The cholinesterase inhibitor, phenserine, improves Morris water maze performance of scopolamine-treated rats

Male Fischer-344 rats (n = 38) at 5 months old were tested in a Morris water maze to determine if treatment with the cholinesterase inhibitor, phenserine (PHEN), would overcome a learning impairment induced by scopolamine (SCOP), a muscarinic cholinergic receptor antagonist. Each rat was randomly assigned to one of five groups to receive two intraperitoneal injections 60 and 30 min, prior to testing, respectively, as follows: (1) saline-saline (SAL); (2) saline-1.0 mg/kg (SCOP); (3) 2 mg/kg PHEN- SCOP (PHEN2); (4) 4 mg/kg PHEN-SCOP (PHEN4); and (5) 1 mg/kg PHEN-SAL (PHEN1). Maze testing occurred across 5 days with 4 days of acquisition trials (4 trials per day) and a fifth day consisting of a single 120 sec probe trial. PHEN1 and SAL were combined into one control (CON) group for purposes of statistical analysis for both acquisition and probe trials as comparison of the two groups revealed that they did not significantly differ on any measure. SCOP-treated rats were significantly impaired compared to CON in learning the location of the submerged platform as measured by latency to locate the platform and the distance traversed to find the platform across days of testing. The PHEN4 group had significantly lower latencies and traveled a shorter distance to reach the submerged platform when compared to SCOP on the fourth day of trials while the PHEN2 group traveled more directly to the submerged platform but did not have shorter latencies than the SCOP group. For probe trials, CON rats swam closer to the target area (a measure of proximity to the removed platform) than did all other groups, and the PHEN4 group swam in an area more proximate to the target area than did the SCOP-treated group. These findings demonstrate the ability of this drug to improve learning when cholinergic function has been impaired in a spatial memory task.     

Suppression of uracil-DNA glycosylase induces neuronal apoptosis

A chronic imbalance in DNA precursors, caused by one-carbon metabolism impairment, can result in a deficiency of DNA repair and increased DNA damage. Although indirect evidence suggests that DNA damage plays a role in neuronal apoptosis and in the pathogenesis of neurodegenerative disorders, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in neuronal survival. To test the hypothesis that repair of DNA damage associated with uracil misincorporation is critical for neuronal survival, we employed an antisense (AS) oligonucleotide directed against uracil-DNA glycosylase encoded by the UNG gene to deplete UNG in cultured rat hippocampal neurons. AS, but not a scrambled control oligonucleotide, induced apoptosis, which was associated with DNA damage analyzed by comet assay and up-regulation of p53. UNG mRNA and protein levels were decreased within 30 min and were undetectable within 6-9 h of exposure to the UNG AS oligonucleotide. Whereas UNG expression is significantly higher in proliferating as compared with nonproliferating cells, such as neurons, the levels of UNG mRNA were increased in brains of cystathionine beta-synthase knockout mice, a model for hyperhomocysteinemia, suggesting that one-carbon metabolism impairment and uracil misincorporation can induce the up-regulation of UNG expression.     

2,3-Diarylthiophenes as selective EP1 receptor antagonists

The synthesis and the EP(1) receptor binding affinity of 2,3-diarylthiophene derivatives are described. The evaluation of the structure-activity relationship (SAR) in this series led to the identification of compounds 4, 7, and 12a, which exhibit high affinity for the human EP(1) receptor and a selectivity greater than 100-fold against the EP(2), EP(3), EP(4), DP, FP, and IP receptors and greater than 25-fold versus the TP receptor. These three antagonists present good pharmacokinetics in rats and significant differences in the way they are distributed in the brain.     

Protein-inhibitor complexes analyzed by alkaline capillary LC-MS

Liquid chromatography-mass spectrometry (LC-MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein-ligand complexes. We have successfully developed LC-MS method for protein molecular weight measurement using small-bore and capillary LC-MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein-ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.     

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella

This study examines how the dynamics of fungus–insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well‐studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme‐based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4–7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.     

Invasion of novel habitats uncouples haplo‐diplontic life cycles

Baker's Law predicts uniparental reproduction will facilitate colonization success in novel habitats. While evidence supports this prediction among colonizing plants and animals, few studies have investigated shifts in reproductive mode in haplo‐diplontic species in which both prolonged haploid and diploid stages separate meiosis and fertilization in time and space. Due to this separation, asexual reproduction can yield the dominance of one of the ploidy stages in colonizing populations. We tested for shifts in ploidy and reproductive mode across native and introduced populations of the red seaweed Gracilaria vermiculophylla. Native populations in the northwest Pacific Ocean were nearly always attached by holdfasts to hard substrata and, as is characteristic of the genus, haploid–diploid ratios were slightly diploid‐biased. In contrast, along North American and European coastlines, introduced populations nearly always floated atop soft‐sediment mudflats and were overwhelmingly dominated by diploid thalli without holdfasts. Introduced populations exhibited population genetic signals consistent with extensive vegetative fragmentation, while native populations did not. Thus, the ecological shift from attached to unattached thalli, ostensibly necessitated by the invasion of soft‐sediment habitats, correlated with shifts from sexual to asexual reproduction and slight to strong diploid bias. We extend Baker's Law by predicting other colonizing haplo‐diplontic species will show similar increases in asexuality that correlate with the dominance of one ploidy stage. Labile mating systems likely facilitate colonization success and subsequent range expansion, but for haplo‐diplontic species, the long‐term eco‐evolutionary impacts will depend on which ploidy stage is lost and the degree to which asexual reproduction is canalized.     

Design,synthesis and biological assessment of N-adamantyl,substituted adamantyl and noradamantyl phthalimidines for nitrite,TNF-α and angiogenesis inhibitory activities

A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.     

Identification of a low molecular weight form of epithelial transforming growth factor

We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.     

Characterization and partial purification of human epithelial transforming growth factor

A polypeptide growth factor has been partially purified from medium conditioned by the human adrenocortical carcinoma cell line SW13. This factor, designated h-TGFe, stimulates anchorage-independent growth of the SW13 cells. Similar activity was observed in human milk, and in conditioned media from seven of 14 epithelial cell lines. The SW13-derived activity is stable to low pH and 8M urea but labile to dithiothreitol and 2% sodium dodecyl sulfate. Human TGFe does not bind to heparin and fails to stimulate growth of endothelial cells in monolayer culture. The apparent molecular weight of h-TGFe is 59k by size exclusion chromatography in the presence of 8M urea and the activity binds strongly to cation exchangers. The activity elutes at 15-30% acetonitrile from a C18 reverse-phase column and has been partially purified by using a four-step chromatographic procedure. TGFe appears to be a novel growth factor produced by many epithelial cells and tissues.     

Value of antenatal diagnosis of abnormalities of the urinary tract.

OBJECTIVE--To assess the value of antenatal diagnosis of abnormalities of the urinary tract on ultrasonography. DESIGN--Retrospective study. SETTING--Two obstetric units in Glasgow. SUBJECTS--62 Fetuses in which renal abnormalities were diagnosed on antenatal ultrasonography. INTERVENTIONS--Six fetuses had their bladders aspirated to determine renal function. Fifteen pregnancies were terminated on the basis of the findings on antenatal ultrasonography, and if possible necropsy was performed on the fetuses. In babies who were born alive the final diagnosis was made by postnatal ultrasonography, intravenous urography, radionuclide scanning, cystography, and, in those who died in the early neonatal period, necropsy. Neonates who were referred with a known obstructed kidney had nephrostomy or pyeloplasty. END POINT--Assessment of the value of antenatal diagnosis of renal abnormalities on ultrasonography for babies who had no clinical evidence of disease postnatally. MAIN RESULTS--Eighteen fetuses did not survive birth; the antenatal diagnosis was accurate in all 18. Of the 44 babies born alive, five had normal urinary tracts, in two of whom antenatal ultrasonography had probably indicated a false positive diagnosis. Fourteen babies died during the early neonatal period. Twenty five babies with renal abnormalities were followed up; the antenatal diagnosis was inaccurate for 10 of them, the commonest misdiagnosis being hydronephrosis for multicystic kidney and vice versa, and there was one false positive diagnosis. The initial clinical findings in 14 babies would have led to the early detection of a urological abnormality. In the 30 babies with no clinical evidence of disease the antenatal diagnosis was of definite value in eight, probable value in 15, and marginal value in seven. Overall, an accurate antenatal diagnosis was made in 46 of the 62 cases (74%); in 12 cases renal disease was detected but its specific nature was not determined; and in four cases the diagnosis was misleading. CONCLUSIONS--The overall value of antenatal diagnosis is that it indicates early termination of fetuses with fatal renal disease, prepares parents and medical staff for the likelihood of serious neonatal problems, and shows abnormalities of the urinary tract that may not be detected postnatally.