Survey of Soybean Insect Pollinators: Community Identification and Sampling Method Analysis.

Soybean, Glycine max (L.) Merrill, flowers can be a source of nectar and pollen for honey bees, Apis mellifera L. (Hymenoptera: Apidae), wild social and solitary bees (Hymenoptera: Apoidea), and flower-visiting flies (Diptera). Our objectives were to describe the pollinator community in soybean fields, determine which sampling method is most appropriate for characterizing their abundance and diversity, and gain insight into which pollinator taxa may contact soybean pollen. We compared modified pan traps (i.e., bee bowls), yellow sticky traps, and sweep nets for trapping pollinators in Iowa soybean fields when soybeans were blooming (i.e., reproductive stages R1-R6) during 2011 and 2012. When all trap type captures were combined, we collected 5,368 individuals and at least 50 species. Per trap type, the most pollinators were captured in bee bowls (3,644 individuals, 44 species), yellow sticky traps (1,652 individuals, 32 species), and sweep nets (66 individuals, 10 species). The most abundant species collected include Agapostemon virescens F. and Lasioglossum (Dialictus) species (Hymenoptera: Halictidae), Melissodes bimaculata Lepeletier (Hymenoptera: Apidae), and Toxomerus marginatus Say (Diptera: Syrphidae). To determine if these pollinators were foraging on soybean flowers, we looked for soybean pollen on the most abundant bee species collected that had visible pollen loads. We found soybean pollen alone or intermixed with pollen grains from other plant species on 29 and 38% of the bees examined in 2011 and 2012, respectively. Our data suggest a diverse community of pollinators-composed of mostly native, solitary bees-visit soybean fields and forage on their flowers within Iowa.

Exploring the diversity and metabolic potential of actinomycetes from temperate marine sediments from Newfoundland, Canada.

Marine sediments from Newfoundland, Canada were explored for biotechnologically promising Actinobacteria using culture-independent and culture-dependent approaches. Culture-independent pyrosequencing analyses uncovered significant actinobacterial diversity (H'-2.45 to 3.76), although the taxonomic diversity of biotechnologically important actinomycetes could not be fully elucidated due to limited sampling depth. Assessment of culturable actinomycete diversity resulted in the isolation of 360 actinomycetes representing 59 operational taxonomic units, the majority of which (94 %) were Streptomyces. The biotechnological potential of actinomycetes from NL sediments was assessed by bioactivity and metabolomics-based screening of 32 representative isolates. Bioactivity was exhibited by 41 % of isolates, while 11 % exhibited unique chemical signatures in metabolomics screening. Chemical analysis of two isolates resulted in the isolation of the cytotoxic metabolite 1-isopentadecanoyl-3ß-D-glucopyranosyl-X-glycerol from Actinoalloteichus sp. 2L868 and sungsanpin from Streptomyces sp. 8LB7. These results demonstrate the potential for the discovery of novel bioactive metabolites from actinomycetes isolated from Atlantic Canadian marine sediments.

Quality over quantity: buffer strips can be improved with select native plant species.

Native plants attractive to beneficial insects may improve the value of buffer strips by increasing biodiversity and enhancing the delivery of insect-derived ecosystem services. In a 2-yr field experiment, we measured the response of insect communities across nine buffers that varied in plant diversity. We constructed buffers with plants commonly found in buffers of USDA-certified organic farms in Iowa (typically a single species), recommended for prairie reconstruction, or recommended for attracting beneficial insects. We hypothesized that the diversity and abundance of beneficial insects will be 1) greatest in buffers composed of diverse plant communities with continuous availability of floral resources, 2) intermediate in buffers with reduced species richness and availability of floral resources, and 3) lowest in buffers composed of a single species. We observed a significant positive relationship between the diversity and abundance of beneficial insects with plant community diversity and the number of flowers. More beneficial insects were collected in buffers composed of species selected for their attractiveness to beneficial insects than a community recommended for prairie restoration. These differences suggest 1) plant communities that dominate existing buffers are not optimal for attracting beneficial insects, 2) adding flowering perennial species could improve buffers as habitat for beneficial insects, 3) buffers can be optimized by intentionally combining the most attractive native species even at modest levels of plant diversity, and 4) plant communities recommended for prairie reconstruction may not contain the optimal species or density of the most attractive species necessary to support beneficial insects from multiple guilds.

Combined vertical reduction rectoplasty and sacral nerve stimulation for rectal evacuatory dysfunction and faecal incontinence associated with previous anorectal malformation.

Functional outcomes following surgery for anorectal malformation are variable, with many children experiencing persisting anorectal dysfunction. We describe a 34-year-old female with previous vestibular fistula who experienced lifelong rectal evacuatory dysfunction and faecal incontinence; she was treated in a two stage process producing efficient defecation and almost total continence.

Health benefits of dietary fat reduction by a novel fat replacer: Mimix.

The primary goals of this study were to identify any health benefits of the replacement of dietary fat with a novel fat replacer, Mimix, and to assure that the consumption of this fat replacer did not convey any deleterious health effects. Male, weanling, Fischer 344 rats were fed one of six diets containing between 5 and 20% w/w as fat for 8 weeks. These diets included two high fat diets (safflower oil or lard), a low fat diet and three diets where 15% of the fat in the high fat diets was replaced with various amounts of Mimix. When animals were fed a diet rich in saturated fat they consumed significantly more energy than other diet groups. When 15% saturated fat (lard) was replaced with safflower oil animals adjusted their food intake so that no difference in energy intake was observed between the high safflower diet and the low fat and Mimix diets. When the various Mimix fat replacements were compared to animals fed a high fat lard diet there was incomplete compensation of energy intake. Animals fed the high fat lard diet also had higher glucose and total serum cholesterol than their low fat and fat replacement counterparts. Feeding a high fat safflower oil diet to rats resulted in a significantly lower total serum cholesterol and serum triglyceride than all other diets. Replacement of dietary fat with Mimix demonstrated no deleterious effects on the heart, liver and intestinal tract that were all of normal weight, morphology and colour compared to other diet groups. Body composition analysis demonstrated that animals fed high fat diets had higher body fat mass at the expense of lean body mass. This was most obvious for animals fed high fat lard diets who had heavier epididymal fat pads. These data demonstrate that the replacement of dietary fat with the novel fat replacer Mimix can convey a number of health benefits in the absence of any deleterious effects.

All trans retinoic acid induces apoptosis in acute promyelocytic NB4 cells when combined with isoquinolinediol, a poly(ADP-ribose) polymerase inhibitor.

NB4 cells, a model of acute promyelocytic leukemia have been shown to undergo granulocytic differentiation in response to all trans retinoic acid (ATRA), or monocytic differentiation in response to 1alpha,25 dihydroxyvitamin D(3) (1,25 D(3)) and phorbol ester. We have shown previously that the expression of poly(ADP-ribose) polymerase (PARP) is dramatically increased during monocytic differentiation and completely down-regulated during neutrophilic differentiation. Here we show that NB4 cells simultaneously treated with ATRA and isoquinolinediol (Iso-Q), a specific PARP inhibitor, fail to differentiate into neutrophils. Nitroblue tetrazolium (NBT) dye reduction was inhibited by 48% and neutrophil formation was reduced by 75%. NB4 cells treated with ATRA and Iso-Q instead showed features of apoptosis including nuclear condensation, DNA fragmentation, and PARP cleavage yielding a 85 kDa fragment. NB4 cells treated with ATRA in combination with 1,25 D(3), a monocytic differentiation inducer, while continuing to reduce NBT also failed to mature into neutrophils or monocytes and again showed features of apoptosis. Down-regulation of Bcl-2 protein expression was evident in NB4 cells treated with ATRA and ATRA in combination with Iso-Q or 1,25 D(3), but not in cells treated with a classic chemotherapeutic agent, arabinosycytosine, suggesting that Bcl-2 down-regulation is neither necessary, nor specific for apoptosis in this model.

Both (n-3) and (n-6) fatty acids stimulate wound healing in the rat intestinal epithelial cell line, IEC-6.

The control of proliferation and epithelial restitution are processes that are poorly understood. The effects of (n-3), (n-6) and trans fatty acids on proliferation of subconfluent IEC-6 cultures and restitution of wounded IEC-6 monolayers were investigated. Incorporation of supplemented fatty acids into cellular phospholipid was also assessed. Sulforhodamine B protein dye binding assay was utilized to assess the proliferative effects of fatty acids on growth of IEC-6 cultures. Incorporation of supplemental fatty acids into cellular phospholipid was examined by thin-layer chromatography combined with gas chromatography. The modulation of epithelial restitution was examined by razor blade wounding confluent IEC-6 monolayers grown in media supplemented with various fatty acids. Inhibition of eicosanoid synthesis by indomethacin during the wounding assay was also assessed. Both (n-3) and (n-6) fatty acids significantly inhibited growth of this intestinal epithelial cell model at concentrations above 125 micromol/L. The trans fatty acid, linoelaidate 18:2(n-6)trans, inhibited growth of IEC-6 cells at concentrations above 250 micromol/L. Another trans fatty acid, elaidate 18:1(n-9)trans, was well-tolerated at concentrations as high as 500 micromol/L. Eicosapentanoic 20:5(n-3), linoleic 18:2(n-6), alpha-linolenic 18:3(n-3), gamma-linolenic 18:3(n-6) and arachidonic 20:4(n-6) acids all significantly enhanced cellular migration in the IEC-6 model of wound healing. Eicosapentanoate, linoleate, alpha-linolenate, gamma-linolenate and arachidonate are all capable of improving reconstitution of epithelial integrity following mucosal injury. Inhibition of eicosanoid synthesis reduced the enhancement of restitution by n-6 fatty acids back to control levels.

Vitamin D analogs, 20-Epi-22-oxa-24a,26a,27a,-trihomo-1alpha,25(OH)2-vitamin D3, 1,24(OH)2-22-ene-24-cyclopropyl-vitamin D3 and 1alpha,25(OH)2-lumisterol3 prime NB4 leukemia cells for monocytic differentiation via nongenomic signaling pathways, involving calcium and calpain.

Side-chain modified vitamin D analogs including 20-Epi-22-oxa-24a,26a,27a-trihomo-1alpha,2 5-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were originally designed to aid in the treatment of hyperproliferative disorders including psoriasis and cancer. Here we demonstrate that these analogs, as well as the 6-cis-locked conformer, 1alpha,25-dihydroxy-lumisterol3 (JN) prime NB4 cells for monocytic differentiation. Previously, the action of MC903 and KH1060 was presumed to be mediated by the nuclear vitamin D receptor (VDRnuc). Differentiation in response to all analogs was shown to be inhibited by 1beta,25-dihydroxyvitamin D3 (HL), the antagonist to the nongenomic activities of 1,25D3. These data suggest that although MC903 and KH1060 may bind the VDRnuc, that the differentiative activities of these agents requires nongenomic signaling pathways. Here we show that 1alpha,25(OH)2-d5-previtamin D3 (HF), JN, KH1060, and MC903 induce expression of PKC alpha and PKC delta and translocation of both isoforms to the particulate fraction, and PKC alpha to the nuclear fraction. The full differentiation response with combinations of analogs and TPA was inhibited 50% by the membrane permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) or calpain inhibitor I. These data demonstrate that intracellular free calcium and the calcium-dependent protease, calpain play critical roles in monocytic differentiation. Intracellular calcium appears to be most critical in the 1,25D3-priming stage of differentiation, while calpain is essential in the TPA maturation response.

Characterization of a novel Na+-dependent, guanosine-specific, nitrobenzylthioinosine-sensitive transporter in acute promyelocytic leukemia cells.

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na+, guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na+ buffer decreased by 75% with the addition of 1 microM NBMPR and the IC50 for NBMPR inhibition was 0.7 +/- 0.1 nM. Replacement of Na+ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na+-dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na+ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mM competing nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of the csg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.

Incorporation of long-chain n-3 fatty acids in tissues and enhanced bone marrow cellularity with docosahexaenoic acid feeding in post-weanling Fischer 344 rats.

We wanted to examine the effects of an oil rich in docosahexaenoic acid (DHA), without eicosapentaenoic acid, on the composition of membrane phospholipid in a variety of tissues. Our in vitro studies had previously shown that DHA could modify glucose and nucleoside transport in cells in culture and also increase selectivity of the nucleoside drug, arabinosylcytosine (araC) toward tumor cells. Here we wanted to examine what effect DHA supplementation would have in the whole animal in terms of the chemosensitivity of normal bone marrow, the dose-limiting tissue during chemotherapy, to araC. The purpose was to determine whether fatty acid supplementation might be useful as an adjuvant to chemotherapy. We fed diets containing 5% (w/w) low fat-corn oil (LF-CO group), 10% moderate fat-safflower oil (MF-SO group), or 10% DHASCO (MF-DHA group) to weanling Fischer 344 rats for 8-9 wk. Feed intake and growth were not different between the different diets. Similarly, treatment of animals with the chemotherapeutic drug araC did not differentially affect growth, feed intake, or tissue fatty acid composition for the different diet groups. Fatty acid compositions of bone marrow, liver, red blood cells, plasma phospholipid and triglyceride, as well as skeletal and cardiac muscle, were substantially different between the dietary groups. The DHASCO oil contained 46% DHA (22:6n-3) and resulted in profound incorporation of DHA in all tissues examined. The most dramatic response was seen in skeletal muscle of MF-DHA fed animals where DHA represented 46% of membrane phospholipid fatty acids. This is likely to have consequences to muscle function. Although DHASCO contains a similar level of saturated fatty acids (42%), few differences in saturates were noted between the various dietary groups for most of the tissues examined. Both LF-CO and MF-SO diets were hypercholesterolemic, and the LF-CO was also hypertriglyceridemic compared to the chow-fed animals. Animals fed the MF-DHA diet had the lowest triglyceride levels of any of the treatment groups and cholesterol levels comparable to chow-fed animals. MF-DHA had substantially higher numbers of colony-forming units-granulocyte macrophage (CFU-GM) as reflected in a twofold higher bone marrow cellularity than either chow or LF-CO animals, suggesting expansion of the bone marrow compartment with DHA feeding. Although higher than LF-SO, the number of CFU-GM in MF-SO animals was not significantly higher than animals fed chow. Bone marrow from LF-CO animals appeared to be more resistant to araC treatment than either MF group. Thus, DHA, fed as DHASCO, has advantages over low or moderate n-6 diets and chow as it is has both hypolipidemic- and bone marrow-enhancing properties in weanling Fischer 344 rats. This suggests that DHA supplementation may be useful in adjuvant chemotherapy.

DHA feeding provides host protection and prevents fibrosarcoma-induced hyperlipidemia while maintaining the tumor response to araC in Fischer 344 rats.

Fischer 344 rats were inoculated with fibrosarcoma tumor cells and fed diets containing 5% or 10% (wt/wt) safflower oil or 10% oil containing docosahexaenoic acid (DHA). Animals were then treated with arabinosylcytosine (araC) or saline for six days. Tumor weights were highest in animals fed 10% safflower oil and treated with saline, intermediate in animals fed oil containing DHA and 5% safflower oil and treated with saline, and lowest in araC-treated animals from all diets. Plasma cholesterol and triglyceride levels correlated highly with final tumor size, regardless of diet or treatment group. Animals fed safflower oil had lower intestinal weights than those fed DHA, which histology demonstrated to be a result of differences in villus height and crypt depth. Substantial loss of bone marrow cells occurred in all dietary groups treated with araC; however, the proportion of granulocyte-macrophage precursors remaining in the DHA animals was higher than in saline-treated animals and twofold higher than in the animals fed 10% safflower oil and treated with araC. These data suggest that, even in the face of rapid tumor growth and chemotherapeutic challenge, consumption of a diet rich in DHA can slow tumor growth, prevent hyperlipidemia, enhance bone marrow cellularity, and promote intestinal growth compared with a moderate-fat n--6-rich diet.

1,25-Dihydroxyvitamin D3 stimulates expression and translocation of protein kinase Calpha and Cdelta via a nongenomic mechanism and rapidly induces phosphorylation of a 33-kDa protein in acute promyelocytic NB4 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) primes NB4 cells for 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation in a dose- and sequence-dependent fashion. Experiments utilizing 1,25-(OH)2D3 analogues and kinase/phosphatase inhibitors suggested that tyrosine kinase and serine/threonine phosphorylation cascades, rather than vitamin D3 receptor-mediated signals, were involved in 1,25-(OH)2D3 action. Here we show that NB4 cells express the alpha and delta (but not the beta, epsilon, and theta) isoforms of protein kinase C (PKC). Both authentic 1, 25-(OH)2D3 and the nongenomic analogue 1alpha,25-dihydroxyprevitamin D3 (HF) increased expression of PKCalpha and PKCdelta. PKCalpha and PKCdelta were translocated to the nucleus of the cell in response to 1,25-(OH)2D3 or HF. The effects of HF were attenuated by the nongenomic antagonist 1beta,25-dihydroxyvitamin D3, suggesting that changes in PKC expression are mediated by a nongenomic signaling pathway. Consistent with the involvement of serine, threonine, and tyrosine phosphorylation cascades mediating 1,25-(OH)2D3 action, enhanced phosphorylation of a variety of cellular proteins at serine and threonine residues and the specific enhanced phosphotyrosyl content of a 33-kDa protein (vdrp33) were observed immediately after 1,25-(OH)2D3 addition. We propose that 1,25-(OH)2D3 primes NB4 cells for 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation by increasing the expression of specific PKC isoforms and inducing the specific phosphorylation of key protein signaling intermediates.

A retrovirus isolated from cell lines derived from neurofibromas in bicolor damselfish (Pomacentrus partitus).

Damselfish neurofibromatosis (DNF) is a naturally occurring, neoplastic disease affecting bicolor damselfish (Pomacentrus partitus) living on coral reefs in southern Florida, USA. The disease consists of multiple neurofibromas, neurofibrosarcomas and chromatophoromas and has been proposed as an animal model for neurofibromatosis type 1 in humans. DNF is transmissible by injection of crude tumour homogenates, cell-free filtrates of homogenates or cells from tumour cell lines. An analysis of tumorigenic cell lines derived from fish with spontaneous or experimentally induced DNF revealed virus particles budding from cells and present in conditioned media. The 90-110 nm particles resembled type C retroviruses. This virus exhibited a buoyant density of 1.14-1.17 g/cm2 in sucrose, at least six virus proteins of 15 to 80 kDa and reverse transcriptase (RT) activity. RT activity was maximized with a poly(rC).oligo(dG) template.primer combination and Mn2+ at a concentration of 0.5-1.0 mM. The optimum temperature for RT was determined to be 20 degrees C, a finding consistent with the ambient temperatures encountered by this species. This retrovirus, tentatively named damselfish neurofibromatosis virus (DNFV) may be the aetiological agent of DNF. Whether DNFV or another, as yet unidentified, virus is the cause of DNF, this agent may be unique in virus oncogenesis; neoplastic transformation of the cell types involved in DNF, Schwann cells and chromatophores, has not been documented in any other transmissible tumour.

Omega-3 polyunsaturated fatty acids increase purine but not pyrimidine transport in L1210 leukaemia cells.

Here we show that in vitro supplementation of L1210 murine lymphoblastic leukaemia cells with n-3 polyunsaturated fatty acids results in considerable changes in the fatty acid composition of membrane phospholipids. Incubations for 48 h with 30 microM eicosapentaenoic acid (20:5, n-3; EPA) or docosahexaenoic acid (22:6, n-3; DHA) results primarily in substitution of long chain n-6 fatty acids with long-chain n-3 fatty acids. This results in a decrease in the n-6/n-3 ratio from 6.9 in unsupplemented cultures to 1.2 or 1.6 for EPA and DHA supplemented cultures, respectively. Coincident with these changes in membrane fatty acid composition, we observed a 5-fold increase in the rate of adenosine (5 microM) uptake via a nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter in EPA- and DHA-supplemented L1210 cells, relative to unsupplemented cells. This seemed to result from a decrease in the Km for adenosine from 12.5 microM in unsupplemented cultures to 5.1 microM in DHA-treated cultures. Guanosine (50 microM) transport was similarly affected by DHA with a 3.5-fold increase in the initial rate of uptake. In contrast, pyrimidine transport, as measured by uptake of thymidine and cytidine, was not similarly affected, suggesting that substrate recognition had been altered by fatty acid supplementation. Studies using [(3)H]NBMPR showed that there was no effect of EPA or DHA on either the number of NBMPR-binding sites or the affinity of these sites for NBMPR. This observation suggests that the increases in adenosine and guanosine transport were not due to increases in the number of transported sites but rather that EPA and DHA directly or indirectly modulate transporter function.

Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells.

Nucleoside transport processes can be classified by the transport mechanism, e=equilibrative and c=concentrative, by the sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), s=sensitive and i=insensitive, and also by permeant selectivity. To characterize nucleoside transport in acute promyelocytic NB4 cells, nucleoside transport was resolved into different components by selective elimination of transport processes with NBMPR and with Na+-deficient media. Initial transport rates were estimated from time course experiments. For adenosine, uridine, and formycin B, equilibrative transport accounted for approximately 60% of their uptake, with ei and es transport contributing almost equally, and Na+-dependent transport accounting for the remaining 40% of the total uptake. Thymidine uptake was mediated exclusively by equilibrative systems with ei and es systems each contributing 50% to total uptake. Adenosine accumulated above equilibrative concentrations, suggesting that a concentrative transport process was active and/or that metabolism led to adenosine's accumulation. Formycin B, a nonmetabolizable analog, also accumulated in the cells, supporting the concentrative potential of the Na+-dependent transporter. Kinetic analyses also provided evidence for three distinct high affinity transport mechanisms. NBMPR binding assays indicated the presence of two high affinity (Km 0.10 and 0.35 nM) binding sites. In conclusion, NB4 cells express ei and es transport, as well as a large ci transport component, which appears to correspond to cif (f=formycin B or purine selective) nucleoside transport, not previously described in human cells.

1,25-dihydroxyvitamin D3 primes acute promyelocytic cells for TPA-induced monocytic differentiation through both PKC and tyrosine phosphorylation cascades.

NB4 cells are the only in vitro model of differentiation in acute promyelocytic leukemia (APL). Although these cells respond to all-trans-retinoic acid to form neutrophils, our group has recently shown that these cells are capable of terminal monocytic differentiation in response to combined treatment with 1,25-dihydroxyvitamin D3 (1,25 D3) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that the agents need not be present simultaneously, but may be added sequentially. TPA treatment prior to 1,25 D3 led to the appearance of adherent cells; however, when 1,25 D3 treatment preceded TPA treatment cells expressed all differentiation markers reflective of terminal differentiation. This priming effect of 1,25 D3 was both dose and time dependent. Increasing the interval between 1,25 D3 and TPA treatment caused a decrease in this priming potential indicative of limited commitment inducing capacity of 1,25 D3. In order to characterize the mechanism of action of 1,25 D3 and TPA, chemical inhibitors of phosphorylation were used. Staurosporine and bisindolymaleimide GF 109203X treatment prior to and during 1,25 D3 treatment or TPA treatment caused attenuation of the differentiation response. Experiments utilizing tyrosine kinase and phosphatase inhibitors supported the hypothesis that 1,25 D3 signaling was mediated by both serine/threonine and tyrosine phosphorylation cascades. Results from this study provide evidence to support the hypothesis that 1,25 D3 signaling occurs via nongenomic mechanisms which when combined with the signaling effects of TPA, allow for the terminal differentiation of APL cells. This model should be used to develop new differentiation therapies for APL and other leukemias.

Overexpression of poly(ADP-ribose) polymerase promotes cell cycle arrest and inhibits neutrophilic differentiation of NB4 acute promyelocytic leukemia cells.

The t(15;17) translocation causes a disruption of the retinoic acid receptor alpha (RAR-alpha) and allows for the expression of the PML-RAR alpha fusion protein considered to be responsible for the differentiation block in acute promyelocytic leukemia (APL). Patients being treated with all-trans retinoic acid (ATRA) undergo remission due to the differentiation of leukemic cells to functional neutrophils but relapse due to subsequent ATRA resistance. Our group has shown recently that NB4 cells, the only in vitro model of APL, are capable of monocytic differentiation in response to 1,25-dihydroxyvitamin D3 and 12-O-tetradecanoylphorbol-13-acetate in addition to the neutrophilic differentiation response that occurs with ATRA treatment. Poly(ADP-ribose) polymerase (PARP) is a ubiquitous protein that plays a role in DNA metabolism and repair. We have shown that, unlike HL-60 cells, NB4 cells completely down-regulate PARP in the neutrophilic lineage and up-regulate PARP 90-fold in the monocytic lineage. To ascertain whether PARP is an active participant in the bipotent differentiation of APL cells, NB4 cells were transiently transfected by lipid-mediated gene transfer with the human PARP gene under the control of the human metallothionein promoter. A 4-fold overexpression of PARP, in response to 8 microM CdCl2, promoted arrest of NB4 cells in the S phase of the cell cycle. Overexpression of PARP alone had no effect on cell viability or induction of phenotypic markers in the monocytic or neutrophilic lineages. However, increased PARP expression did result in an increase in the number of cells in the subdiploid population likely to include apoptotic cells. Overexpression of PARP, alone with 12-O-tetradecanoylphorbol-13-acetate (200 nM), 1,25-dihydroxyvitamin D3 (200 nM), or a suboptimal dose of the combined agents, did not alter the expected monocytic differentiation marker profile over cells transfected with control plasmid (pSV2Neo). In contrast, PARP overexpression blocked the appearance of phenotypic markers of terminally differentiated neutrophils in 85% of the transfected population in response to 1 microM ATRA. Comparable to wild-type NB4 cells, 90% of cells transfected with pSV2Neo developed neutrophilic differentiation markers (nitroblue tetrazolium-positive and multi-lobed nuclei) in response to 1 microM ATRA. These data suggest that overexpression of PARP arrests APL cells and blocks ATRA-induced terminal neutrophilic differentiation. We propose that normal down-regulation of PARP in NB4 cells is a requirement for neutrophilic maturation.

Monocytic differentiation of acute promyelocytic leukemia cells in response to 1,25-dihydroxyvitamin D3 is independent of nuclear receptor binding.

We have shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) primes NB4 cells, the only available acute promyelocytic leukemia cell line, for 12-O-tetradecanoyl-phorbol-13-acetate-induced monocytic differentiation. Here, we have used isomers of 1,25(OH)2D3 to investigate the role of 1,25(OH)2D3 and its putative nuclear receptor (VDR) in NB4 cell monocytic differentiation. 1 beta,25-dihydroxyvitamin D3 (HL), a specific antagonist of only the nongenomic signals of 1,25(OH)2D3, attenuated the priming effect of 1,25(OH)2D3. The 6-cis conformer of 1,25(OH)2D3 (HF), which is unable to bind to VDR, was 20 times more potent than 1,25(OH)2D3 as a priming agent for monocytic differentiation. This response was also blocked by the HL antagonist. Unlike myelocytic HL-60 cells, which respond to 1,25(OH)2D3 with increases in VDR expression and monocytic differentiation, neither HF nor 1,25(OH)2D3 regulated VDR expression in NB4 cells. In the monocytic differentiation of acute promyelocytic leukemia cells, 1,25(OH)2D3 appears to signal through a pathway independent of VDR/VDRE action.

Modulation of poly(ADP-ribose) polymerase during neutrophilic and monocytic differentiation of promyelocytic (NB4) and myelocytic (HL-60) leukaemia cells.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.

Characterization of Schwann cells from normal nerves and from neurofibromas in the bicolour damselfish.

Schwann cells are an important component of neurofibromas, one of the primary lesions encountered in neurofibromatosis type 1 in man. A central question in studies of neurofibromatosis type 1 has been whether the Schwann cells present in these tumours are intrinsically abnormal or exhibit abnormal phenotypes in response to stimuli from other cell types in these tumours. Damselfish neurofibromatosis is a naturally occurring disease in a species of marine fish, the bicolour damselfish, that is being developed as an animal model of neurofibromatosis type 1. Affected fish exhibit multiple neurofibromas and neurofibrosarcomas (malignant schwannomas). The present study compares the morphology, antigen expression and proliferative capacity in vitro of Schwann cells derived from peripheral nerves of normal, healthy fish with cells isolated from both spontaneously occurring and experimentally induced neurofibromas. Schwann cells from normal nerves expressed S100 antigens but not fibronectin or glial fibrillary acidic protein antigens and were similar in morphology and proliferative capacity to Schwann cells isolated from mammalian peripheral nerves. Tumour-derived cultures contained variable proportions (27-79%) of S100-positive cells that were identified as Schwann cells based on this feature. These tumour-derived Schwann cells exhibited a different morphology than normal Schwann cells, usually exhibited an increased reactivity to anti-S100 antibodies and were able to proliferate in vitro without added mitogens. Repeated subculturing of tumour-derived cultures led to the production of six cell lines all of which were composed exclusively of Schwann cells as indicated by S100 expression. These findings show that Schwann cells are an important component of tumours in Damselfish neurofibromatosis and that these cells are morphologically and physiologically altered in this disease. Observations of cell lines also suggest that tumour-derived Schwann cells are intrinsically abnormal and that this phenotype is not a result of stimuli from other cell types in the tumours.