Synchronous melanosis of upper and lower urinary tract.

Urothelial melanosis is an exceptionally rare diagnosis, with less than 25 cases being reported in the literature. Melanosis of the urothelium is characterized by abnormal melanin deposition within tissues, producing a black, velvety appearance to the urothelial mucosa. We present a 67-year-old male undergoing cystoscopy during a routine percutaneous nephrolithotomy (PCNL), who was found to have diffuse bladder melanosis extending up the ureter and into the renal pelvis. To our knowledge, this is the first reported case of synchronous melanosis of upper and lower urinary tract.

Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds.

Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.

Cytochrome c oxidase oxygen reduction reaction induced by cytochrome c on nickel-coordination surfaces based on graphene oxide in suspension.

BACKGROUND: The electrochemical and spectroscopic investigation of bacterial electron-transfer proteins stabilized on solid state electrodes has provided an effective approach for functional respiratory enzyme studies. METHODS: We assess the biocompatibility of carboxylated graphene oxide (CGO) functionalized with Nickel nitrilotriacetic groups (CGO-NiNTA) ccordinating His-tagged cytochrome c oxidase (CcO) from Rhodobacter sphaeroides. RESULTS: Kinetic studies employing UV-visible absorption spectroscopy confirmed that the immobilized CcO oxidized horse-heart cytochrome c (Cyt c) albeit at a slower rate than isolated CcO. The oxygen reduction reaction as catalyzed by immobilized CcO could be clearly distinguished from that arising from CGO-NiNTA in the presence of Cyt c and dithiothreitol (DTT) as a sacrificial reducing agent. Our findings indicate that while the protein content is about 3.7‰ by mass with respect to the support, the contribution to the oxygen consumption activity averaged at 56.3%. CONCLUSIONS: The CGO-based support stabilizes the free enzyme which, while capable of Cyt c oxidation, is unable to carry out oxygen consumption in solution on its own under our conditions. The turnover rate for the immobilized CcO was as high as 240 O2 molecules per second per CcO unit. GENERAL SIGNIFICANCE: In vitro investigations of electron flow on isolated components of bacterial electron-transfer enzymes immobilized on the surface of CGO in suspension are expected to shed new light on microbial bioenergetic functions, that could ultimately contribute toward the improvement of performance in living organisms.

Computed Tomography Imaging Characteristics of Histologically Confirmed Papillary Renal Cell Carcinoma-Implications for Ancillary Imaging.

Low-attenuation renal lesions on non-contrast computed tomography (CT) are often considered to be benign cysts without need for further imaging. However, the papillary subtype of renal cell carcinoma (RCC) may have similar radiographic characteristics. A single-center retrospective review was therefore performed to identify extirpated papillary RCC (pRCC) specimens with correlation made to preoperative tumor imaging characteristics. A total of 108 pRCC specimens were identified of which 84 (27 type I, 17 type 2, 40 unspecified) had CT imaging available for review. Non-contrast CT was available for 73 tumors with 16 (22%) demonstrating Hounsfield units (HU) measurements fewer than 20 at baseline without differences between papillary subtypes. Mean attenuation following contrast administration was similar between papillary subtypes (45 HU for type 1 pRCC and 49 HU for type 2). This study highlights that pathologically proven pRCC is a heterogeneous entity in terms of density on preoperative CT imaging. A non-contrast CT scan with HU fewer than 20 may not be an adequate evaluation for incidental renal masses, as over 1 in 5 pRCCs demonstrate lower attenuation than this cutoff. Further study is needed to identify the appropriate role of ancillary imaging in the workup of seemingly benign-appearing renal lesions.

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

BACKGROUND: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity. METHODS: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. RESULTS: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. CONCLUSION: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. GENERAL SIGNIFICANCE: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

Improving mitochondrial bioenergetics under ischemic conditions increases warm ischemia tolerance in the kidney.

Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.

The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.

Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.

Antiviral activity of geneticin against dengue virus.

The aminoglycoside, geneticin (G418), was recently shown to have antiviral activity against bovine viral diarrhea virus (BVDV). Since BVDV, dengue virus (DENV) and yellow fever virus (YFV) all belong to the Flaviviridae family, it seemed possible that a common step in their life cycle might be affected by this aminoglycoside. Here it is shown that geneticin prevented the cytopathic effect (CPE) resulting from DENV-2 infection of BHK cells, in a dose-dependent manner with an 50% effective concentration (EC(50)) value of 3+/-0.4microg/ml. Geneticin had no detectable effect on CPE caused by YFV in BHK cells. Geneticin also inhibited DENV-2 viral yield with an EC(50) value of 2+/-0.1microg/ml and an EC(90) value of 20+/-2microg/ml. With a CC(50) value of 165+/-5microg/ml, the selectivity index of anti-DENV activity of geneticin in BHK cells was established to be 66. Furthermore, 25microg/ml of geneticin nearly completely blocked plaque formation induced by DENV-2, but not YFV. In addition, geneticin, inhibited DENV-2 viral RNA replication and viral translation. Gentamicin, kanamycin, and the guanidinylated geneticin showed no anti-DENV activity. Neomycin and paromomycin demonstrated weak antiviral activity at high concentrations. Finally, aminoglycoside-3'-phosphotransferase activity of neomycin-resistant gene abolished antiviral activity of geneticin.

Antiviral activity of geneticin against bovine viral diarrhoea virus.

BACKGROUND: Aminoglycoside G418 is commonly used to generate stable replicons for RNA viruses, such as hepatitis C virus, West Nile virus, and bovine viral diarrhoea virus (BVDV). This precludes testing 6418's own antiviral activities against those viruses. Here, we report antiviral activity of 6418 against BVDV. METHODS: Cell viability and virus yield reduction assays were used to investigate antiviral effects of G418 against BVDV. The expression of viral proteins and RNA were determined by western blot and real-time quantitive PCR, respectively. RESULTS: We demonstrated that G418 (50% cytotoxicity concentration of 400 microg/ml) improved cell viability of Madin-Darby bovine kidney cells infected with a cytopathic strain of BVDV (NADL) in a dose-dependent manner with 50% effective concentration of 4 microg/ml. Interestingly, close structural analogues with known properties as translation inhibitors similar to G418 - kanamycin and gentamicin - had no antiviral activity against BVDV. In addition, 6418 inhibits virus yield of two different strains of BVDV (NADL and NY-1) without affecting viral RNA replication and translation or viral NS3 protein processing. CONCLUSION: Our data indicate that antiviral activity of G418 could result from interference with either the assembly or release of active virus, rather than the regulation of viral translation and replication. Thus, we propose the use of chemical analogues of G418 as antiviral therapeutics for treatment of viral diseases associated with the Flaviviridae family, such as hepatitis C virus, dengue virus, yellow fever virus, West Nile virus and others.

Cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus.

Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7+/-2.2% to 18.8+/-2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis.