Heterogeneous Effects of Direct Hypoxia Pathway Activation in Kidney Cancer

General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects.     

Characterization of Glutaminase from Triticale

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticalesp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH⁺ ₄). A monocharged anion (Cl^–) and a multicharged anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (r_s = 0.69; p<0.05) and C3c/C3 activation ratio (r_s = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway activation in driving hepatic inflammation in NASH. Therefore, alternative pathway factors may be considered attractive targets for treating NASH by inhibiting complement activation.     

Distribution of Integrin Subunits in Normal Human Kidney

We evaluated on serial sections the distribution of a large number of integrin α and β chains in normal adult human kidney: 1) the β1 chain and its corresponding a subunits (α1, α2, α3, α4, α5, α6), 2) αv and β3 chains, 3) the β2 chain and its corresponding α chains (αX, αM, αL), and 4) the β4 chain. We also evaluated ICAM-1, VCAM and ELAM and the major extracellular matrix components (ECM). A three step immunoperoxidase technique was used on frozen sections. Each cell of the kidney shows a specific distribution of these molecules. The relation with ECM and some of their ligands was evaluated. This immunohistochemical study shows that there is no strict colocalisation of a given ECM component with its specific receptor.     

Involvement of the Kynurenine Pathway in Human Glioma Pathophysiology

The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD⁺). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD⁺, which is necessary for energy production and DNA repair.     

Glutaminase catalyzes reaction of Glutamate to GABA

Here, for the first time, we report an NMR spectroscopy study of l-Glutamine (Gln) conversion by Glutaminase (Glnase), which shows that the reaction involves two distinct steps. In the first step, Glnase rapidly hydrolyzes Gln to Glutamate (Glu) (∼16.87 μmol of Gln/min/mg of Glnase) and in the second step, Glu generated in the first step is decarboxylated into gamma-amino butyric acid (GABA) with a much slower rate (∼0.185 μmol/min/mg). When Glnase was added to the sample containing l-Glu alone, it was also converted to GABA, at a similar rate as in the second step mentioned above. The rate of Glu decarboxylation into GABA by Glnase is about an order of magnitude lower than that by commonly known enzyme, Glutamate decarboxylase. Potential impact of these findings, on the mechanistic aspects of Gln–Glu shuttle in neuroscience and glutaminolysis in tumors, is discussed.     

Epigenetic Disruption of the PIWI Pathway in Human Spermatogenic Disorders

Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.     

Evidence for Compartmentation of Synaptosomal Phosphate-Activated Glutaminase

Abstract: Phosphate-activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40–60% by the sulphydryl group reagent N -ethylmaleimide (NEM), forming the basis for distinction between NEM-sensitive and NEM-insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate-activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM-sensitive and NEM-insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0–7.6) and the phosphate concentration (5–10 mM) affect chiefly NEM-sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p -hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate-activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM-sensitive glutaminase is localized to the outer face and NEM-insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.     

The Role of Phospholipase D in Modulating the MTOR Signaling Pathway in Polycystic Kidney Disease

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.     

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34^th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms'' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms'' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM^neg and EpCAM^dim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cells and predominantly co-express EpCAM^bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM⁺EpCAM^- and to a lesser extent in NCAM⁺EpCAM⁺ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells (NCAM⁺EpCAM^-FZD7⁺) or both (NCAM⁺FZD7⁺). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.