Electron Microscopy Analysis of Early Localization of Cisplatin in Ovarian Carcinoma Cells

Taking advantage of the electron-dense nature of platinum, in this study the authors used an electron microscopy approach to investigate the cellular localization of cisplatin in an ovarian carcinoma cell line. Platinum spots were detected in contact with the plasma membrane and the nuclear envelope as well as in the cytoplasm and nuclear matrices. Contact with the plasma membrane was through a single blunt contact or spanning through the membrane. No sequestration in intracellular vescicles was observed, thereby supporting that phagocytosis and receptor-mediated endocytosis were not occurring. A molecular analysis indicated lack of expression of aquaporin 9, thus excluding its involvement in the membrane translocation of cisplatin. The present data suggest that cisplatin rapidly accumulates in the cell through endocytosis-independent membrane translocation and are consistent with passive diffusion.     

AMP-activated protein kinase regulates hERG potassium channel

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Ca²⁺ channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), or catalytically inactive ^αK45RAMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active ^γR70QAMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. ^γR70QAMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active ^γR70QAMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth.     

Activation of Toll-like receptor 2 increases macrophage resistance to HIV-1 infection

Patients infected with HIV-1, the etiological agent of AIDS, have increased intestinal permeability, which allows for the passage of microbial products, including Toll-like receptor (TLR) ligands, into circulation. The exposure of HIV-1-infected cells to certain TLR agonists affects viral replication, but studies associating viral production with the activation of TLR2 in HIV-1-infected cells are rare and controversial. Here, we report that the TLR2 ligands Zymosan and Pam3CSK4 potently inhibit HIV-1 replication in acutely infected monocyte-derived macrophages and the exposure to TLR2 ligands prior to infection renders macrophages refractory to HIV-1 production. Macrophage treatment with Pam3CSK4 did not change the cellular expression of the HIV-1 entry receptors CD4 and CCR5. Both TLR2 ligands increased the macrophage production of β-chemokines and IL-10, and the blockage of these soluble factors prevented the inhibitory effect of TLR2 activation on HIV-1 replication. Our findings show that the direct engagement of TLR2 in HIV-1-infected macrophages increase cellular resistance to HIV-1 infection, and that controlling HIV-1 replication with agonists for TLR2 might have implications for the development of antiretroviral therapies.     

The significance of enhancer of zeste homolog 2 (EZH2) expression in spindle cell lesions of the breast

Spindle cell lesions of the breast are rare entities and pose a diagnostic challenge for pathologists due to overlapping morphologic and immunohistochemical features. We evaluated EZH2 expression in various benign (fibromatosis (n = 8), myofibroblastoma (n = 7), neurofibroma (n = 1), nodular fasciitis (n = 5), benign phyllodes tumor (n = 18)) and malignant (malignant phyllodes tumor (n = 8), metaplastic breast carcinoma (n = 16) and angiosarcoma (n = 8)) spindle cell lesions as a potential diagnostic and therapeutic marker. The EZH2 expression was evaluated semi-quantitatively to categorize the cases as ‘low’ and ‘high’ expression. All benign lesions showed low EZH2 expression, whereas high EZH2 expression was observed in the majority (28/32; 88%) of malignant lesions. The study results suggest that EZH2 may be used both as an additional diagnostic tool to reach an accurate diagnosis of the spindle cell lesions of the breast and as a therapeutic target for the malignant lesions.     

Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele‐Specific Expression

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication‐based mechanisms such fork stalling and template switching or microhomology‐mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele‐specific LMNB1 expression levels.     

Right Ventricular Cardiomyopathy Meeting the Arrhythmogenic Right Ventricular Dysplasia Revised Criteria? Don't Forget Sarcoidosis!

A 53-year-old woman was referred for ventricular fibrillation with resuscitation. A CT-angiography showed signs of a right ventricular enlargement without obvious cause. A cardiac MRI demonstrated a dilated and hypokinetic right ventricle with extensive late gadolinium enhancement. Arrhythmogenic right ventricular dysplasia (ARVD) was suspected according to the "revised ARVD task force criteria". An endomyocardial biopsy was inconclusive. The patient developed purulent pericarditis after epicardial ablation therapy and died of toxic shock syndrome. The post-mortem pathologic examination demonstrated sarcoidosis involving the heart, lungs, and thyroid gland.     

Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD⁺) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD⁺ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m²) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD⁺ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD⁺ boosters can also directly affect skeletal muscle mitochondrial function in humans.