Old-growth Acer macrophyllum trees host a unique suite of arbuscular mycorrhizal fungi and other root-associated fungal taxa in their canopy soil environment.

Canopy soils occur on tree branches throughout the temperate rainforests of the Pacific Northwest Coast and are recognized as a defining characteristic of these ecosystems. Certain tree species extend adventitious roots into these canopy soil environments. Yet, research on adventitious root-associated fungi remains limited. Our study used microscopy to compare fungal colonization intensity between canopy and forest floor roots of old-growth bigleaf maple (Acer macrophyllum) trees. Subsequently, two high-throughput sequencing platforms were used to explore the spatial and seasonal variation of root-associated fungi between the two soil environments over one year. We found that canopy and forest floor roots had similar colonization intensity and were associating with a diversity of arbuscular mycorrhizal fungi and other potential symbionts, many of which were resolved to species level. Soil environment and seasonality affected root-associated fungal community composition, and several fungal species were indicative of the canopy soil environment. In Washington State's (USA) temperate old-growth rainforests, these canopy soil environments host a unique suite of root-associated fungi. The presence of arbuscular mycorrhizae provides further evidence that adventitious roots form fungal associations to exploit canopy soils for resources, and there may be novel relationships forming with other fungi. These soils may be providing a redundancy compartment (i.e., "nutrient reserve"), imparting a resiliency to disturbances for certain old-growth trees.

ACU193, a Monoclonal Antibody that Selectively Binds Soluble Aß Oligomers: Development Rationale, Phase 1 Trial Design, and Clinical Development Plan.

BACKGROUND: Alzheimer's disease is a large and growing unmet medical need. Clinical trial designs need to assess disease-related outcomes earlier to accelerate the development of better treatments for Alzheimer's disease. ACU193 is a monoclonal antibody that selectively targets amyloid ß oligomers, thought to be the most toxic species of Aß that accumulates early in AD and contributes to downstream pathological effects. Nonclinical data indicate that ACU193 can reduce the toxic effects of amyloid ß oligomers. ACU193 is currently being investigated in a phase 1 clinical trial designed with the properties described in this report. This phase 1 trial is designed to provide data to enable a go/no-go decision regarding the initiation of a subsequent phase 2/3 study. OBJECTIVES: To design a phase 1 study that assesses target engagement and incorporates novel measures to support more rapid development of a potential disease-modifying treatment for Alzheimer's disease. DESIGN: The INTERCEPT-AD trial for ACU193 is an ongoing randomized, placebo-controlled phase 1a/b study that assesses safety, tolerability, pharmacokinetics, target engagement, clinical measures, and several Alzheimer's disease biomarkers, including novel digital and imaging biomarkers. SETTING: For INTERCEPT-AD, brief inpatient stays for patients in the single ascending dose portion of the study, with the remainder of the evaluations being performed as outpatients at multiple clinical trial sites in the U.S. PARTICIPANTS: Patients with early Alzheimer's disease (mild cognitive impairment or mild dementia with a positive florbetapir positron emission tomography scan). INTERVENTION: ACU193 administered intravenously at doses of 2- 60 mg/kg. MEASUREMENTS: Safety assessments including magnetic resonance imaging for the presence of amyloid-related imaging abnormalities, clinical assessments for Alzheimer's disease including the Alzheimer's Disease Rating Scale-cognition and Clinical Dementia Rating scale, pharmacokinetics, a measure of target engagement, and digital and imaging biomarkers, including a computerized cognitive test battery and a measure of cerebral blood flow using arterial spin labelling magnetic resonance imaging. RESULTS: A phase 1 study design was developed for ACU193 that allows collection of data that will enable a go/no-go decision for initiation of a subsequent adaptive phase 2/3 study. CONCLUSIONS: A phase 1a/b trial and an overall clinical development plan for an Alzheimer's disease treatment can be designed that maintains patient safety, allows informed decision-making, and achieves an accelerated timeline by using novel biomarkers and adaptive study designs.

NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype.

Cervical cancer is associated with human papilloma virus infection. However, this infection is insufficient to induce transformation and progression. Loss of heterozygosity analyses suggest the presence of a tumor suppressor gene (TSG) on chromosome 6p21.3-p25. Here we report the cloning NOL7, its mapping to chromosome band 6p23, and localization of the protein to the nucleolus. Fluorescence in situ hybridization analysis demonstrated an allelic loss of an NOL7 in cultured tumor cells and human tumor samples. Transfection of NOL7 into cervical carcinoma cells inhibited their growth in mouse xenografts, confirming its in vivo tumor suppressor activity. The induction of tumor dormancy correlated with an angiogenic switch caused by a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These data suggest that NOL7 may function as a TSG in part by modulating the expression of the angiogenic phenotype.

Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity.

Exposure to topoisomerase II inhibitors is linked to the generation of leukemia involving translocations of the MLL gene, normally restricted to an 8.3 kbp tract, the breakpoint cluster region (BCR). Using an in vitro assay, apoptotic activators, including radiation and anti-CD95 antibody, trigger site-specific cleavage adjacent to exon 12 within the MLL BCR and promote translocation of the MLL gene in cells that can survive. To explore the mechanism of cleavage and rearrangement in more detail, the entire MLL BCR was placed into the pREP4 episomal vector and transfected into human lymphoblastoid TK6 cells. Episomes containing either the MLL BCR, or deletion constructs of 367 bp or larger, were cleaved at the same position as genomic MLL after exposure to apoptotic stimuli. Further analysis of sequence motifs surrounding the cleaved region of MLL showed the presence of both a predicted nuclear matrix attachment sequence and a potential strong binding site for topoisomerase II, flanking the site of cleavage. Inactivation of topoisomerase II by the catalytic inhibitor merbarone did not inhibit MLL cleavage, suggesting that the initial cleavage step for MLL rearrangement is not mediated by topoisomerase II.

Does proximity to mature trees influence ectomycorrhizal fungus communities of Douglas-fir seedlings?

The influence of mature trees on colonization of Douglas-fir (Pseudotsuga menziesii) seedlings by ectomycorrhizal fungi (EMF) is not well understood. Here, the EMF communities of seedlings planted near and far from trees are compared with each other, with EMF of seedlings potted in field soils and with EMF of mature trees. Seedlings were planted within 6 m, or beyond 16 m, from residual Douglas-fir trees in recently harvested green-tree retention units in Washington State, USA, or potted in soils gathered from near each residual tree. Mature tree roots were sampled by partly excavating the root system. The EMF communities were assessed by polymerase chain reaction-restriction fragment length polymorphism and sequence analysis of ribosomal RNA genes. Seedlings near trees had higher species richness and diversity of EMF communities compared with seedlings far from trees. The EMF communities of seedlings near trees were more similar to those of mature trees, while seedlings far from trees were more similar to glasshouse seedlings. By enhancing the EMF diversity of seedlings, residual trees may maintain or accelerate the re-establishment of mycorrhizal communities associated with mature forests.

Effects of intra-striatal GDNF on motor coordination and striatal electrophysiology in aged F344 rats.

The purpose of this study was to examine whether improvement in motor function could be demonstrated in old rats, and to see if GDNF affected post-synaptic DA function. Aged (20 month old) versus young rats were tested following GDNF treatment for postural control by using an inclined balance beam and a wire grip strength test. Rats were also examined electrophysiologically for spontaneous striatal cell firing rate alone and in the presence of DA receptor agonists, and histologically for the intensity of striatal TH staining, and number of DA containing nigral cells. Behavior was significantly improved in the aged animals who received central GDNF infusions, although the extent of improvement was less than what has been observed in 16-month-old rats. There was no effect of GDNF treatment in the aged animals on spontaneous firing rate in the striatum, or on the post synaptic response to locally applied D(1) and D(2) receptor family agonists. However, there was an effect of age alone on firing rate, and on the response to locally applied SKF 38393 and quinpirole. By using unbiased cell counting we observed no age-related decline in the number of TH positive cells in the substantia nigra. There was no effect of GDNF on the number of TH positive cells in the substantia nigra in either young or aged rats, although there were morphological improvements in DA neurons of the GDNF treated aged rats. These results replicate earlier studies showing an effect of age on striatal firing rate and dopamine receptor function, and suggest that the GDNF mediated improvement in behavior may be located other than post synaptically within the striatum.

Effects of ethanol on striatal dopamine overflow and clearance: an in vivo electrochemical study.

Previous studies have shown that the neurotransmitter dopamine (DA) is implicated in the reinforcing effects of ethanol and other abused drugs. Ethanol also alters DA overflow and uptake in vivo. Further studies of the role of DA in the behavioral and neurochemical effects of ethanol may help explain the pharmacological mechanisms by which these effects are produced. In these studies we used in vivo electrochemical recordings to investigate the effects of ethanol (EtOH) on the dynamics of evoked DA overflow and DA uptake in rat dorsal striatum. Local applications of EtOH from a multibarrel micropipette did not produce detectable changes in extracellular levels of endogenous DA in the dorsal striatum. EtOH application did attenuate potassium (K+)-evoked overflow of DA in a time-dependent fashion. In contrast, tyramine-induced DA overflow, a calcium-independent process thought to be carrier mediated, was not altered by local EtOH application in the dorsal striatum. Striatal uptake of locally applied exogenous DA was decreased by nomifensine, an effect that was attenuated by locally applied EtOH. Taken together, these data suggest that one of the effects of EtOH on DA-containing nerve endings in the rat striatum involves functional changes in the high-affinity DA transporter associated with these nerve endings.

Caloric restriction enhances evoked DA overflow in striatum and nucleus accumbens of aged Fischer 344 rats.

Previous studies have shown deficits in DA neuronal systems in senescence. Other studies indicate that prolonged dietary restriction can attenuate many of the detrimental effects of age. We have shown previously using in vivo electrochemistry that K+-evoked striatal DA overflow decreases as a function of age. This was a regional effect that appeared to be due to functional changes in DA neurons, rather than a decrease in the storage and synthesis of DA. In the present studies, we used in vivo electrochemistry to investigate the effects of caloric restriction on age related decreases in K+-evoked DA overflow along a dorsal to ventral axis in the striatum of aged female Fischer 344 rats. Aged (26-28-month-old) diet restricted animals (DRF) showed evoked DA overflow that was significantly greater in amplitude and duration compared to aged (26-28-month-old) ad lib fed animals (ALF). These results provide additional evidence that decreased DA neuronal function resulting from age is improved by caloric restriction.

Effects of ethanol and nomifensine on NE clearance in the cerebellum of young and aged Fischer 344 rats.

Rapid chronoamperometric recordings coupled with local application of drugs by pressure ejection were used to investigate the effects of nomifensine and ethanol (EtOH) on exogenous norepinephrine (NE) clearance in the cerebellum of young (5-month-old) and aged (24-26-month-old) male Fischer 344 rats. In the young rats, local nomifensine application prolonged exogenous NE clearance, indicating transporter mediated uptake inhibition. NE clearance was modestly but significantly prolonged in the aged rats as compared to the young rats, suggesting less efficient uptake. Consistent with this, there was little effect of nomifensine on NE clearance in the aged rats. In contrast to the effect of nomifensine, EtOH inhibited NE clearance in both young and aged rats. These data further support the hypothesis that one effect of EtOH in cerebellar NE systems is inhibition of NE uptake into NE-containing nerve terminals, and they also demonstrate that the effect of nomifensine on exogenous NE clearance in vivo in the cerebellum is altered by the aging process, while the effect of EtOH is not.

Toxic effects of the novel protein UpI from the sea anemone Urticina piscivora.

UpI is a basic protein, with molecular mass (approximately 28 kDa) and a pI > 9.4, isolated from the sea anemone Urticina piscivora. It is a potent cardiac stimulatory protein with the partial amino acid sequence D1ENEN5LYGPN10ENKAK15AKDLT20AGASY25LTKEA30GCTKL35QAGCT40MYQAY45N [1]. The toxic effects of UpI and the crude extract from which it was isolated have been investigated on three tumour cell lines: KB, L1210, and HEL 299 cells. UpI, however, was less potent on each cell line than the crude extract. Since previous experiments had shown extracts of U. piscivora to be haemolytic on erythrocytes of rat, guinea pig and dog, the haemolytic action of UpI was investigated. It was found to be a potent haemolysin on erythrocytes of rat, guinea pig, dog, pig and human, causing haemolysis on erythrocytes of each species tested at concentrations as low as 10(-10) M. Haemolysis was inhibited in a concentration-dependent manner by the phospholipid sphingomyelin but not cholesterol. Using scanning electron microscopy, it is now being shown that UpI produces significant structural damage to membranes of erythrocytes from rat and guinea pig. It proved to be a potent ichthyotoxin. These data suggest that sea anemone toxin not only possess different pharmacological activities but that UpI, one of the active constituents, could be responsible for the different pharmacological effects exhibited by the crude extract.

Medial dorsal striatum is more sensitive than lateral dorsal striatum to cocaine inhibition of exogenous dopamine clearance: relation to [3H]mazindol binding, but not striosome/matrix.

Previous studies have shown that acute systemic cocaine inhibits dopamine (DA) transport and thereby produces dose-dependent changes in exogenous DA clearance in the brain. This measure reflects the dynamic activity of the DA transporter. There are also differential effects of cocaine on DA clearance in dorsal and ventral striatum, and evidence from many studies suggests that even within the dorsal striatum, the interaction of cocaine with the DA transporter may not be homogeneous. A greater understanding of how cocaine interacts with the striatal DA transporter will help clarify the role of the striatum in mediating effects of cocaine. In these studies we used in vivo electrochemical recording to examine the effect of ip cocaine on exogenous DA clearance in the medial and lateral dorsal striatum with respect to both DA transporter binding sites and electrode localization to striosomes or matrix. Baseline exogenous DA clearance was different in medial and lateral dorsal striatum in the absence of cocaine. Systemic cocaine produced a more pronounced inhibition of DA clearance in medial dorsal striatum than in lateral dorsal striatum. [3H]Mazindol binding to DA transporters was lower in medial dorsal striatum, but was not in register with striosome or matrix compartments. There was no notable difference between cocaine's effects on DA clearance in striosome or matrix that was due to this compartmentalization. Others have demonstrated that medial dorsal striatum receives proportionally more innervation from the mesolimbic, as opposed to the nigrostriatal, DA system. Taken together, these and previous in vivo results suggest that this differential sensitivity to cocaine reflects the unique properties of the ascending mesolimbic DA projection and the lower density of DA transporters associated with it.

Occupancy of the serotonin transporter by fluoxetine, paroxetine, and sertraline: in vivo studies with [125I]RTI-55.

[125I]RTI-55 was used tracer doses to label serotonin (5-HT) transporters in vivo in the mouse brain. Fluoxetine, paroxetine, and sertraline, potent antidepressants and selective inhibitors of serotonin transporter sites, were administered in various doses and at various times. The doses and times that result in significant binding of the drugs to transporters correspond to doses and times where they are reported to have physiological effects. Estimates of occupancy rate and duration of binding to serotonin transporters were made. The rate of occupancy of the 5-HT transporter site was fastest for sertraline, intermediate for paroxetine and slowest for fluoxetine. Similarly, the duration of occupancy was significantly shorter for sertraline and paroxetine (approximately 10 h) than for fluoxetine (approximately 50 h). The results indicate that in competition studies, [125I]RTI-55 can be used to identify doses of drugs that are physiologically effective, to determine their relative rate of occupancy, and most importantly, to measure the residency time on the central serotonin transporter in vivo.

Repeated intravenous cocaine administration to rats produces behavioral sensitization without changing brain cocaine levels.

Repeated intraperitoneal (i.p.) administration of cocaine to rats results in behavioral sensitization. However, augmented brain cocaine levels are also produced by this treatment. In the present study, a 43% increase in cocaine levels was observed in striatum in response to eight once-daily i.p. injections of cocaine (10 mg/kg). It has been suggested that this dispositional change does not occur with intravenous (i.v.) cocaine administration. In agreement with this suggestion, the striatal cocaine levels observed following either a single i.v. injection or eight once-daily i.v. injections of cocaine (1 mg/kg) were similar. Nonetheless, the rats became behaviorally sensitized in response to the repeated i.v. cocaine administration. These results suggest that the increased brain levels of cocaine observed following repeated i.p. cocaine administration cannot completely account for behavioral sensitization.

In vivo binding of [125I]RTI-55 to dopamine transporters: pharmacology and regional distribution with autoradiography.

Previous studies have demonstrated that para-substituted WIN 35,065-2 analogs of cocaine show high binding affinity for dopamine uptake sites both in vitro and in vivo, and inhibit DA uptake in vitro. These analogs also produce potent cocaine-like behavioral effects in various procedures. The purpose of the present studies was to evaluate the iodinated WIN 35,065-2 analog [125I]RTI-55 as an in vivo ligand for the DA transporter. Following intravenous injection in mice, [125I]RTI-55 showed highest accumulation in areas with high densities of dopamine uptake sites. Light microscopic autoradiography was used to examine binding with higher resolution. Displacement studies demonstrated that [125I]RTI-55 binding in dopamine containing regions, striatum and olfactory tubercles, was saturable and inhibited by other cocaine analogs. GBR 12909 and WIN 35,428 significantly inhibited [125I]RTI-55 binding in striatum, while paroxetine significantly inhibited hypothalamic binding but had little effect in striatum. The latter finding suggests that [125I]RTI-55 also binds to the serotonin transporter. Haloperidol had no effect on [125I]RTI-55 binding in any brain region measured. In addition, treatment of animals with the dopamine neurotoxin MPTP caused significant reductions in striatal [125I]RTI-55 binding. The results of these studies indicate that [125I]RTI-55 binds primarily to the dopamine transporter in the mouse striatum in vivo.

[123/125I]RTI-55, an in vivo label for the serotonin transporter.

[123I]RTI-55, an iodinated derivative of the cocaine analog 3 beta-phenyltropane-2 beta-carboxylic acid methyl ester, was evaluated as an agent for in vivo labeling of the serotonin transporter. Labeling of the precursor of RTI-55 with I-123 was efficient and yielded a high specific activity product. After intravenous injection of [123I]RTI-55 into rats, the tracer accumulated in regions with high densities of serotonin and dopamine uptake sites. The distribution of [123I]RTI-55 binding in areas rich in serotonin uptake sites correlated with [3H]serotonin uptake measured in vitro in the same regions. Specific [123I]RTI-55 binding to serotonin uptake sites was inhibited by paroxetine but not by GBR 12,909. Treatment of rats with neurotoxic doses of fenfluramine caused decreases of 66% (in the hypothalamus) to 83% (in the superior colliculi) of specific [125I]RTI-55 binding in all areas except in the striatum and the olfactory tubercles (regions rich in dopamine transporters). These results indicate that [123/125I]RTI-55 binds, although not selectively, to the serotonin transporter in vivo. Furthermore, they suggest that [123I]RTI-55 holds promise as a SPECT imaging agent for the study of the serotonin transporter in humans in health and disease.

Stimulus generalization from cocaine to analogs with high in vitro affinity for dopamine uptake sites.

Previous research has shown that phenyltropane derivatives of cocaine are very potent ligands for dopamine transporters in in vitro binding and uptake, and in in vivo binding assays. In the present study, these analogs were tested for their ability to substitute for cocaine in rats trained to discriminate cocaine from saline. Results indicate that these compounds are from 6-13 times more potent than cocaine in producing cocaine-appropriate responding. This provides further evidence in support of the importance of dopamine uptake inhibition for the behavioral effects of cocaine, and suggests utility of these compounds in understanding cocaine abuse.

Behavioral effects of novel cocaine analogs: a comparison with in vivo receptor binding potency.

Several novel cocaine analogs, previously shown to be very potent in in vitro binding studies, have been examined for their stimulatory effects on locomotor activity and for their ability to displace [3H]WIN 35,428 binding in vivo in mice. These compounds, like WIN 35,428, lack an ester link between the phenyl group and the tropane ring and have para-substitutions on the phenyl ring. They were much more potent than (-)-cocaine in producing increases in locomotor activity. In addition, they were more potent than (-)-cocaine in inhibiting [3H]WIN 35,428 binding in vivo in mouse striatum. Thus, these compounds demonstrate similar high potency in behavioral tests and in receptor binding assays, both in vivo and in vitro. Results support the hypothesis of a relationship between binding at the dopamine transporter and the behavioral effects of cocaine-like drugs. Further, assuming that maximal occupancy occurs with total displacement of [3H]WIN 35,428 binding in vivo, the data suggest that maximal locomotor effects occur with near total occupancy of transporter binding sites.

Terephthalamidine: past and future.

Terephthalamidine (NSC 57155) is one of 800 terephthalanilides and related compounds which were synthesized and tested preclinically in the late 1950's and early 1960's. Based upon their activity against murine leukemias, some of these agents were tested briefly in clinical trials at that time. Despite the observation of responses, the compounds were dropped because of severe and unusual neurotoxicity. More recently, terephthalamidine has been screened for antitumor activity and chosen for further clinical investigation by the NCI's Project for the Review of Old Drugs (P.R.O.D.) because of its novel structure and spectrum of preclinical activity. The current availability of a plasma assay for the drug permits further study of its clinical pharmacokinetics and pharmacodynamics and, perhaps, the development of improved scheduling strategies.

In vitro antibacterial activities of three plants used in traditional medicine in Sierra Leone.

The antibacterial activities of the methanol and hot and cold aqueous extracts of the leaves of Aspilia africana, Ficus exasperata and Mareya micrantha were bioassayed against three Gram-negative and three Gram-positive bacterial species: Aerobacter aerogenes, Agrobacterium tumefaciens, Bacillus subtilis, Clostridium sporogenes, Escherichia coli and Staphylococcus aureus. The methanol and aqueous extracts of the leaves of Aspilia africana and Mareya micrantha and the undiluted oil of M. micrantha exhibited differential antibacterial activities on both Gram-positive and Gram-negative bacterial species at concentrations ranging from 0.1 to 0.5 g/ml. Extracts of Ficus exasperata leaves were inactive at all concentrations tested.