The β-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic β-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for β-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-β-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K. aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K. aerogenes. The possible evolutionary implications of the results are discussed

Di-(p-chlorophenyl)dithiophosphinato Derivatives of Fe(III), Co(III) & Ru(III)

623-62

Reversion of pH-Induced Physiological Drug Resistance: A Novel Function of Copolymeric Nanoparticles

The extracellular pH of cancer cells is lower than the intracellular pH. Weakly basic anticancer drugs will be protonated extracellularly and display a decreased intracellular concentration. In this study, we show that copolymeric nanoparticles (NPs) are able to overcome this “pH-induced physiological drug resistance” (PIPDR) by delivering drugs to the cancer cells via endocytosis rather than passive diffussion.As a model nanoparticle, Tetradrine (Tet, Pka 7.80) was incorporated into mPEG-PCL. The effectiveness of free Tet and Tet-NPs were compared at different extracellular pHs (pH values 6.8 and 7.4, respectively) by MTT assay, morphological observation and apoptotic analysis in vitro and on a murine model by tumor volume measurement, PET-CT scanning and side effect evaluation in vivo.<0.05) when the extracellular pH decreased from 7.4 to 6.8. Meanwhile, the cytotoxicity of Tet-NPs was not significantly influenced by reduced pH. In vivo experiment also revealed that Tet-NPs reversed PIPDR more effectively than other existing methods and with much less side effects.The reversion of PIPDR is a new discovered mechanism of copolymeric NPs. This study emphasized the importance of cancer microenvironmental factors in anticancer drug resistance and revealed the superiority of nanoscale drug carrier from a different aspect

Bicarbonate and dichloroacetate: Evaluating pH altering therapies in a mouse model for metastatic breast cancer

BACKGROUND:The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.METHODS:We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.RESULTS:Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.CONCLUSIONS:DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Novel functions and regulation of cryptic cellobiose operons in Escherichia coli

Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (beta-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolismopen1

Reduction of metastasis using a non-volatile buffer

The tumor microenvironment is acidic as a consequence of upregulated glycolysis and poor perfusion and this acidity, in turn, promotes invasion and metastasis. We have recently demonstrated that chronic consumption of sodium bicarbonate increased tumor pH and reduced spontaneous and experimental metastases. This occurred without affecting systemic pH, which was compensated. Additionally, these prior data did not rule out the possibility that bicarbonate was working though effects on carbonic anhydrase, and not as a buffer per se. Here, we present evidence that chronic ingestion of a non-volatile buffer, 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) with a pKa of 6.9 also reduced metastasis in an experimental PC3M prostate cancer mouse model. Animals (n = 30) were injected with luciferase expressing PC3M prostate cancer cells either subcutaneously (s.c., n = 10) or intravenously (i.v., n = 20). Four days prior to inoculations, half of the animals for each experiment were provided drinking water containing 200 mM IEPA buffer. Animals were imaged weekly to follow metastasis, and these data showed that animals treated with IEPA had significantly fewer experimental lung metastasis compared to control groups (P < 0.04). Consistent with prior work, the pH of treated tumors was elevated compared to controls. IEPA is observable by in vivo magnetic resonance spectroscopy and this was used to measure the presence of IEPA in the bladder, confirming that it was orally available. The results of this study indicate that metastasis can be reduced by non-volatile buffers as well as bicarbonate and thus the effect appears to be due to pH buffering per se

Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma

Hypoxia and pH influence gene expression in tumours, and it is becoming increasingly clear that the pattern of genes expressed by a tumour determines its growth and survival characteristics. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the cellular response to hypoxia and high HIF-1 expression has been identified as a poor prognostic factor in tumours. Recently, we identified the tumour-associated carbonic anhydrases (CA), CA9 and CA12 as hypoxia-inducible in tumour cell lines. Furthermore, we identified CA IX to be a poor prognostic factor in breast cancer. The aim of this study was to assess the prognostic significance of CA XII. CA XII expression was studied by immunohistochemistry in a series of 103 cases of invasive breast cancer and any association with recognised prognostic factors or relation with the outcome was examined. CA XII expression was present in 77 out of 103 (75%) cases and was associated with lower grade (P=0.001), positive estrogen receptor status (P&lt;0.001), and negative epidermal growth factor receptor status (P&lt;0.001). Furthermore, although CA XII expression was associated with an absence of necrosis (P&lt;0.001), expression of CA XII in some high-grade tumours was induced in regions directly adjacent to morphological necrosis. Additionally, using univariate analysis, CA XII positive tumours were associated with a lower relapse rate (P=0.04) and a better overall survival (P=0.01). In conclusion, CA XII expression is influenced both by factors related to differentiation and hypoxia in breast cancer in vivo and CA XII expression is associated with a better prognosis in an unselected series of invasive breast carcinoma patients

Quantitative cardiovascular magnetic resonance for molecular imaging

Cardiovascular magnetic resonance (CMR) molecular imaging aims to identify and map the expression of important biomarkers on a cellular scale utilizing contrast agents that are specifically targeted to the biochemical signatures of disease and are capable of generating sufficient image contrast. In some cases, the contrast agents may be designed to carry a drug payload or to be sensitive to important physiological factors, such as pH, temperature or oxygenation. In this review, examples will be presented that utilize a number of different molecular imaging quantification techniques, including measuring signal changes, calculating the area of contrast enhancement, mapping relaxation time changes or direct detection of contrast agents through multi-nuclear imaging or spectroscopy. The clinical application of CMR molecular imaging could offer far reaching benefits to patient populations, including early detection of therapeutic response, localizing ruptured atherosclerotic plaques, stratifying patients based on biochemical disease markers, tissue-specific drug delivery, confirmation and quantification of end-organ drug uptake, and noninvasive monitoring of disease recurrence. Eventually, such agents may play a leading role in reducing the human burden of cardiovascular disease, by providing early diagnosis, noninvasive monitoring and effective therapy with reduced side effects