Metarhizin A suppresses cell proliferation by inhibiting cytochrome c oxidase activity

Aims

Metarhizin A was originally isolated from Metarhizium flavoviride as a potent inhibitor of the growth of insect and mammalian cells. In this study, we aimed to understand the molecular targets of metarhizin A involved in its anti-proliferative activity against human cells.

Main methods

Cell cycle regulators and signaling molecules were examined by immunoblotting using specific antibodies. A mitochondria-enriched fraction was prepared from mouse liver, and mitochondrial activity was monitored using an oxygen electrode. Enzyme activity was measured using purified cytochrome c oxidase and permeabilized cells.

Key findings

Metarhizin A inhibits the growth of MCF-7 cells with an IC₅₀ value of ~ 0.2 μM and other cells in a similar manner; a cell cycle-dependent kinase inhibitor, p21, is selectively induced. Significant amounts of reactive oxygen species (ROS) are generated and ERK1/2 is activated in cells treated with metarhizin A. Metarhizin A completely suppresses oxygen consumption by mitochondria, and potently inhibits the activity of cytochrome c oxidase. It induces cell death when MCF-7 cells are cultured under limiting conditions.

Significance

Metarhizin A is a potent inhibitor of cytochrome c oxidase and activates the MAPK pathway through the generation of ROS, which induces growth arrest of cells, and, under some conditions, enhances cell death. The cytochrome c oxidase system is a possible molecular target of metarhizin A.     

Routine Eye Screening by an Ophthalmologist Is Clinically Useful for HIV-1-Infected Patients with CD4 Count Less than 200 /μL

Objective

To investigate whether routine eye screening by an ophthalmologist in patients with HIV-1 infection is clinically useful.

Methods

A single-center, retrospective study in Tokyo, Japan. HIV-1-infected patients aged over 17 years who visited our clinic for the first time between January 2004 and December 2013 and underwent full ophthalmologic examination were enrolled. At our clinic, ophthalmologic examination, including dilated retinal examination by indirect ophthalmoscopy was routinely conducted by ophthalmologists on the first visit. The prevalence of ophthalmologic diseases and associated factors including the existence of ocular symptoms were analyzed.

Results

Of the 1,515 study patients, cytomegalovirus retinitis (CMV-R) was diagnosed in 24 (2%) patients, HIV retinopathy (HIV-R) in 127 (8%), cataract in 31 (2%), ocular syphilis in 4 (0.3%), and uveitis with unknown cause in 8 (0.5%). Other ocular diseases were diagnosed in 14 patients. The CD4 count was <200 /μL in all CMV-R cases and 87% of HIV-R. The prevalence of any ocular diseases, CMV-R, and HIV-R in patients with CD4 <200 /μL were 22%, 3%, and 15%, respectively, whereas for those with CD4 ≥200 /μL were 5%, 0%, and 2%, respectively. No ocular symptoms were reported by 71% of CMV-R cases and 82% of patients with any ocular diseases.

Conclusions

Routine ophthalmologic screening is recommended for HIV-1-infected patients with CD4 <200 /μL in resource-rich settings based on the high prevalence of ocular diseases within this CD4 count category and because most patients with ocular diseases, including those with CMV-R, were free of ocular symptoms.     

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Excessive ingestion of long-chain polyunsaturated fatty acids during developmental stage causes strain- and sex-dependent eye abnormalities in mice

The eyes are riched in long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid [ARA; 20:4 (n−6)] and docosahexaenoic acid [DHA; 22:6 (n−3)]. Despite their abundance in the eyes, ARA and DHA cannot be sufficiently synthesized de novo in mammals. During gestation, eye development is exceptionally rapid, and substantial amounts of LC-PUFAs are needed to ensure proper eye development. Here, we studied the influences of dietary LC-PUFAs in dams (C57BL/6 and C3H/He) on the eye morphogenesis and organogenesis of their pups. Intriguingly, fetuses and newborn mice from C57BL/6 dams fed an LC-PUFA (particularly ARA)-enriched diet displayed a much higher incidence of eye abnormalities such as microphthalmia (small eye) and corneal opacity than those from dams fed an LC-PUFA-poor diet. The effects of LC-PUFAs on eye anomalies were evident only in the female pups of C57BL/6 inbred mice, not in those of C3H/He mice or male C57BL/6 mice. These results demonstrate a gene-by-environment (GxE) interaction in eye development in mice. Furthermore, our molecular analysis suggested the potential roles of Pitx3 and Pax6 in the above interaction involving ARA.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80

Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert -butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K _m value was estimated to be 44.4 μ M for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS–PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30–90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.