Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

Mobilization of Neural Stem Cells and Generation of New Neurons in 6-OHDA�Clesioned Rats by Intracerebroventricular Infusion of Liver Growth Factor

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson''s disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine–lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases. (J Histochem Cytochem 57:491–502, 2009)     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.     

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

Introduction

Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region.

Methods

To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry.

Results

Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region.

Conclusions

Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.     

Update on skin repigmentation therapies in vitiligo

Treatment for vitiligo is difficult and prolonged. Nevertheless, at present considerable knowledge accumulated during several decades on the pathogenic mechanisms, revealed important clues for designing new strategies to improve vitiligo depigmentation. With available medical therapies, high repigmentation percentages mostly on facial and neck lesions are achieved, although they are less effective on trunk and limbs and poor on the acral parts of the extremities. Narrow band UVB and psoralens and UVA are the two most important treatments for generalized vitiligo affecting more than 10–20% of the cutaneous surface, and topical corticosteroids, or calcineurin inhibitors are the most valuable treatments for localized vitiligo. Persistence of achieved regimentation is variable and an undefined percentage of patients may have variable recurrence. When vitiligo becomes refractory, surgical methods may improve depigmentation as effectively as with medical therapy; in segmental (unilateral) or long standing, non‐segmental (bilateral) stable vitiligo, repigmentation with surgical methods is usually permanent.     

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

Background

Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV''s natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.

Methodology/Principal Findings

In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.

Conclusion/Significance

These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?     

A Nanoconjugate Apaf-1 Inhibitor Protects Mesothelial Cells from Cytokine-Induced Injury

Background

Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology

Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma.Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion

Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.