Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: modifications of essential pyrrolidinone ring substituents

We recently reported the first benzoic acid, 1-[4-carboxy-2-(3-pentylamino)phenyl]-5,5-bis(hydroxymethyl)pyrrolidin-2-one (8), that is a potent inhibitor of avian influenza A neuraminidase (N9) and, unlike other reported potent neuraminidase inhibitors, does not contain a basic aliphatic amine or guanidine nor a simple N-acetyl grouping. However, 8 was a poor inhibitor of influenza B neuraminidase. In the present study we further evaluated 8 as an inhibitor of human influenza A NA isolates, and it was effective against N2NA but found to be 160-fold less active against N1NA. We also synthesized analogues of 8 involving moderate modifications of essential substituents on the pyrrolidinone ring. Specifically, the aminomethyl (9), hydroxyethyl (10), and aminoethyl (11) analogues were prepared. Only the most conservative change (compound 9) resulted in continued effective inhibition of influenza A, in addition to a noteworthy increase in the activity of 9 for N1NA. The effectiveness of 9 against influenza B neuraminidase was furthermore improved 10-fold relative to 8, but this activity remained 50-fold poorer than for type A NA.     

Behavioral characterization of striatal‐enriched protein tyrosine phosphatase (STEP) knockout mice

Striatal‐enriched protein tyrosine phosphatase (STEP) has been described as a regulator of multiple kinases and glutamate receptor subunits critical for synaptic plasticity. Published behavioral and biochemical characterization from the founder line of STEP knockout (KO) mice revealed superior cognitive performance, with enhanced phosphorylation of substrates such as ERK, Fyn and GluN2B; suggesting that inhibitors of STEP may have potential as therapeutic agents for the treatment of neuropsychiatric disorders. The objectives of this work aimed to replicate and extend the previously reported behavioral consequences of STEP knockout. Consistent with previous reported data, STEP KO mice demonstrated exploratory activity levels and similar motor coordination relative to WT littermate controls as well as intact memory in a Y‐maze spatial novelty test. Interestingly, KO mice demonstrated deficits in pre‐pulse inhibition as well as reduced seizure threshold relative to WT controls. Immunohistochemical staining of brains revealed the expected gene‐dependent reduction in STEP protein confirming knockout in the mice. The present data confirm expression and localization of STEP and the absence in KO mice, and describe functional downstream implications of reducing STEP levels in vivo.     

Ectomycorrhizal Specificity Patterns in a Mixed Pinus contorta and Picea engelmannii Forest in Yellowstone National Park

We used molecular genetic methods to test two hypotheses, (i) that host plant specificity among ectomycorrhizal fungi would be common in a closed-canopy, mixed Pinus contorta-Picea engelmannii forest in Yellowstone National Park and (ii) that specificity would be more common in the early successional tree species, P. contorta, than in the invader, P. engelmannii. We identified 28 ectomycorrhizal fungal species collected from 27 soil cores. The proportion of P. engelmannii to P. contorta ectomycorrhizae was nearly equal (52 and 48%, respectively). Of the 28 fungal species, 18 composed greater than 95% of the fungal community. No species was associated exclusively with P. contorta, but four species, each found in only one core, and one species found in two cores were associated exclusively with P. engelmannii. These fungi composed less than 5% of the total ectomycorrhizae. Thus, neither hypothesis was supported, and hypothesized benefits of ectomycorrhizal specificity to both trees and fungi probably do not exist in this system.     

Possible spread of bronchogenic carcinoma to the chest wall after a transthoracic fine needle aspiration biopsy. A case report

Spread of tumor along the needle tract following aspiration biopsy with the so-called "fine" needles (thinner than 18 gauge) is recognized to be an extremely uncommon complication of this technique. A case is presented in which such a spread appears to have occurred to the chest wall 13 months after a transthoracic aspiration biopsy of a bronchogenic tumor.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

High resolution cytogenetic evaluation of couples with recurring fetal wastage

Summary Both high resolution and routine chromosome analyses were used to study couples with a history of two or more spontaneous abortions in early pregnancy. In the 20 couples studied, two of the women were found to have an inversion. One paracentric inversion, (13)(q13.1q22.3), was apparent on routine analysis. The small pericentric inversion (11)(p11.12q12.3), was only detected by high resolution techniques. Given the low yield and increased cost and effort involved, we do not believe that high resolution studies are justified for screening couples with repeated abortions.     

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.     

Mechanistic insight into activation of MAPK signaling by pro-angiogenic factors

Background

Angiogenesis is important in physiological and pathological conditions, as blood vessels provide nutrients and oxygen needed for tissue growth and survival. Therefore, targeting angiogenesis is a prominent strategy in both tissue engineering and cancer treatment. However, not all of the approaches to promote or inhibit angiogenesis lead to successful outcomes. Angiogenesis-based therapies primarily target pro-angiogenic factors such as vascular endothelial growth factor-A (VEGF) or fibroblast growth factor (FGF) in isolation. However, pre-clinical and clinical evidence shows these therapies often have limited effects. To improve therapeutic strategies, including targeting FGF and VEGF in combination, we need a quantitative understanding of the how the promoters combine to stimulate angiogenesis.

Results

In this study, we trained and validated a detailed mathematical model to quantitatively characterize the crosstalk of FGF and VEGF intracellular signaling. This signaling is initiated by FGF binding to the FGF receptor 1 (FGFR1) and heparan sulfate glycosaminoglycans (HSGAGs) or VEGF binding to VEGF receptor 2 (VEGFR2) to promote downstream signaling. The model focuses on FGF- and VEGF-induced mitogen-activated protein kinase (MAPK) signaling and phosphorylation of extracellular regulated kinase (ERK), which promotes cell proliferation. We apply the model to predict the dynamics of phosphorylated ERK (pERK) in response to the stimulation by FGF and VEGF individually and in combination. The model predicts that FGF and VEGF have differential effects on pERK. Additionally, since VEGFR2 upregulation has been observed in pathological conditions, we apply the model to investigate the effects of VEGFR2 density and trafficking parameters. The model predictions show that these parameters significantly influence the response to VEGF stimulation.

Conclusions

The model agrees with experimental data and is a framework to synthesize and quantitatively explain experimental studies. Ultimately, the model provides mechanistic insight into FGF and VEGF interactions needed to identify potential targets for pro- or anti-angiogenic therapies.