Small Molecule Channels Harness Membrane Potential to Concentrate Potassium in trk1Δtrk2Δ Yeast.

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome, and proximal renal tubular acidosis. Using yeast as a eukaryotic model system, we asked whether, in the context of a protein ion channel deficiency in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. Trk potassium transporters use membrane potential to move potassium from a relatively low concentration outside cells (∼15 mM) to one of >10× higher inside (150-500 mM); trk1Δtrk2Δ are unable to concentrate potassium or grow in standard media. Here we show that potassium-permeable, but not potassium-selective, small-molecule ion channels formed by amphotericin B can harness membrane potential to concentrate potassium and thereby restore trk1Δtrk2Δ growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach.

Comparison of the Pharmacokinetic Properties of Triamcinolone and Dexamethasone for Local Therapy of the Inner Ear.

Some forms of triamcinolone may provide alternate options for local therapy of the inner ear in addition to the steroids currently in use. We compared the perilymph pharmacokinetics of triamcinolone-acetonide, triamcinolone, and dexamethasone, each delivered as crystalline suspensions to guinea pigs. Triamcinolone-acetonide is a widely used form of the drug with molecular properties that allow it to readily permeate biological barriers. When applied intratympanically triamcinolone-acetonide entered perilymph rapidly but was also found to be eliminated rapidly from perilymph. The rapid rate of elimination severely limits the apical distribution of the drug when applied locally, making it unsuitable for use in the ear. In contrast, triamcinolone, rather than triamcinolone-acetonide, is a more polar form of the molecule, with higher aqueous solubility but calculated to pass less-readily through biological boundaries. Perilymph concentrations generated with intratympanic applications of triamcinolone were comparable to those with triamcinolone-acetonide but elimination measurements showed that triamcinolone was retained in perilymph longer than triamcinolone-acetonide or dexamethasone. The slower elimination is projected to result in improved distribution of triamcinolone toward the cochlear apex, potentially allowing higher drug levels to reach the speech frequency regions of the human ear. These measurements show that triamcinolone could constitute an attractive additional treatment option for local therapy of auditory disorders.

Lactosylceramide synthase ß-1,4-GalT-V: A novel target for the diagnosis and therapy of human colorectal cancer.

Little is known about an oncogenic signal transducer ß-1,4-galactosyltransferase-V (ß-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that ß-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as ß-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that ß-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease.

Dexamethasone and Dexamethasone Phosphate Entry into Perilymph Compared for Middle Ear Applications in Guinea Pigs.

Dexamethasone phosphate is widely used for intratympanic therapy in humans. We assessed the pharmacokinetics of dexamethasone entry into perilymph when administered as a dexamethasone phosphate solution or as a micronized dexamethasone suspension, with and without inclusion of poloxamer gel in the medium. After a 1-h application to guinea pigs, 10 independent samples of perilymph were collected from the lateral semicircular canal of each animal, allowing entry at the round window and stapes to be independently assessed. Both forms of dexamethasone entered the perilymph predominantly at the round window (73%), with a lower proportion entering at the stapes (22%). When normalized by applied concentration, dexamethasone phosphate was found to enter perilymph far more slowly than dexamethasone, in accordance with its calculated lipid solubility and polar surface area properties. Dexamethasone phosphate therefore has a problematic combination of kinetic properties when used for local therapy of the ear. It is relatively impermeable and enters perilymph only slowly from the middle ear. It is then metabolized in the ear to dexamethasone, which is more permeable through tissue boundaries and is rapidly lost from perilymph. Understanding the influence of molecular properties on the distribution of drugs in perilymph provides a new level of understanding which may help optimize drug therapies of the ear.

Restored Physiology in Protein-Deficient Yeast by a Small Molecule Channel.

Deficiencies of protein ion channels underlie many currently incurable human diseases. Robust networks of pumps and channels are usually responsible for the directional movement of specific ions in organisms ranging from microbes to humans. We thus questioned whether minimally selective small molecule mimics of missing protein channels might be capable of collaborating with the corresponding protein ion pumps to restore physiology. Here we report vigorous and sustainable restoration of yeast cell growth by replacing missing protein ion transporters with imperfect small molecule mimics. We further provide evidence that this tolerance for imperfect mimicry is attributable to collaboration between the channel-forming small molecule and protein ion pumps. These results illuminate a mechanistic framework for pursuing small molecule replacements for deficient protein ion channels that underlie a range of challenging human diseases.

Black salve and bloodroot extract in dermatologic conditions.

Due to extensive advertising of black salve's effectiveness in "curing" skin cancers and healing other skin conditions, many patients are turning to self-treating with black salve. Although black salve has not been proven to have anticancer properties, application of black salve has been shown to cause damage to healthy tissue and the need for further treatment. We describe a 35-year-old woman whose one-time application of black salve to a healing biopsy site resulted in skin erosion and formation of a dermatitic plaque with subsequent scarring. Dermatologists and other health professionals need to be aware of this increasingly popular product to be able to better inform and treat their patients.

Five-year outcomes of wide excision and Mohs micrographic surgery for primary lentigo maligna in an academic practice cohort.

BACKGROUND: Wide local excision with 5-mm margins is the standard of care for lentigo maligna (LM). Mohs micrographic surgery (MMS) is used increasingly to treat this tumor. OBJECTIVE: To study the authors' experience with these 2 approaches. MATERIALS AND METHODS: Primary LM cases treated at the authors' institution from January 1, 1995, through December 31, 2005, were studied retrospectively. Main outcome measures were recurrence and outcomes after treatment for recurrence. RESULTS: In total, 423 LM lesions were treated in 407 patients: 269 (64%) with wide excision and 154 (36%) with MMS. In the MMS group (primarily larger head and neck lesions with indistinct clinical margins), recurrence rates were 3 of 154 (1.9%). In the wide excision group (primarily smaller, nonhead and neck, or more distinct lesions), recurrence rates were 16 of 269 (5.9%). Each of the 16 recurrences was biopsy proven and treated surgically: 6 by standard excision and 10 by MMS. CONCLUSION: This follow-up study of LM surgical treatments shows excellent outcomes for wide excision and MMS. Because this is a nonrandomized retrospective study, no direct comparisons between the 2 treatments can be made. When recurrences occurred, repeat surgery, either standard excision or MMS, was usually sufficient to provide definitive cure.

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

Temporal dynamics of microbial rhodopsin fluorescence reports absolute membrane voltage.

Plasma membrane voltage is a fundamentally important property of a living cell; its value is tightly coupled to membrane transport, the dynamics of transmembrane proteins, and to intercellular communication. Accurate measurement of the membrane voltage could elucidate subtle changes in cellular physiology, but existing genetically encoded fluorescent voltage reporters are better at reporting relative changes than absolute numbers. We developed an Archaerhodopsin-based fluorescent voltage sensor whose time-domain response to a stepwise change in illumination encodes the absolute membrane voltage. We validated this sensor in human embryonic kidney cells. Measurements were robust to variation in imaging parameters and in gene expression levels, and reported voltage with an absolute accuracy of 10 mV. With further improvements in membrane trafficking and signal amplitude, time-domain encoding of absolute voltage could be applied to investigate many important and previously intractable bioelectric phenomena.

Characteristics of sebaceous carcinoma and early outcomes of treatment using Mohs micrographic surgery versus wide local excision: an update of the Mayo Clinic experience over the past 2 decades.

BACKGROUND: Sebaceous carcinoma (SC) is a rare cutaneous neoplasm. OBJECTIVE: To characterize SC and treatment approaches and outcomes. METHODS AND MATERIALS: We retrospectively reviewed records of patients with SC from 1992 through 2012. Recurrence-free survival was estimated and compared between groups. RESULTS: We identified 52 patients with SC (39, 75.0% male). Mean age ± standard deviation at diagnosis was 72.7 ± 10.8. Forty-nine patients (94.2%) were white. Twenty-one (of 29 with known status) had a diagnosis of Muir-Torre syndrome. Six had multiple primary SCs (total of 73 SCs in 52 patients). The most common locations for SC were the back (20.5%), cheek (13.7%), nose (11.0%), and eye (9.6%). Treatment was recorded for 70 SCs; 35 (50.0%) were treated using Mohs micrographic surgery (MMS) and 26 (37.1%) using wide local excision (WLE). Of the 45 patients (66 SCs) with clinical follow-up, three (6.7% of patients; 4.8% of SCs) had documented recurrence. CONCLUSION: MMS and WLE are effective treatments for SC. Further research is warranted to determine whether one treatment is more efficacious than the other.

Incidence of lentigo maligna in Olmsted County, Minnesota, 1970 to 2007.

BACKGROUND: The incidence of lentigo maligna (LM) may be increasing, but no population-based epidemiologic studies have been performed to our knowledge. OBJECTIVE: We sought to determine the incidence of LM in Olmsted County, Minnesota, along with overall and recurrence-free survival. METHODS: Using the Rochester Epidemiology Project, we identified all adult residents of Olmsted County, Minnesota, with a first lifetime diagnosis of LM between 1970 and 2007. Medical records were reviewed to determine demographic, clinical, and surgical data, and incidence and survival were calculated. RESULTS: Among 145 patients identified, median (range) age at diagnosis of LM was 70 (33-97) years. Treatment changed over time, with Mohs micrographic surgery becoming available after 1986. No patients died of LM; 5 had local recurrence. Estimated local recurrence-free survival at 5, 10, 15, and 20 years after diagnosis was 98%, 96%, 92%, and 92%, respectively. Overall age- and sex-adjusted incidence of LM among adults was 6.3 per 100,000 person-years, increasing from 2.2 between 1970 and 1989 to 13.7 between 2004 and 2007. LIMITATIONS: Retrospective study design and homogeneous population are limitations. CONCLUSION: The incidence of LM increased significantly among residents of Olmsted County, Minnesota, over an extended time frame, with incidence being significantly higher among men than women and increasing with age.

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes.

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20-80min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA-primaquine conjugate (TriGalNAc-PGA-PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250ng per million cells of the conjugate bound to one million rat hepatocytes at 0°C, and approximately 2µg per million cells of the conjugate was taken up over 7h incubation at 37°C. Furthermore, our results suggested that TriGalNAc-PGA-PQ was almost completely degraded over 24h, and small degradation products were secreted into cell culture medium. The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.

Use of a glycolipid inhibitor to ameliorate renal cancer in a mouse model.

In a xenograft model wherein, live renal cancer cells were implanted under the kidney capsule in mice, revealed a 30-fold increase in tumor volume over a period of 26 days and this was accompanied with a 32-fold increase in the level of lactosylceramide (LacCer). Mice fed D- threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase (LCS: ß-1,4-GalT-V), showed marked reduction in tumor volume. This was accompanied by a decrease in the mass of lactosylceramide and an increase in glucosylceramide (GlcCer) level. Mechanistic studies revealed that D-PDMP inhibited cell proliferation and angiogenesis by inhibiting p44MAPK, p-AKT-1 pathway and mammalian target for rapamycin (mTOR). By linking glycosphingolipid synthesis with tumor growth, renal cancer progression and regression can be evaluated. Thus inhibiting glycosphingolipid synthesis can be a bonafide target to prevent the progression of other types of cancer.

Human HDL containing a novel apoC-I isoform induces smooth muscle cell apoptosis.

AIMS: We discovered that some adults with coronary heart disease (CHD) have a high density lipoprotein (HDL) subclass which induces human aortic smooth muscle cell (ASMC) apoptosis in vitro. The purpose of this investigation was to determine what properties differentiate apoptotic and non-apoptotic HDL subclasses in adults with and without CHD. METHODS AND RESULTS: Density gradient ultracentrifugation was used to measure the particle density distribution and to isolate two HDL subclass fractions, HDL2 and HDL3, from 21 individuals, including 12 without CHD. The HDL fractions were incubated with ASMCs for 24 h; apoptosis was quantitated relative to C2-ceramide and tumour necrosis factor-alpha (TNF-α). The observed effect of some HDL subclasses on apoptosis was ∼6-fold greater than TNF-α and ∼16-fold greater than the cell medium. We observed that apoptotic HDL was (i) predominately associated with the HDL2 subclass; (ii) almost exclusively found in individuals with a higher apoC-I serum level and a novel, higher molecular weight isoform of apoC-I; and (iii) more common in adults with CHD, the majority of whom had high (>60 mg/dL) HDL-C levels. CONCLUSIONS: Some HDL subclasses enriched in a novel isoform of apoC-I induce extensive ASMC apoptosis in vitro. Individuals with this apoptotic HDL phenotype generally have higher apoC-I and HDL-C levels consistent with an inhibitory effect of apoC-I on cholesteryl ester transfer protein activity. The association of this phenotype with processes that can promote plaque rupture may explain a source of CHD risk not accounted for by the classical risk factors.

The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease.

The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA(6). Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate α-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the protease.

Distinct structural elements of the adaptor ClpS are required for regulating degradation by ClpAP.

Adaptor proteins modify substrate recognition by AAA+ ATPases. We examined how the adaptor ClpS regulates substrate choice by the Escherichia coli protease ClpAP. Binding of six ClpS molecules to a ClpA hexamer enhanced N-end-rule substrate degradation and inhibited ssrA-tagged protein proteolysis. Substoichiometric ClpS binding allowed intermediate degradation of both substrate types, revealing that adaptor stoichiometry influences substrate choice. ClpS controls substrate selection using distinct mechanisms. The N-terminal segment is essential for delivering N-end-rule substrates but dispensable for ssrA-protein inhibition. We tested existing models for ClpS action and found that ClpS does not block recognition of ssrA-tagged substrates by steric occlusion and that adaptor-mediated tethering of N-end-rule substrates to ClpAP was insufficient to explain facilitated delivery. We propose that ClpS functions, at least in part, as an allosteric effector of ClpAP, broadening our understanding of how AAA+ adaptors control substrate selection.

The relationship between evolutionary and physiological variation in hemoglobin.

Physiological and evolutionary adaptations operate at very different time scales. Nevertheless, there are reasons to believe there should be a strong relationship between the two, as together they modify the phenotype. Physiological adaptations change phenotype by altering certain microscopic parameters; evolutionary adaptation can either alter genetically these same parameters or others to achieve distinct or similar ends. Although qualitative discussions of this relationship abound, there has been very little quantitative analysis. Here, we use the hemoglobin molecule as a model system to quantify the relationship between physiological and evolutionary adaptations. We compare measurements of oxygen saturation curves of 25 mammals with those of human hemoglobin under a wide range of physiological conditions. We fit the data sets to the Monod-Wyman-Changeux model to extract microscopic parameters. Our analysis demonstrates that physiological and evolutionary change act on different parameters. The main parameter that changes in the physiology of hemoglobin is relatively constant in evolution, whereas the main parameter that changes in the evolution of hemoglobin is relatively constant in physiology. This orthogonality suggests continued selection for physiological adaptability and hints at a role for this adaptability in evolutionary change.

Monolayer transistor using a highly ordered conjugated polymer as the channel.

Field-effect transistor structures based on polydiacetylene (PDA) derivatives have been fabricated. Monolayer channels of UV polymerized pentacosa-10,12-diynoic ethanolamide exhibit modulation of source-drain current on application of a gating voltage. Comparison of the two-dimensional crystal morphology of this material with several closely related derivatives that show no gating suggests that a high degree of alignment and order in the polymer chains is necessary for the observed transistor action.