The occurrence of bone and joint cancers and their association with rural living and radon exposure in Iowa.

Primary bone and joint cancers are rare and understudied, yet these neoplasms are difficult to treat and impact all age groups. To explore the long-term changes in the occurrence of bone and joint cancers, patients diagnosed with these neoplasms between 1975 and 2016 were identified in the Surveillance Epidemiology and End Results of the National Cancer Institute of the USA. The age-adjusted incidence (AAIR) and mortality (AAMR) rates were calculated for three decades and compared to AAIR and AAMR in years 1975-1984. By using the population-based cancer registries of the USA, Iowa was identified as a state with increased cases of bone and joint malignancies. The bone and joint cancer cases in Iowa were correlated with the percentage of rural population, the average farmland size, or the residential radon levels. Results demonstrated that the mean AAIR of bone and joint cancers for US female and male patients (< 50 years of age) increased from 0.57 (95% C.I. 0.55-0.63) and 0.76 (95% C.I. 0.69-0.82) for years 1975-1984 to 0.71 (95% C.I. 0.66-0.76) and 0.94 (95% C.I. 0.87-1.07) for years 2005-2014, respectively. The increase in bone and joint cancer cases in Iowa positively correlated with the percentage rural population (R = 0.222, P < 0.02), and the average farmland size (R = 0.236, P < 0.02) but not the radon levels (R = - 0.038, P < 0.7). The findings revealed that patients younger than 50 years of age and those who resided in rural areas and engaged in farming were more likely to be diagnosed with primary bone and joint cancers.

Leucine and branched-chain amino acid metabolism contribute to the growth of bone sarcomas by regulating AMPK and mTORC1 signaling.

Osteosarcoma and chondrosarcoma are sarcomas of the bone and the cartilage that are primarily treated by surgical intervention combined with high toxicity chemotherapy. In search of alternative metabolic approaches to address the challenges in treating bone sarcomas, we assessed the growth dependence of these cancers on leucine, one of the branched-chain amino acids (BCAAs), and BCAA metabolism. Tumor biopsies from bone sarcoma patients revealed differential expression of BCAA metabolic enzymes. The cytosolic branched-chain aminotransferase (BCATc) that is commonly overexpressed in cancer cells, was down-regulated in chondrosarcoma (SW1353) in contrast with osteosarcoma (143B) cells that expressed both BCATc and its mitochondrial isoform BCATm. Treating SW1353 cells with gabapentin, a selective inhibitor of BCATc, further revealed that these cells failed to respond to gabapentin. Application of the structural analog of leucine, N-acetyl-leucine amide (NALA) to disrupt leucine uptake, indicated that all bone sarcoma cells used leucine to support their energy metabolism and biosynthetic demands. This was evident from the increased activity of the energy sensor AMP-activated protein kinase (AMPK), down-regulation of complex 1 of the mammalian target of rapamycin (mTORC1), and reduced cell viability in response to NALA. The observed changes were most profound in the 143B cells, which appeared highly dependent on cytosolic and mitochondrial BCAA metabolism. This study thus demonstrates that bone sarcomas rely on leucine and BCAA metabolism for energy and growth; however, the differential expression of BCAA enzymes and the presence of other carbon sources may dictate how efficiently these cancer cells take advantage of BCAA metabolism.

Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma.

BACKGROUND: The mitochondrial branched-chain aminotransferase (BCATm) is a recently discovered cancer marker with a poorly defined role in tumour progression. METHODS: To understand how a loss of function of BCATm affects cancer, the global knockout mouse BCATmKO was challenged with EL-4 lymphoma under different diet compositions with varying amounts of branched-chain amino acids (BCAAs). Next, the growth and metabolism of EL-4 cells were studied in the presence of different leucine concentrations in the growth medium. RESULTS: BCATmKO mice experienced delayed tumour growth when fed standard rodent chow or a normal BCAA diet. Tumour suppression correlated with 37.6- and 18.9-fold increases in plasma and tumour BCAAs, 37.5% and 30.4% decreases in tumour glutamine and alanine, and a 3.5-fold increase in the phosphorylation of tumour AMPK in BCATmKO mice on standard rodent chow. Similar results were obtained with a normal but not with a choice BCAA diet. CONCLUSIONS: Global deletion of BCATm caused a dramatic build-up of BCAAs, which could not be utilised for energy or amino acid synthesis, ultimately delaying the growth of lymphoma tumours. Furthermore, physiological, but not high, leucine concentrations promoted the growth of EL-4 cells. BCATm and BCAA metabolism were identified as attractive targets for anti-lymphoma therapy.

Boric acid destabilizes the hyphal cytoskeleton and inhibits invasive growth of Candida albicans.

Exposure of Candida albicans to sub-lethal concentrations of boric acid (BA) restricts the dimorphic fungus to its yeast morphology and prevents the formation of invasive hyphae on solid substrates. Exposure to BA causes a rapid and reversible disappearance of polarisome and Spitzenkörper in growing hyphae. In BA-treated hyphae of C. albicans, actin quickly reorganizes from cytoplasmic cables to cortical patches and cell wall growth switches from an apical to an isotropic pattern. As a result of the cytoskeletal changes, the hyphal tips broaden and directional growth of hyphae ceases in the presence of BA. An analysis of homozygous deletion strains showed that mutants with constitutive or enhanced hyphal growth (tup1, nrg1, ssn6, rbf1) are BA-sensitive, demonstrating that cellular morphology is a major determinant of BA tolerance. The screening of deletion mutants also showed that deficiencies of the main activator of hyphal gene expression, Efg1, and the Rim101-signalling cascade, leading to Efg1 activation, cause BA resistance. Taken together, the data presented show that the selective inhibitory effect on BA on C. albicans hyphae is rooted in a disruption of apical cytoskeletal elements of growing hyphae.

Expression and characterization of a ß-fructofuranosidase from the parasitic protist Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative ß-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present. RESULTS: We amplified the ß-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known ß-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar K(m) and V(max) to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin. CONCLUSIONS: T. vaginalis has the coding capacity to produce an active ß-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect ß-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.

Role of Hog1, Tps1 and Sod1 in boric acid tolerance of Saccharomyces cerevisiae.

In order to identify genetic contributions to boric acid (BA) resistance, a yeast knockout collection was screened for BA-sensitive mutants. Prominent among the BA-sensitive mutants were strains with defects in the cytoplasmic part of the high osmolarity/glycerol (HOG) signalling pathway, the trehalose-synthesis pathway (TPS1/TPS2) and the copper-zinc superoxide dismutase SOD1. An analysis of HOG-pathway mutants and fluorescence microscopy of Hog1-GFP fusions showed that the non-redundant cytoplasmic components of the pathway, Pbs2p and Hog1p, are required to maintain BA resistance, but that import of the activated Hog1p kinase into the nucleus neither occurs during BA stress nor is necessary for wild-type-like BA tolerance. Pbs2p and Hog1p are also required to support normal morphogenesis during BA stress as their absence leads to BA-induced hyperpolarized growth. An analysis of Sod1p and Tps1p expression revealed that BA stress induces superoxide dismutase and increases trehalose synthesis activity, albeit only after a 7 h delay. We conclude that normal BA resistance of Saccharomyces cerevisiae depends on the functioning of HOG signalling, the trehalose synthesis pathway and superoxide dismutase activity.

The subcellular localization of yeast glycogen synthase is dependent upon glycogen content.

The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilized Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase was located within cells. We demonstrated that the localization pattern of Gsy2-GFP depended upon the glycogen content of the cell. When glycogen was abundant, Gsy2-GFP was found uniformly throughout the cytoplasm, but under low glycogen conditions, Gsy2-GFP localized to discrete spots within cells. Gsy2p is known to bind to glycogen, and we propose that the subcellular distribution of Gsy2-GFP reflects the distribution of glycogen particles. In the absence of glycogen, Gsy2p translocates into the nucleus. We hypothesize that Gsy2p is normally retained in the cytoplasm through its interaction with glycogen particles. When glycogen levels are reduced, Gsy2p loses this anchor and can traffic into the nucleus.

Boric Acid Disturbs Cell Wall Synthesis in Saccharomyces cerevisiae.

Boric acid (BA) has broad antimicrobial activity that makes it a popular treatment for yeast vaginitis in complementary and alternative medicine. In the model yeast S. cerevisiae, BA disturbs the cytoskeleton at the bud neck and impairs the assembly of the septation apparatus. BA treatment causes cells to form irregular septa and leads to the synthesis of irregular cell wall protuberances that extend far into the cytoplasm. The thick, chitin-rich septa that are formed during BA exposure prevent separation of cells after abscission and cause the formation of cell chains and clumps. As a response to the BA insult, cells signal cell wall stress through the Slt2p pathway and increase chitin synthesis, presumably to repair cell wall damage.

Inhibition of Candida albicans by Fluvastatin Is Dependent on pH.

The cholesterol-lowering drug fluvastatin (FS) has an inhibitory effect on the growth of the pathogenic yeast Candida albicans that is dependent on the pH of the medium. At the low pH value of the vagina, FS is growth inhibitory at low and at high concentrations, while at intermediate concentrations (1-10 mM), it has no inhibitory effect. Examination of the effect of the common antifungal drug fluconazole in combination with FS demonstrates drug interactions in the low concentration range. Determination of intracellular stress and the activity of the FS target enzyme HMG-CoA reductase confirm our hypothesis that in the intermediate dose range adjustments to the sterol biosynthesis pathway can compensate for the action of FS. We conclude that the pH dependent uptake of FS across yeast membranes might make FS combination therapy an attractive possibility for treatment of vaginal C. albicans infections.

Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae.

The mannosyltransferase mutants mnn9 and mnn10 were isolated in a genetic screen for septation defects in Saccharomyces cerevisiae. Ultrastructural examination of mutant cell walls revealed markedly thin septal structures and occasional failure to construct trilaminar septa, which then led to the formation of bulky default septa at the bud neck. In the absence of a functional septation apparatus, mnn10 mutants are unable to complete cytokinesis and die as cell chains with incompletely separated cytoplasms, indicating that mannosylation defects impair the ability to form remedial septa. We could not detect N-linked glycosylation of the beta(1,3)glucan synthase Fks1p and mnn10 defects do not change the molecular weight or abundance of the protein. We discuss a model explaining the pleiotropic effects of impaired N-linked protein glycosylation on septation in S. cerevisiae.