoid some of the undesirable side effects associated with MC4R activation, new pharmacological approaches for modulating the MC system have been investigated. In this article, we provide a review of the MC4R patent landscape from 2008 to 2014 and analyze the physicochemical properties of compounds described herein.

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SM sub m02) or the entire m02 gene family (SM sub m02-16) grew like their wild-type parent in cultured cells. However, whereas SM sub m02 was as pathogenic as the wild-type virus, SM sub m02-16 was markedly less virulent. SM sub m02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient γ c /Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.

Successful reproduction is a determining factor for species survival. Pollution may impair reproductive success of adults through effects on gamete quality. Reproductive impairment of the sea urchin Anthocidaris crassispina upon chronic (4 weeks) exposure to 0.01 and 0.1 mg l−1 Cd2+ was investigated. Criteria used for assessing gamete quality included sperm motility, egg morphology, fertilization rate and dynamics of first cleavage. A dose–response relationship was found between Cd2+ levels and changes in sperm motility, and percentage fertilization. Sperm motility, measured by computer-assisted sperm analysis, indicated that percent motile sperm, velocities, and percent sperm with normal trajectory were significantly affected by chronic exposure to ⩾0.1 mg l−1 Cd2+. A decline in sperm motility was also accompanied by a decrease in fertilization success of sea urchin sperm. Width/height ratio of sea urchin eggs was not affected by cadmium, but larger egg sizes were found when sea urchins were exposed to 0.1 mg l−1 Cd2+. Male sea urchins exposed to Cd2+ produced poorer quality sperm, as indicated by a lower percent fertilization and lower cleavage rate, implying that male sea urchins were more sensitive than females to chronic Cd2+ exposure. Results of the present study provide an explanation of reproductive impairment in marine invertebrates upon chronic exposure to Cd2+.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.

Identification of orally active, small molecule antagonists of the glucagon receptor represents a novel treatment paradigm for the management of type 2 diabetes mellitus. The present work discloses novel glucagon receptor antagonists, identified via conformational constraint of current existing literature antagonists. Optimization of lipophilic ligand efficiency (LLE or LipE) culminated in enantiomers (+)-trans-26 and (-)-trans-27 which exhibit good physicochemical and in vitro drug metabolism profiles. In vivo, significant pharmacokinetic differences were noted with the two enantiomers, which were primarily driven through differences in clearance rates. Enantioselective oxidation by cytochrome P450 was ruled out as a causative factor for pharmacokinetic differences.

Diagnosis of feline lungworm, Aelurostrongylus abstrusus, is typically achieved by identifying larvae in feces following concentration through flotation or using the Baermann technique. This work presents observations on the usefulness of an indirect immunofluorescence antibody assay for detection of antibodies to this parasite in the sera of infected cats. Using first-stage larvae of A abstrusus and sera from both experimentally and naturally infected cats, it was determined that the test was fairly sensitive and did not cross-react with serum from an Ancylostoma braziliense (hookworm)-infected cat.

The cAMP signaling system can trigger precise physiological cellular responses that depend on the fidelity of many protein–protein interactions, which act to bring together signaling intermediates at defined locations within cells. In the heart, cAMP participates in the fine control of excitation–contraction coupling, hence, any disregulation of this signaling cascade can lead to cardiac disease. Due to the ubiquitous nature of the cAMP pathway, general inhibitors of cAMP signaling proteins such as PKA, EPAC and PDEs would act non-specifically and universally, increasing the likelihood of serious ‘off target’ effects. Recent advances in the discovery of peptides and small molecules that disrupt the protein–protein interactions that underpin cellular targeting of cAMP signaling proteins are described and discussed.

Many regulatory agencies require that the efficacy of veterinary anthelmintic medications be evaluated by enumerating parasites in treated and untreated animals after necropsy. Current ethical considerations, i.e., the 3 Rs of research, call for the replacement of this method with less invasive techniques that would not require animal sacrifice. This study tested standard gastrointestinal endoscopy as an in vivo method of quantifying the intestinal hookworm, Ancylostoma caninum . Worm counts were compared with those from gold standard necropsy. Thirteen dogs inoculated with third-stage A. caninum larvae underwent endoscopy 4–6 weeks post-infection, just prior to necropsy. Two-thirds of the adult hookworms were located in the middle section of the small intestine that could not be reached for endoscopic examination. Not surprisingly, the total worm counts obtained by endoscopy did not correlate with those from necropsy ( R 2 = 0.05, p = 0.464). One method to increase small intestinal access would be to use specialized balloon or spiral endoscopes developed for this purpose in human gastroenterology. Based on the results of this study, standard endoscopy alone is unsuitable for quantification of A. caninum in the small intestine. Parasites in more accessible sites, such as whipworms in the cecum and colon, might be more appropriate targets for endoscopic counting.

Methotrexate (MTX) suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. The intracellular level of phosphoribosyl 5-pyrophosphate (PRPP) of EAT cells increased in a dose-dependent manner in response to MTX treatment. At the same time, the rate of glucose transport was lowered. Hypoxanthine reversed both these effects of MTX and partially rescued EAT cell growth. Under all conditions tested, changes in rate of glucose transport were shown to be the result of alterations in the number of glucose transporter (Vmax) rather than ligand affinity (Km). The dual action of MTX as a chemotherapeutic agent is discussed in this light.

Dopamine receptors in the pars reticulata of the substantia nigra were inactivated following bilateral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Loss of D1, but not D2, receptors was associated with a significant decrease in the incidence of stereotyped head-down sniffing elicited by the mixed D1/D2 receptor agonist, apomorphine. The results support the hypothesis that D1 receptors in the substantia nigra are necessary for the expression of dopamine mediated stereotyped behaviour.

Isolation of a specific Ancylostoma species typically requires death of the source animal, or holding an animal long enough to collect feces after treatment, for worm recovery and identification. The reason for collecting worms is that the eggs are not easy to distinguish morphologically. In keeping with the 3 Rs of laboratory animal research (reduction, refinement, replacement), the objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time field-collected samples of canine feces without the need for killing the host. During a collection trip to Florida, fecal samples (n = 148) were collected and identified as containing eggs of Ancylostoma species (n = 64) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 2 samples were identified as A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Larval cultures were initiated from these samples, and larvae from the cultures were returned to New York and used to inoculate a purpose-bred kitten with the goal of inhibiting the growth of any contaminating Ancylostoma caninum that might be present in the culture. The infection was patent at 15 days, and eggs were identified as A. braziliense by RFLP and DNA sequencing. Using forceps during endoscopy, 2 adult worms (1 male, 1 female) were recovered from the cat and identified morphologically as A. braziliense . Larvae were cultured from the feces of this cat and used to infect a laboratory-reared beagle dog. Additionally, worms recovered from the feces of the cat post-treatment were confirmed to be A. braziliense , except for 1 female A. caninum containing infertile eggs. The dog (patent 14 days post-infection) was also infected with A. braziliense as determined by RFLP and DNA sequencing of eggs and cultured larvae. Both the cat and dog were treated, verified to be no longer shedding eggs, and then placed into adoptive homes.

Reported in this communication is a unusually rapid growth of Lymnaea viridis , the intermediate host of Faciola hepatica , maintained in the laboratory for experimental purposes. When the snails were fed on blue-green algae at about 20°C, they attained a shell length of 12 mm in 37 days from the date of hatching. The snails began laying eggs from 18 to 24 days following hatching.

The current accepted standard for evaluating the efficacy of gastrointestinal anthelmintic drugs is necropsy of infected animals followed by a comparison of worm counts between treated and non-treated groups. In this study capsule endoscopy, a minimally invasive method of imaging the small intestine of humans, is evaluated as a possible alternative to necropsy for the purposes of worm quantification in dogs. Eighteen Beagle dogs were included in this study. These dogs were part of a separate trial intended to determine the efficacy of various candidate parasiticides against Ancylostoma caninum via the necropsy standard. Dogs were inoculated with A. caninum L3s 4 weeks prior to treatment with one of the candidate compounds; a control group (n=8) received no treatment. Capsule endoscopy was performed 6-14 days post-treatment, followed by necropsy the following day. Seventeen dogs had complete examinations, i.e. the capsule traversed the small intestine and reached the colon within the battery life of the capsule. A strong correlation (r(s)=0.87, P

The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin–rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM. Cancer Res; 70(11); 4590–601. ©2010 AACR.

Toxocaracanis and Toxocara cati of dogs and cats, respectively, can cause significant disease in people. Human seroprevalence for Toxocara antibodies varies with factors such as geographic location, socio-economic status, and dietary habits. Risk factors for infection include geophagia and low-level education. Toxocara canis is better recognized as a cause of human toxocariasis, but Toxocara cati should not be overlooked. In addition, patent infections with Baylisascaris procyonis, the raccoon ascarid, have been increasingly recognized in dogs. Pet owners need to be properly educated about zoonotic risks, and veterinarians should institute regular parasite screening and treatment for all pets. Establishment of national surveillance programs to determine the incidence and specific etiological agent in human larva migrans patients would aid in the development of targeted intervention strategies.

Fifteen Angus bulls and 15 Angus steers 9 months of age and 275 kg of body weight were bled at 20-min intervals over a 6-hr period and serum GH and IGF-I concentrations were measured by RIA. There were no differences between bulls and steers in the mean GH concentration, pulse frequency and amplitude when analyzed by the computer program PULSAR. Mean IGF-I concentration was not different between the two sex phenotypes, nor was there a significant correlation between the integrated IGF-I and GH concentrations. Subsequently, five bulls and five steers were selected from the 30 animals, full-fed a diet for growth in individual pens for 3 months and bled at 15-min intervals over a 24-hr period. Bulls tended to show a greater weight gain and feed conversion efficiency (P less than .10) than steers during the 3-month period. Serum GH concentrations had a pulsatile pattern in all animals with no apparent diurnal rhythm during the 24-hr bleeding. Although mean GH concentration was not different between the two sex phenotypes, bulls tended to have lower baseline levels (P less than .10) and greater peak amplitudes than steers. Serum IGF-I concentrations fluctuated within a two-fold concentration range, with no obvious pulsatility similar to that of GH. Mean IGF-I concentrations of each of the 10 animals were correlated with mean peak GH amplitudes (r = .79), but not with mean GH. These results suggest that gonadal hormone(s) modulates the GH secretory pattern and increases IGF-I secretion which may be related to the greater growth rate of bulls compared with steers.

N. Sugimoto, Y.-M. Chen, S.-Y. Lee, M. Matsuda and C.-Y. Lee. Pathodynamics of intoxication in rats and mice by enterotoxin of Clostridium perfringens type A. Toxicon29, 751–759, 1991.—The pathodynamics of lethal intoxication in rats and mice by i.v. administration of enterotoxin of Clostridium perfringens type A was studied using whole animals and isolated organs. A lethal i.v. dose (50 μg/kg) of enterotoxin killed anesthetized rats and mice within 4–15 min. Rapid changes of ECG pattern suggestive of hyperpotassemia, rapid fall of blood pressure and transient hyperpnea followed by respiratory depression were observed. Analysis of plasma levels of cations revealed hyperpotassemia in both animal species. On the other hand, enterotoxin (up to 100 μg) showed little direct cardiotoxicity on the isolated heart. ECG changes produced by i.v. injection of KCl (0.5 ml of 50 mM) mimicked the ECG changes observed in the intoxicated rats injected with a lethal dose of enterotoxin. Perfusion of rat isolated organs showed that potassium concentration in the eluent from the liver (but not lungs or lower extremities) increased markedly within 1–2min after the administration of enterotoxin. The amount of potassium liberated from a rat liver was about 133 μmoles, which is sufficient to increase the plasma level of potassium to more than 10 mM. In addition to potassium, cytoplasmic enzymes, such as glutamate oxalacetate transaminase, glutamate pyruvate transaminase and lactate dehydrogenase, were also liberated from the intoxicated liver, indicating that potassium was liberated from hepatocytes by the change in membrane permeability produced by enterotoxin. It is concluded that hyperpotassemia elicited by the cytotoxic action of enterotoxin on hepatocytes caused cardiac failure leading to the death of the intoxicated animals.

A 2-year-old female domestic shorthair cat on the island of Saipan was presented to a local veterinarian for headshaking. Otoscopic examination showed mild erythema of the right tympanic membrane, but was otherwise unremarkable. Headshaking resolved with topical gentamicin/betamethasone/clotrimazole therapy; however, erythema persisted. Further otoscopy revealed movement of the erythematous region, which was in fact the red-colored strongylid nematode, Mammomonogamus auris, residing within the middle ear. Myringotomy and a saline flush were performed under heavy sedation. A silastic tube was inserted into the incision and the worms were retrieved by applying negative pressure. Follow-up treatment included topical thiabendazole/dexamethasone/neomycin ointment as well as selamectin. Mammomonogamus auris has previously been documented only three times, once each in China, Sri Lanka and Japan. This is the first report of M auris in cats from Saipan.

Fluorescence microscopic imaging (FMI) is one of the fastest growing and most powerful techniques to study cellular activities in a living single cell. FMI has been widely used to monitor the temporal and spatial changes of many important intracellular messengers such as Ca2+, H+ and cAMP. In the course of our study of cellular responses with confocal scanning fluorescence microscopy, we detected two sources of artifacts which may render experimental observations invalid. First, the water content of the DMSO used could affect the efficiency of loading of the fluorescence indicator into cells and also give rise to spurious fluorescence spots. Secondly, apparently spontaneous temperature-dependent oscillations of BCECF fluorescence and cellular pulsations were recorded in cells which might be misinterpreted as natural rhythmic behavior. These were later shown to be artifacts arising from changes in refractive indices of the immersion oil due to small fluctuations in temperature, which in turn leads to random shifts of the focal plane erroneously manifest as signal oscillations. Based on these observations, certain recommendations for the control and elimination of false images are presented.

Multiple hyperthermia was found to exert an additive antitumor effect when combined with the in vivo production of tumor necrosis factor alpha (TNF-alpha) in mice bearing Ehrlich ascites tumor (EAT). TNF-alpha was produced in EAT-bearing mice by priming the animals with zymosan and subsequently challenging with lipopolysaccharide (LPS). Mice were pretreated with sulindac and D-mannoheptulose to alleviate the toxic side effects of LPS. While the ability of these tumor-bearing mice to produce TNF-alpha remained unchanged under hyperthermia, the EAT cell number was suppressed in the combined-treatment group compared with groups treated with TNF-alpha or hyperthermia alone. In the same comparison, the life span of EAT-bearing mice in the combined-treatment group was prolonged.

Three groups of Lymnaea viridis snails were infected with one, three or five Fasciola hepatica miracidia, and their metacercarial production was observed. In all groups cercarial shedding commenced in some snails as early as day 27 post-infection. The duration and pattern of cercarial shedding varied from snail to snail in each group. Snails infected with three or five miracidia produced more metacercariae than those infected with a single miracidium. The overall metacercarial production was influenced by the temperature at which the snails were kept during infection.

Waglerin-I, a lethal toxin isolated from the venom of Trimeresurus wagleri, consists of 22 amino acid residues with a proline-rich sequence. In the present study, we investigated the cause of death and the possible site of action of this toxin. In anesthetized mice, i.v. administration of waglerin-I at 0.5 microgram/g produced respiratory failure within 5 min. The blood pressure could be maintained by the application of artificial respiration immediately after respiratory arrest. Waglerin-I at 4 microM reversibly blocked the indirect twitch of the mouse phrenic nerve-hemidiaphragm preparation, but had no effect on the direct twitch. It is concluded that respiratory failure, resulting from neuromuscular block, is the primary cause of death due to waglerin-I in mice. In contrast to the results obtained in mice, in anesthetized rats no toxic effects on respiration and blood pressure were observed except for a transient hypotension up to 10 micrograms/g (i.v.). In accordance with the in vivo experiments, waglerin-I at concentrations up to 40 microM did not block the indirect twitch of the rat diaphragm. It thus appears that rats are resistant to the toxic effects of waglerin-I.

The histologic and ultrastructural features of a time sequence study of the development, evolution, and healing of acetic acid-induced experimental duodenal ulcer are presented. Duodenal ulcers produced by serosal application of acetic acid featured microvascular injury with progressive disintegration of the tips of the mucosal villi and subtotal necrosis of the duodenal wall. At 3 days ulcers transformed into a chronic state with regenerating epithelium originating from the crypts of the intact bordering mucosa extending toward the center of the ulcers. By 21 days healed ulcers were covered by distorted duodenal surface mucosa. We conclude that this reproducible and standardized model of duodenal ulcer features vascular injury as the earliest microscopic event, that ischemic necrosis leads to ulceration, and that the chronic phase bears morphologic resemblance to human duodenal ulcer.

The developmental expression of the individual components of the insulin-like growth factor (IGF) system in pigs was examined. Serum IGF-I and IGF-binding protein-3 (IGFBP-3) levels were low during fetal life and increased during postnatal development. Levels of mRNAs encoding these proteins were not greater for liver than for nonhepatic tissues (skeletal muscle, lung, kidney) and did not increase during the postnatal period, whereas hepatic growth hormone (GH) receptor mRNA expression was increased postnatally. Serum IGF-II levels exceeded IGF-I levels at all developmental stages examined and both exhibited postnatal increases. IGF-II mRNA abundance, in contrast, was high in the fetal tissues with the exception of lung and declined during the perinatal transition. Hepatic IGFBP-2 mRNA and serum IGFBP-2 levels increased during the latter half of gestation and then declined postnatally. The levels in muscle and liver of type I IGF receptors and the corresponding mRNAs also exhibited postnatal decreases. The discordance of changes in hepatic IGF-I and IGF-II mRNA abundance with serum IGF levels during the postnatal period does not support the concept that liver is the primary endocrine source of IGFs in the young pig. The postnatal increases in serum IGF levels may reflect decreased plasma clearance rates of these peptides which may be related to the transition in IGFBP type from IGFBP-2 to IGFBP-3 in blood and the reduced tissue expression of IGF receptors.

Helicobacter pylori has been implicated in the genesis of human gastritis, dyspepsia, and peptic ulcers. However, its influence in the quality of experimental gastric ulcer healing has not been previously investigated. Standardized gastric fundic ulcers were produced in 50 male Sprague-Dawley rats (150–200 g) by a 4 mm in diameter focal, serosal application of 100% acetic acid. Thirty rats were administered 2 ml H. pylori suspension (urease producing, ATCC 43504) in normal saline (10 8 CFU/ml) 2×/day for 7 days. Twenty rats (controls) received 2 ml normal saline 2×/day for 7 days. Gastric ulcer surface area was measured under a dissecting microscope and mucosal specimens were obtained for qualitative and quantitative histology. No gross or microscopic duodenal abnormalities were identified at sacrifice. Ninety percent of control rats showed grossly and microscopically entirely healed ulcers. The remaining 10% showed partially reepithelialized ulcers (area, 0.78 to 1.77 mm 2 ; mean, 1.27 ± 0.7 mm 2 ). The grossly “healed” mucosa demonstrated marked dilatation of gastric glands lined with mature surface epithelial cells. Parietal cells were scanty (5–10% of all cells). One hundred percent of the H. pylori -exposed rats showed persistence of chronic active ulcers (area, 1.76 to 19.63 mm 2 ; mean, 8.95 ± 6.15 mm 2 ). The ulcer beds were infiltrated by acute and chronic inflammatory cells, abundant fibroblasts, and capillary networks. The raised ulcer borders were characterized by dilated glands lined by mature surface epithelial cells. Various special stains demonstrated the presence of H. pylori in the surface mucus and within the crypts. We conclude that (1) H. pylori delays healing of experimental gastric ulcers distorting the restoration of mucosal architecture and (2) this animal model is extremely useful for the study of the role of H. pylori in the healing of experimental peptic ulcers.

Effects on transmembrane potentials of cardiotoxin isolated from Formosan cobra venom were studied on rabbit atrial cells by means of electrophysiological techniques. It was found that cardiotoxin at a concentration of 10 −5 g per ml caused a progressive and irreversible decrease of the resting potential in myocardial cells starting 5 min after addition of the toxin. The decrease in maximum diastolic repolarization of the pacemaker type cells occurred much later and to a much less extent. Associated with the decrease of resting potential, the magnitude and rate of rise of the action potential, the overshoot, the time to 80% repolarization and the spike ionic conductance were also decreased. The effect of the toxin on the membrane potential was not altered by either tetrodotoxin or sodium removal but was inhibited by high calcium. It is suggested that disintegration of the membrane structure is responsible for the membrane depolarization.

By means of Sephadex G-75 column chromatography, Vipera russelli venom was separated into five fractions. The phospholipase A2 (PLA2) activity was concentrated in Frs. II and III. These two PLA2 fractions were rechromatographed on CM-Sephadex C-50. Several subfractions of Fr. II and Fr. III contained PLA2 activities. Frs. III-3, III-6 and III-10 showed single bands in SDS-polyacrylamide gel electrophoresis with molecular weights estimated to be 15,054, 15,167 and 15,029, and isoelectric points of 4.15, 8.80 and greater than 10, respectively. Fr. III-3 had the most potent neuromuscular blocking action on the chick biventer cervicis nerve-muscle preparation, causing a complete neuromuscular blockade at 3 micrograms/ml. The response of the muscle to ACh, tested after complete blockade, was not altered. Most PLA2 subfractions had hypotensive actions in rats at 0.1 mg/kg. In the guinea-pig lung, Frs. II-5, II-7, III-3, III-6 and III-10 increased the perfusion pressure, which may account for part of their hypotensive actions.

Bacterial lipopolysaccharide (LPS) induces the release of tumour necrosis factor (TNF) into serum of mice previously infected with Listeria monocytogenes or immunized with formalin-killed Corynebacterium parvum. This release is greatly reduced by neutralisation of lipid A of LPS with the antibiotic polymyxin B sulfate. The effect is dose-dependent. Base-hydrolysed LPS, which is devoid of lipid A, cannot induce TNF release. Crude lipid A still retains its ability to induce TNF release but is significantly less effective than native LPS molecules. LPS neutralised by polymyxin B also loses its ability to cause high mortality in C. parvum primed mice. These results suggest that lipid A of LPS molecule is important in causing lethality and TNF release in vivo while the polysaccharide portion may be involved in delivering the lipid A moiety to TNF-producing cells.

Wan Wan Lin , C. Y. Lee and J. W. Burnett . Effect of sea nettle ( Chrysaora quinquecirrha ) venom on isolated rat aorta. Toxicon 26, 1209–1212, 1988.—The venom from sea nettle ( Chrysaora quinquecirrha ) (1–10μg/ml) produced an irreversible contraction of the isolated rat aortic ring that was slow in onset, increased with time, and reached maximum in about 10–20 min. The contraction was not inhibited by pretreatment with phenoxybenzamine, atropine, indomethacin, tetrodotoxin, ouabain, low Na + or Na + -free medium, however, it was markedly decreased by the Ca 2+ channel blockers, nifedipine and verapamil. In Ca 2+ -free medium, no increase in tension was produced by the venom. It is concluded that sea nettle venom causes a contraction of the rat aortic ring by increasing Ca 2+ influx through the voltage-dependent Ca 2+ channels.

A basic phospholipase A2 purified from the venom of a crotalid snake (Trimeresurus mucrosquamatus) was applied to chick and mouse neuromuscular preparations. The enzyme at lower concentrations (1-3 micrograms/ml) blocks neuromuscular transmission at a presynaptic site, while at higher concentrations (10-30 micrograms/ml) it causes contracture and depolarization of the muscle as well. It is concluded that the mode of action of the enzyme on vertebrate nerve-muscle transmission is similar to that of notexin and ceruleotoxin from elapid snake venoms.

The ability of Ehrlich ascites tumor cells to take up glucose increased progressively during the course of tumor development. Simultaneously as the rate of uptake rose, the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface also increased. In its stereospecificity requirement toward competing sugars and in its sensitivity to phloretin and diethylstilbestrol, this class of binding sites resembled the putative glucose carriers identified in various other cell systems and may represent the glucose transporter in Ehrlich ascites cells. Work with methotrexate (MTX) substantiated this view. Methotrexate arrested tumor growth, inhibited glucose uptake, and reduced the number of cytochalasin B binding sites. In both MTX-treated and untreated cells, the magnitude of changes in number of cytochalasin B binding sites closely paralleled and sufficiently accounted for the magnitude of changes in glucose uptake. Qualitative changes in the turnover and affinity for substrate of the putative glucose carrier need not be invoked.

Four highly toxic fractions (I-B-2, I-C, III-A and III-B) were obtained from the venom of the Indian krait (Bungarus caeruleus) by CM-cellulose column chromatography, followed by various steps of gel filtration on Sephadex G-50 and rechromatography on CM-Sephadex C-25. Among them, fractions III-A and III-B proved to be homogeneous on disc-electrophoresis. Based on the ld 50 in mice, time to death at higher doses, and especially the effect on acetylcholine response of the rat denervated diaphragm as well as the chick biventer cervicis muscle, it is concluded that fractions I-B-2 and I-C belong to postsynaptically acting neurotoxins, similar to α-bungarotoxin, whereas fractions III-A and III-B belong to presynaptically acting neurotoxins, similar to β-bungarotoxin.

Three neurotoxic components (W-III, W-IV and W-V) were isolated from the venom of Walterinnesia aegyptia by CM-cellulose column chromatography, followed by gel filtration on a Sephadex G-50 column. The ld 50 in mice (i.p.) was 0.95 μg per g body wt for W-III, 0.14 μg per g for W-IV and 0.2 μg per g for W-V, while in baby chicks (i.m.) it was over 0.5 μg per g for W-III, 0.12 μg per g for W-IV and 0.23 μg per g for W-V. All of the three toxins blocked the neuromuscular transmission of the isolated rat phrenic nerve-diaphragm and the chick biventer cervicis muscle and inhibited the response of the latter to acetylcholine, just like other postsynaptically acting snake neurotoxins. The neuromuscular block produced by W-III was reversible in both muscle preparations, whereas that by W-IV as well as W-V was irreversible in the chick biventer cervicis but partially reversible in the rat diaphragm.

Both cobra cardiotoxin (CTX) and scorpion toxin II induce contracture of the guinea-pig taenia coli, the latter being more potent than the former. Tachyphylaxis was observed with each toxin. Acetylcholine (ACh) contracture was enhanced by toxin II but not by CTX. High K + (152 mM) abolished toxin II contracture but only partially inhibited CTX and ACh contractures. Ca 2+ -free medium, especially in the presence of 1 mM EGTA, inhibited CTX- and toxin II-contracture by more than 60%, but abolished ACh contracture. On the other hand, high Ca 2+ (12 mM) was without effect on ACh contracture but inhibited both CTX- and toxin II-contracture, and enhanced K + contracture. Contracture was produced after washout of Ca 2+ -free medium and toxins but not after washout of high Ca 2+ medium, suggesting that the binding of toxins was inhibited by high Ca 2+ . Tetrodotoxin (TTX) abolished toxin II contracture, but only partially inhibited CTX contracture. Atropine markedly inhibited toxin II contracture but did not significantly affect CTX contracture. Procaine potentiated CTX contracture but abolished toxin II contracture. The spontaneous contraction induced by toxin II was also inhibited by TTX, atropine and procaine. ACh contracture was abolished by atropine and potentiated by procaine, but unaffected by TTX. The rate of decline of K + (60 mM) contracture in Ca 2+ -free medium was enhanced by CTX but unaffected by toxin II. 45 Ca 2+ efflux was increased by CTX more than by toxin II. Both toxins also significantly increased 45 C 2+ uptake. These findings indicate that CTX induces contracture of taenia coli mainly by releasing the Ca 2+ pool of the muscle membrane, whereas toxin II does so mainly by releasing acetylcholine from nerve terminals and partially by acting on the muscle membrane as a result of an increase of the membrane Na + permeability.

As shown previously, β-bungarotoxin (β-BuTX) mimicks surgical denervation in inhibiting growth of the chick embryo, in causing skeletal muscle atrophy, fat displacement of the muscle fibers, ankylosis and a shorter upper beak. In this paper, effects of β-BuTX on acetylcholinesterase activity and ultrastructure of the skeletal muscle were studied by histochemical and electron microscopic methods. Acetylcholinesterase activity of the β-BuTX treated muscle decreased markedly. The longitudinal section of thigh muscle of 16-day-old chick embryo treated with β-BuTX showed that actin and myosin myofilaments became fewer and disruptive. Unlike those of the normal control muscle, A, I, H and M bands disappeared in the treated muscle. This change became more severe in those of the 19-day-old chick embryo. On the other hand, the cross section of thigh muscle of a 19-day-old chick embryo showed that the hexagonal arrays of actin and myosin filaments were disorganized. Moreover, the sarcolemma of the toxin-treated muscle was fragmented and the trilamellar organization of the membrane became obscure. This result is correlated with our previous findings that the membrane bound enzymic activities (acetylcholinesterase, Ca 2+ -ATPase, Na + -K + -ATPase and Mg 2+ -ATPase) and acetylcholine receptors decreased in the toxin treated muscle. Phospholipase A activity of β-BuTX may play an important role in the destruction of the membrane.