Contributions of recombination and repair proteins to telomere maintenance in telomerase‐positive and negative Ustilago maydis

Homologous recombination and repair factors are known to promote both telomere replication and recombination‐based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase‐positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase‐negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi‐solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II‐like as well as ALT‐like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I‐like telomere features. In addition, we observed direct physical interactions between Blm and two telomere‐binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.     

Identification of DK419, a potent inhibitor of Wnt/β-catenin signaling and colorectal cancer growth

The Wnt signaling pathway is critical for normal tissue development and is an underlying mechanism of disease when dysregulated. Previously, we reported that the drug Niclosamide inhibits Wnt/β-catenin signaling by decreasing the cytosolic levels of Dishevelled and β-catenin, and inhibits the growth of colon cancers both in vitro and in vivo. Since the discovery of Niclosamide’s anthelmintic activity, a growing body of literature indicates that Niclosamide is a multifunctional drug. In an effort to identify derivatives of Niclosamide with improved pharmacokinetic properties that maintain the multifunctional drug activity of Niclosamide for clinical evaluation, we designed DK419, a derivative containing a 1H-benzo[d]imidazole-4-carboxamide substructure, using the structure-activity relationships (SAR) of the Niclosamide salicylanilide chemotype. Similar to Niclosamide, we found DK419 inhibited Wnt/β-catenin signaling, altered cellular oxygen consumption rate and induced production of pAMPK. Moreover, we found DK419 inhibited the growth of CRC tumor cells in vitro, had good plasma exposure when dosed orally, and inhibited the growth of patient derived CRC240 tumor explants in mice dosed orally. DK419, a derivative of Niclosamide with multifunctional activity and improved pharmacokinetic properties, is a promising agent to treat colorectal cancer, Wnt-related diseases and other diseases in which Niclosamide has demonstrated functional activity.     

Lipid metabolism of phenol-tolerant <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> strains for lignin bioconversion

Background

Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated.

Results

We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified?>?100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition.

Conclusions

This work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.

     

Melanoma in patients with GATA2 deficiency

GATA2 deficiency is a recently described genetic disorder affecting hematopoietic stem cells and is associated with immunodeficiency, hematologic malignancy, and various cutaneous pathologies including cutaneous tumors. To explore the incidence and clinical course of melanoma in patients with germline GATA2 deficiencies, we conducted a retrospective chart review of 71 such patients and identified two with invasive melanoma. One melanoma was diagnosed early because it was associated with pruritus due to a graft‐versus‐tumor effect following bone marrow transplantation. The other one, a lentigo maligna melanoma, was locally excised but progressed to widespread metastasis and death several years later. Our observations and published studies of melanoma biology suggest an association between decreased GATA2 expression and melanoma progression. These findings suggest that GATA2 deficient patients may have an increased risk of melanoma and should be observed closely for new or changing skin lesions.     

Galloway-Mowat syndrome in Taiwan: <Emphasis Type="Italic">OSGEP</Emphasis> mutation and unique clinical phenotype

Background

Galloway-Mowat syndrome (GAMOS) is a rare autosomal recessive disease characterized by the combination of glomerulopathy with early-onset nephrotic syndrome and microcephaly with central nervous system anomalies. Given its clinical heterogeneity, GAMOS is believed to be a genetically heterogenous group of disorders. Recently, it has been reported that mutations in KEOPS-encoding genes, including the OSGEP gene, were responsible for GAMOS.

Results

Overall, 6 patients from 5 different Taiwanese families were included in our study; the patients had an identical OSGEP gene mutation (c.740G?>?A transition) and all exhibited a uniform clinical phenotype with early-onset nephrotic syndrome, craniofacial and skeletal dysmorphism, primary microcephaly with pachygyria, and death before 2?years of age. We reviewed their clinical manifestations, the prenatal and postnatal presentations and ultrasound findings, results of imaging studies, associated anomalies, and outcome on follow-up. All individuals were found to have an “aged face” comprising peculiar facial dysmorphisms. Arachnodactyly or camptodactyly were noted in all patients. Neurological findings consisted of microcephaly, hypotonia, developmental delay, and seizures. Brain imaging studies all showed pachygyria and hypomyelination. All patients developed early-onset nephrotic syndrome. The proteinuria was steroid-resistant and eventually resulted in renal function impairment. Prenatal ultrasound findings included microcephaly, intrauterine growth restriction, and oligohydramnios. Fetal MRI in 2 patients confirmed the gyral and myelin abnormalities.

Conclusions

Our study suggests that a careful review of the facial features can provide useful clues for an early and accurate diagnosis. Prenatal ultrasound findings, fetal MRI, genetic counseling, and mutation analysis may be useful for an early prenatal diagnosis.

     

MicroRNA-223 Suppresses the Canonical NF-κB Pathway in Basal Keratinocytes to Dampen Neutrophilic Inflammation

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Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

NADH-Linked Electron Transfer Induces Cd2+ Movement in Corn Root Plasma Membrane Vesicles

The rate of electron transfer from NADH to ferricyanide associatedwith corn root plasma membrane vesicles was not significantlyaffected by the presence of Ca²⁺, Cd²⁺, Zn²⁺, or Co²⁺. However,unlike Ca²⁺, other tested divalent cations induced an electrontransfer-dependent increase in light scattering by the vesicles.The efficacy in inducing the light scattering increase followedthe order of Cd²⁺>Zn²⁺>Co²⁺. The extent of the light scatteringchange increased as the concentration of added Cd²⁺ increased.Using phase contrast microscopy, the increase in light scatteringwas related to an expansion of the vesicles. Accompanying thevolume increase, a decrease of Cd²⁺ concentration in the externalspace was observed by the use of Arsenazo III, a metallochromophorespecific for divalent cations. Under a specified electron transfercondition, a similar percentage of decrease over a wide rangeof initial Cd²⁺ concentration was detected. The presence ofA23187 [GenBank] decreased the rate and extent of light scattering increaseand its associated Cd²⁺ concentration change. However, A23187 [GenBank] had no effect on scattering changes already established by theelectron transfer and Cd²⁺. These results suggest that the NADHlinked electron transfer reaction may induce a Cd²⁺ uptake bythe vesicles of corn root plasma membrane with inside-out orientation. (Received September 25, 1995; Accepted December 19, 1995)     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.