Galectin-7 in the control of epidermal homeostasis after injury

Galectins, a family of β-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7–deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7–deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7–deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.     

Phylogenetic analysis of the vertebrate galectin family

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.     

Interaction of the family-B DNA polymerase from the archaeon Pyrococcus furiosus with deaminated bases

The interaction of archaeal family B DNA polymerases with deaminated bases has been examined. As determined previously by our group, the polymerase binds tightly to uracil (the deamination product of cytosine), in single-stranded DNA, and stalls replication on encountering this base. DNA polymerisation was also inhibited by the presence of hypoxanthine, the deamination product of adenine. Quantitative binding assays showed that the polymerase bound DNA containing uracil 1.5-4.5-fold more strongly than hypoxanthine and site-directed mutagenesis suggested that the same pocket was used for interaction with both deaminated bases. In contrast the polymerase was insensitive to xanthine, the deamination product of guanine. Traces of uracil and hypoxanthine in DNA can lead to inhibition of the PCR by archaeal DNA polymerases, an important consideration for biotechnology applications. Dual recognition of uracil and hypoxanthine may be facilitated by binding the bases with the glycosidic bond in the anti and syn conformation, respectively.     

Photocrosslinked bovine serum albumin hydrogels with partial retention of esterase activity

Novel biohybrid hydrogels based on bovine serum albumin (BSA) were synthesized by one-pot photopolymerization of chemically modified protein in the presence of N,N'-methylenebisacrylamide (MBA) as cross-linking agent under mild conditions. Two batches of methacrylated albumins were prepared by treating the protein with different amounts of methacrylic anhydride (MAN) and the degree of substitution (DS) of primary amines was quantified via trinitrobenzesulfonic acid (TNBS) colorimetric assay. Hydrogels readily formed when a diluted buffered solution of the modified protein and MBA was exposed to LW-UV in the presence of 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) as the radical initiator. In contrast, no hydrogel was obtained in the absence of a polymerizable BSA, nor when the cross-linker, the radical initiator or UV light exposure was excluded from the reaction, suggesting the critical importance of the combined conditions for hydrogel formation. Hydrogels were characterized via scanning electron microscopy (SEM) and the swelling ratios were monitored at different pHs. The esterolytic activity of the novel biohybrid materials was quantitatively investigated via UV-vis spectroscopy by measuring the release of para-nitrophenol upon incubation with para-nitrophenyl acetate (p-NPA) substrate. The effect of the addition of acrylic acid co-monomer and of the monomer concentration in the catalytic activity and in the swelling behavior was also examined. Finally, the reusability of the materials following one round of catalysis was evaluated.     

Diversity of Arsenate Reductase Genes (<Emphasis Type="Italic">arsC</Emphasis> Genes) from Arsenic-Resistant Environmental Isolates of <Emphasis Type="Italic">E. coli</Emphasis>

A polymerase chain reaction (PCR) approach was used to assess the occurrence and diversity of arsenate reductase gene (arsC gene) in arsenic-resistant environmental E. coli strains. For this purpose, two different sets of primers were designed for the specific amplification of approximately 370-bp fragments from the arsC gene. These primers were used to screen a collection of 25 environmental arsenic-resistant strains isolated from different geographical regions of India, as well as Bangladesh. The PCR results showed that 17 out of the 25 environmental isolates (68%) contained a gene related to the arsC family. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsC genes in the isolated strains. A significant divergence in the DNA sequence was found in the arsC genes among As-resistant environmental E. coli strains from this study, and arsenic resistance, a genetic character, arose from a common ancestral background.     

High Producer Haplotype (CAG) of -863C/A, -308G/A and -238G/A Polymorphisms in the Promoter Region of TNF-α Gene Associate with Enhanced Apoptosis of Lymphocytes in HIV-1 Subtype C Infected Individuals from North India

Introduction

The natural history of HIV-1 infection and its progression towards AIDS vary considerably among individuals. Host genetic factors may be one of the possible reasons for variable HIV-1 disease progression. Single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α gene can influence its production. The aim of the present study was to determine the association of functional TNF-α SNPs and its associated parameters related to apoptosis that may influence the rate of HIV-1 disease progression.

Methods

Therapy naive, 100 HIV slow progressors (SPs), 100 HIV fast progressors (FPs), 50 HIV exposed but seronegative individuals (ESNs) and 260 healthy controls from same ethnic origin were recruited. Genotyping of TNF-α variants (−863C/A, -308G/A and -238G/A) was done using PCR-RFLP. CD4 counts were determined by flow cytometry. Plasma viral load was estimated by COBAS AMPLICOR HIV-1 monitor test. Plasma TNF-α concentration was estimated by Human CBA Th1/Th2 cytokine kit. The lymphocyte mitochondrial membrane potential was measured by JC-1 dye by flow cytometry.

Results

Genotype and allele frequency of TNF-α -238G/A and -863C/A was not significantly different in HIV-1-infected patients when compared to controls, while that of TNF-α -308G/A variant (high TNF-α producer) was significantly higher in FPs compared to SPs (p<0.01, OR = 3.43). Haplotype analyses also showed that carriers of high TNF-α producing haplotype CAG was significantly more common among FPs compared to SPs (p<0.01, OR = 3). The circulating TNF-α levels in blood also correlated well with genotypes. The lymphocyte mitochondrial membrane potential of FPs having CAG haplotype was significantly low as compared to wild type (CGG) haplotype (417±22 vs 571±28, p<0.01).

Conclusion

High producer haplotype, CAG of TNF-α gene associates with enhanced apoptosis of lymphocytes in HIV-1 infected individuals, hence faster progression to AIDS. However, further functional studies are needed to confirm this association and this knowledge may help clinicians to better understand the disease outcome.     

TACE/ADAM-17 phosphorylation by PKC-epsilon mediates premalignant changes in tobacco smoke-exposed lung cells

Background

Tobacco smoke predisposes humans and animals to develop lung tumors, but the molecular events responsible for this are poorly understood. We recently showed that signaling mechanisms triggered by smoke in lung cells could lead to the activation of a growth factor signaling pathway, thereby promoting hyperproliferation of lung epithelial cells. Hyperproliferation is considered a premalignant change in the lung, in that increased rates of DNA synthesis are associated with an increased number of DNA copying errors, events that are exacerbated in the presence of tobacco smoke carcinogens. Despite the existence of DNA repair mechanisms, a small percentage of these errors go unrepaired and can lead to tumorigenic mutations. The results of our previous study showed that an early event following smoke exposure was the generation of oxygen radicals through the activation of NADPH oxidase. Although it was clear that these radicals transduced signals through the epidermal growth factor receptor (EGFR), and that this was mediated by TACE-dependent cleavage of amphiregulin, it remained uncertain how oxygen radicals were able to activate TACE.

Principal Findings

In the present study, we demonstrate for the first time that phosphorylation of TACE at serine/threonine residues by tobacco smoke induces amphiregulin release and EGFR activation. TACE phosphorylation is triggered in smoke-exposed lung cells by the ROS-induced activation of PKC through the action of SRC kinase. Furthermore, we identified PKCε as the PKC isoform involved in smoke-induced TACE activation and hyperproliferation of lung cells.

Conclusions

Our data elucidate new signaling paradigms by which tobacco smoke promotes TACE activation and hyperproliferation of lung cells.     

Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe

The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.