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Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.

Fibroblasts represent a heterogeneous cell population, that in adult body maintains the homeostasis of the extracellular matrix (ECM) and can acquire an immunoregulatory phenotype. Indeed, activated fibroblasts produce large amounts of cyclooxygenase-2 (COX-2) and proinflammatory cytokines (1). The activation of fibroblasts is represented by their differentiation into myofibroblasts. This process, either in wound healing or cancer tissue, is associated with the expression of alpha-smooth muscle actin (alpha-SMA), increased levels of growth factors and ECM-degrading proteases (2). Moreover, myofibroblasts form clusters in wound healing process and hypertrophic scars. In particular, cell clusters of hypertrophic scars are represented by nodules of myofibroblasts (3). It is known that human dermal fibroblasts established from neonatal foreskin, and forced in vitro to form clusters named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process leads to a programmed necrosis, designated “nemosis” (1). In the present study we generated spheroids from human primary myofibroblasts of skin, to evaluate necrotic, inflammation and activation markers during myofibroblasts clustering. Western blotting analysis, showing low levels of COX-2 and a significant decrease of alpha-SMA in protein extracts of spheroids, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is confirmed by cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines, by confocal immunofluorescence analysis of connexin 43 and immunohistochemical evaluation of proliferation marker Ki-67. This work could represent an experimental model to study myofibroblasts deactivation and highlights an alternative process regulating the turnover of myofibroblasts., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Glutathione, the most abundant non-enzymatic cellular thiol, regulates the redox environment through a balance of its reduced (GSH) and oxidised (GSSG) forms. Its antioxidant tasks include the defence against oxidative damages caused by reactive oxygen species (ROS), detoxification from toxic compounds, protection from oxidation of protein sulfhydryls. Hence, this tripeptide affects many cellular functions, such as tran- sport, metabolism, cell cycle, carcinogenesis, apoptosis; furthermore, it is involved in the post-translational regulation of protein functions through the S-glutathionylation of specific cysteine residues. The enzyme me- chanism for GSH biosynthesis is conserved throughout prokaryotes and eukaryotes and includes two sequen- tial steps for the assemblage of glutamate, cysteine and glycine into GSH, the driving force being guaranteed by the hydrolysis of ATP. In most prokaryotes, the two reaction steps are catalysed by two distinct enzymes, γ-glutamyl-cysteine ligase (GshA) and glutathione synthetase (GshB). In this work, the GSH biosynthesis was investigated for the first time in a cold-adapted microorganism, such as Pseudoalteromonas haloplanktis. This psychrophile, perfectly adapted to ROS protection, exhibits intriguing features in the functional role and biosynthesis of GSH. First, some antioxidant enzymes from this source are S-glutathionylated; second, differently from other proka- ryotes containing a single GshA and GshB, P. haloplanktis has two redundant GshA gene products (GshA I and GshA II) besides one GshB. In this study a detailed characterization of the recombinant forms of these enzymes was realised, focusing on the different role played by the redundant GshAs. The cold adaptations of the three enzymes of P. haloplanktis were compared and two distinct complete systems for GSH synthesis were reconstituted and analysed in order to dissect partial and complete reaction rates for GSH biosynthesis, even for potential biotechnological applications.

The organization and physiological functions of multicellular organisms depend also on the presence of connective tissues (1). Fibroblasts are versatile connective tissue cells and represent a heterogeneous cell population (2). They maintain the homeostasis of the extracellular matrix but can also acquire an immunoregulatory phenotype (2). Moreover, fibroblasts can form multicellular aggregates when activated in vivo to myofibroblasts, during wound repair and in fibrotic states (1). It is known that established human dermal fibroblasts, when forced to form multicellular aggregates named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process terminates in a programmed necrosis, designated as nemosis (2). In the present study we analysed spheroids of human primary fibroblasts from skin. To this aim we formed spheroids adapting the hanging-drops and agarose-coated U-bottom well plates methods. Immunohistochemical analysis of spheroids, collected at different times, detected the presence of vimentin, a myofibroblast marker. Moreover, hematoxylin and eosin staining showed very negligible areas of necrosis. Lactate dehydrogenase (LDH) release, associated with loss of membrane integrity, was estimated in conditioned media from fibroblasts grown as spheroids or monolayers. Marginal levels of LDH activity were detected in conditioned media from spheroids and monolayers, although after 96 h an increase of LDH release, more evident in monolayers media, was measured. Western blotting analysis of spheroid extracts showed the absence of COX-2 and the presence of a-smooth muscle actin, a marker of myofibroblast differentiation. Hence, very low levels of LDH activity associated with the absence of COX-2 demonstrate that human primary fibroblasts from skin, cultured as spheroids, don’t undergo nemosis. Furthermore, TUNEL staining of sectioned spheroids showed very few apoptotic cells, positive for DNA breaks. Our study highlights new aspects about fibroblasts biology and interactions., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Cell division cycle 25 (Cdc25) proteins are dual specificity phosphatases involved in the progression of the cell cycle. In particular, mammalian cells express three isoforms of Cdc25, called Cdc25A, -B and -C: Cdc25A mainly controls G1/S progression, whereas Cdc25B and Cdc25C predominantly activate G2/M transition. Moreover, over-expression of Cdc25A and B activities has been frequently observed in a wide variety of human tumors with poor prognosis. Thus, the design and study of small molecules endowed with an inhibitory activity towards Cdc25 proteins represent a promising strategy for the development of new anti-cancer therapies. A previous work from this group described the properties of a set of small molecules, which were able to inhibit Cdc25 functions (Lavecchia et al. J Med Chem 55, 2012, 4142–4158). Among them, compound 11, possessing a quinonic structure, acted as an irreversible inhibitor of Cdc25B and showed a strong antiproliferative action on some cancer cell lines. To expand the structure-activity relationship study and to explore potential new structure analogues of 11, we performed a multiple ligand-based chemoinformatics approach against full ZINC database (~35 million purchasable compounds) and NCI Open database (~260 000 compounds). 25 structures identified from these searches (hereafter called AL1–AL25) were analyzed, and selected compounds were tested in vitro to assess the Cdc25B inhibition activity. In particular, compounds AL11, -12, -13, -14, -15, -16, -23, -24 and -25 maintained a percentage of Cdc25B inhibition comparable to that exerted by compound 11, and some of them were even more powerful. The effect of these active analogues was also evaluated on melanoma cell lines (A2058 and SAN), because of the limited information on the effect of Cdc25 inhibitors in this cellular system. In both melanoma cells, after 48-h treatment with 100 mM of each inhibitor, a significant reduction of cell growth was observed only with AL11, whereas the other active analogues, as well as compound 11, were ineffective. Furthermore, cytofluorimetric analysis and measurements of the activity of caspase-9 and -3 indicated that AL11 exerted a pro-apoptotic effect on melanoma cells. In addition, the cellular treatment with AL11 produced an alteration of the cellular redox state and a reduction of the mitochondrial membrane potential. Studies are in progress to clarify the mechanisms involved in the cytotoxic potential of AL11 and other related molecules in melanoma cell lines, with the aim at the refinement of the inhibitory action of the studied compounds.

Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. Surgery is standard treatment for localized melanoma, while there is no therapy for advanced-stage of melanoma and its metastasis, because of the high resistance of melanoma cells to various anticancer therapies. Human skin is an important metabolic organ in which occurs photo-induced synthesis of vitamin D3 from 7-dehydrocholesterol (7-DHC). The 7-DHC, the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHCderived compounds. The intracellular levels of 7-DHC or its derivatives can have deleterious effects on cellular functionality and viability. In this study we evaluated the effect on melanoma cell lines by 7-DHC as such and for this aim much care to minimize 7-DHC modifications was used. We found that from 12 to 72 hours of treatment 82-86% of 7-DHC entered into the cells, and the levels of 7-DHC-derivative compounds were not significant. At same time ROS production was significantly increased already after 2 hours and, after 24 hours, a reduction of cell viability was observed. Indeed, after 48-72 hours a pro-apoptotic effect of 7-DHC was detected. The cytotoxic effect of 7-DHC was associated with an increase in Bax levels, decrease in Bcl-2/Bax ratio, reduction of mitochondrial membrane potential, increase in apoptosis-inducing factor levels, unchanged caspase-3 activity, and absence of cleavage of PARP-1. These findings could partially explain the mechanism through which 7-DHC exerts its cytotoxic effects. This is the first report in which the biological effects found in melanoma cells are mainly attributable to 7-DHC as such.

Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induces apoptosis in many cell lines through an alteration of the redox state. We have investigated if the pro-apoptotic effect of diclofenac is mediated by an alteration of the redox state, focusing the attention on the involvement of SOD2, an essential mitochondrial antioxidant enzyme. The study was realised using the melanoma cell lines A2058 and SAN, and the human fibroblast immortalized cell line BJ-5ta. Analysis of propidium iodide incorporation and measurement of caspase-3 activity showed an increase of apoptosis after diclofenac treatment in melanoma cells, whereas no effect was observed in BJ-5ta; moreover, the usage of the fluorescent probes DHE and DCFH-DA allowed the demonstration that diclofenac provoked an increase of the intracellular ROS levels only in melanoma cells. Therefore, the effect of diclofenac on the protein levels and enzymatic activity of SOD2 was investigated and only in melanoma cells a significant reduction of these parameters was evident after a 48-h treatment. To investigate if the reduction of SOD2 contributed to the pro-apoptotic effect of diclofenac, the melanoma cells were transfected with siRNAs specific for the SOD2 messenger. In the presence of diclofenac, the SOD2 silencing amplified the proapoptotic effect of the drug, whereas no effect was observed without the drug. To highlight the mechanism by which diclofenac induces apoptosis, we evaluated the mitochondria involvement in this process, through the analysis of the sub-cellular localization of typical mitochondrial proteins. Western blotting analysis showed that in melanoma cells cytochrome c translocated from mitochondria to cytosol after diclofenac treatment. Surprisingly, the drug altered also the sub-cellular distribution of SOD2; indeed, SOD2 appeared in the cytosolic fraction at the expenses of an almost corresponding reduction of this enzyme in the mitochondrial fraction, a feature confirmed by immunofluorescence experiments. In conclusion, the diclofenac exerts its cytotoxic effect selectively in melanoma cells, targeting the mitochondria; in particular, the data suggest that both reduction and cytosolic translocation of SOD2 contribute to the the mithochondrial dysfunction.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) widely used in clinical therapeutics, has cytotoxic effects and induces apoptosis in many cultured cell lines. Many studies suggest that NSAIDs, and in particular the highly selective cyclooxigenase-2 inhibitors, could act as anticancer. We have investigated the involvement of mitochondrial dysfunction in the mechanism of diclofenac-induced apoptosis in the melanoma cell lines, A2058 and SAN, and in the human fibroblasts immortalized cell line BJ5ta. The analysis by phase contrast microscopy of diclofenac-treated cells showed the typical morphologic changes related to apoptosis only in A2058 and SAN cell lines. These effects appeared after a treatment with 150 lM diclofenac suggesting a preferential cytotoxic effect in the two melanoma cell lines. Propidium iodide incorporation followed by cytometric analysis showed an increase of apoptosis during diclofenac treatment. Since the caspase 3 represents the final effector of the caspase dependent apoptotic process we have also analysed its activity following diclofenac treatment. A marked increase of caspase 3 activity in the cell extracts of A2058 and SAN cells was evidenced by spectrofluorimetric analysis as well as the analysis of intracellular levels of bcl-2 confirmed the pro-apoptotic effect of diclofenac only in the melanoma cell lines. Furthermore, the cytotoxic effect of diclofenac in the melanoma cell lines was associated to a decrease of the antioxidant SOD2 protein levels. These preliminary data suggest that mitochondria could represent a diclofenac target and in particular the reduction of SOD2 protein contributes to the mitochondria dysfunction.

Photodynamic therapy (PDT) is based on the uptake of a photosensitizer followed by its photosensitization through a selective irradiation, which induce oxidative stress and cytotoxicity. In this work the effect of 5-aminolevulinic acid (5-ALA) was evaluated during photodynamic treatment. We have used as experimental system four cell lines: COLO 829 (primary melanoma), A2058 (metastatic melanoma), BJ-5ta (human immortalized fibroblasts), SH-SY5Y (human neuroblastoma). 5-ALA is the precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway of heme. PpIX is a potent photosensitizer, thus leading to an ALA-based PDT. When exogenous 5-ALA is added to culture medium, the production of cellular PpIX can be induced. We have verified the intracellular production of PpIX in the above mentioned cell lines after treatment with different concentration of 5-ALA for various times in the dark. Fluorimetric analysis has shown a dose-dependent increase of fluorescence intensity up to 0.3 mM 5-ALA after 6 hours of incubation. Hence, to evaluate the 5-ALA photocytotoxicity effect, the cells were incubated with 0.3 mM 5-ALA for 6 hours in the dark. After incubation, the cells were irradiated with different light doses using a halogen lamp with a 520 nm long pass filter and a fluence rate of 2.6 mW/cm2. After this exposure, the cells were incubated for additional 24 hours and then the photocytotoxicity effect was evaluated. The results showed that the cytotoxic effect was proportional to the light dose and, interestingly, the metastatic melanoma cell line A2058, is more responsive than the other cell lines used. Furthermore, analysis of expression profiles of some antioxidant enzymes, after 5-ALA-based-PDT lead to a reduction of SOD2 protein levels in cellular extracts of A2058 cell. These results may provide further insights for clinical applications of PDT in cancers particularly resistant to conventional therapies.

Systemic sclerosis (SSc) is a multisystemic disease characterized by vascular injury, circulating autoantibodies and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. It is related to increased synthesis of ECM proteins by activated fibroblasts: this process is modulated by many cytokines such as TGF-α and -β, PLGF, EGF, interleukins and TNF -α and -β. Recent studies demonstrated a role of oxidative stress in SSc pathogenesis. In fact, reactive oxigen species (ROS) accumulation is involved in SSc through different mechanisms: autoantibodies production, changes in the protease – antiprotease balance leading to fibrosis, fibroblasts activation by TGF-β and PDGF. The antioxidant enzymes superoxide dismutases (SODs) constitute an ubiquitous metal enzymes family which catalyzes the dismutation of two superoxide radicals into hydrogen peroxide and oxygen. Extra cellular superoxide dismutase (SOD3) is copper and zinc superoxide dismutase, which is expressed in selected tissues and is secreted into the extracellular space. This enzyme is associated to inflammatory responses in several experimental models; furthermore, hydrogen peroxide produced by SOD3 during enzymatic activity, modulates the profile of cytokine secretion in some cell populations and induces increased proliferation and upregulation of collagen I gene in normal fibroblasts. We investigated the expression and the intracellular localization of SOD3 in dermal fibroblasts from both SSc patients and healthy donors and evaluated SOD3 activity in fibroblasts culture medium. RT-PCR analysis demonstrated increased levels of SOD3 mRNA in SSc fibroblasts when compared to control healthy fibroblasts. SOD3 staining by immunofluorescence displayed a characteristic “secretory” pattern in both healthy and SSc fibroblasts. SOD assay, performed with xanthine/xanthine oxidase method, demonstrated SOD3 enzymatic activity in SSc fibroblasts culture medium four times higher than in healthy fibroblasts. Further studies will identify the factors regulating SOD3 expression in SSc fibroblasts and clarify the role of SOD3 enzymatic activity in SSc pathogenesis.

Streptococcus mutans is an eubacterial pathogenic microorganism involved in the development of dental caries, growing either in aerobic or anaerobic conditions. The bacterium lacks cytochromes and catalase, but possesses other anti-oxidant enzymes involved in the control of the intracellular redox potential, such as superoxide dismutase (SOD), the key enzyme scavenging the toxic superoxide anions. Previous researches suggested that SOD from S. mutans (SmSOD) belongs to the cambialistic group, functioning with Fe or Mn in the active site; this feature is probably related to the adaptation of this microorganism to different growth conditions. Recently, the genome of S. mutans has been sequenced, and one SOD gene has been identified. SmSOD was heterologously produced in Escherichia coli, with its C-terminal extremity fused to a His-tag. This recombinant form of the enzyme (rSmSOD) had a specific activity of 1150 U/mg and contained both Fe and Mn (0.45 and 0.07 atoms/subunit, respectively). To improve its metal content, rSmSOD was incubated in the presence of Mn or Fe ions and, after extensive dialysis, metal content and specific activity were redetermined. The amount of Mn in the sample treated with Mn (SmSOD–Mn) raised to 0.21 atoms/subunit, whereas the Fe content remained unchanged. Moreover, the specific activity of SmSOD–Mn improved to 3500 U/mg. Vice versa, in the sample treated with Fe (SmSOD–Fe), the Fe content reached 0.52 atoms/subunit at the expense of Mn; the specific activity of SmSOD-Fe slightly decreased to 1050 U/mg. rSmSOD was half-inactivated after 10 min exposure at 68C. The effect of typical inhibitors/inactivators of Fe- and Mn-SODs was investigated, in terms of a possible regulation of the sensitivity by samples with different metal contents. Sodium azide functioned as a weak inhibitor of SmSOD, the lowest resistance being displayed by SmSOD-Fe. Hydrogen peroxide caused a modest inactivation on either untreated rSmSOD or SmSODFe, whereas it was almost ineffective on SmSOD–Mn. Sodium peroxynitrite provoked a significant inactivation of SmSOD–Mn; the untreated rSmSOD and even more SmSOD–Fe were more resistant to peroxynitrite inactivation. These results confirm the cambialistic nature of SmSOD and prove its regulation by different metal contents. Therefore, the adaptative response of S. mutans during the aerobic exposure in the oral cavity could involve a different metal uptake of SmSOD.

The thioredoxin system, involved in preservation of the reduced state of cytoplasmic proteins, includes two key components of the intracellular redox balance, namely thioredoxin (Trx) and thioredoxin reductase (TrxR). Trx is a small monomeric protein containing the conserved CXXC sequence; a similar motif is also present in the homodimeric TrxR, a NADPH-dependent flavoenzyme. Reduction of protein targets by the Trx/TrxR system involves oscillation between oxidized and reduced form of both cysteine motifs engaged in a reversible disulfide bridge. A previous characterization of Fe-SOD from the psychrophilic eubacterium Pseudoalteromonas haloplkanktis (Ph) proved the high efficient catalysis of this enzyme, another key element of the intracellular redox balance (1). Here we present the properties of the thioredoxin system in P. haloplanktis. PhTrx and PhTrxR were obtained as recombinant proteins and their properties were compared with those of the corresponding endogenous proteins purified from P. haloplanktis. To this aim, genes encoding PhTrx and PhTrxR were cloned in a pET plasmid and as His-tagged recombinant proteins. Exogenous FAD was added in the bacterial total extract to reach a stoichiometric FAD content in the recombinant PhTrxR; under these conditions its activity was almost identical to that measured on endogenous PhTrxR. Kinetic parameters of the dithiobisnitrobenzoate (DTNB) reduction by recombinant and endogenous PhTrxR were determined. Using this assay, thermophilicity and thermostability of the flavoenzyme were also evaluated. Maximum activity was reached at 30°C; however the enzyme showed a marked heat tolerance, its half life being 10 min at 60°C. This latter finding was confirmed by heat denaturation profiles obtained by fluorimetric measurements. The functionality of PhTrx was evaluated by its thiol-disulfide oxidoreductase activity in the presence of DTT as electron donor, and human insulin as substrate. Finally, the combined activity of PhTrxR and PhTrx in a complete thioredoxin system, was also evaluated using NADPH as electron donor and human insulin as the thioredo

The antioxidant function of Fe- and Mn-containing superoxide dismutases (SOD) observed under constraints from extreme rather than mild cellular conditions could reflect an adaptive evolution to oxygen tolerance in the structural organisation of this class of enzymes. For instance, the mitochondrial human Mn-SOD and the hyperthermophilic archaeal Fe-SOD from Sulfolobus solfataricus (SsSOD) share a similar structural organisation. Further studies on members of this ubiquitous enzyme isolated from differently adapted micro–organisms could give useful information on possible adaptive mechanisms in the structure-function relationships of this SOD family. For this reason, this enzyme has been purified and characterised from Pseudoalteromonas haloplanktis, a psychrophilic eubacterium isolated from marine Antarctic sediments. Two chromatographic steps on DEAE-Sepharose and HTP allowed to purify SOD from P. haloplanktis (PhSOD) to homogeneity. The relative molecular weight of the purified enzyme estimated by SDS-PAGE is about 20,000. As SsSOD, also PhSOD shows a homotetrameric structure, as determined by gel filtration. PhSOD has an unusual thermal stability for a psycrophilic enzyme, as evaluated by its half-life of 10 min at 52°C. Similar results were obtained by UV-melting curves. Enzymatic assays showed that PhSOD has a specific activity of 6500 U/mg. The enzyme is inactivated by hydrogen peroxide and it is inhibited by sodium azide, whereas PMSF, a specific inactivator of the archaeal SsSOD, has no effect. Future research plan includes the determination of the metal content and the cloning of the gene encoding PhSOD. To this aim, a molecular probe has been designed on the basis of the amino acid sequence of some fragments of the purified protein.

Superoxide dismutase (SOD) is a metal-enzyme, catalyzing the dismutation of superoxide anion in molecular oxygen and hydrogen peroxide. It is involved in the cellular defence mechanism against the reactive and toxic products of oxygen metabolism. SODs have been classified into two main families, according to their different structural folding and metal content: the Cu/Zn family and that containing Fe or Mn in the active site. SODs isolated from extremophilic organisms are suitable models to study the structure-function relationships and the molecular and evolutive mechanisms for the adaptation of proteins to extreme environments. We have previously isolated a SOD from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD). Its is a Fe-SOD endowed with a remarkable heat stability, its t1/2 being 2 hours at 100°C. Structural ands functional studies on the recombinant SsSOD and the analysis of some mutant and modified forms have explained several aspects about the mechanisms adopted by the enzyme to function at high temperatures. Therefore, it could be interesting to carry out similar studies also in a psychrophilic organism. In this communication we report the purification and the preliminary biochemical characterization of SOD from the psychrophilic eubacterium Pseudoalteromonas haloplanktis, isolated from Antarctic marine sediments and adapted to grow at low temperatures. SOD from P. haloplanktis (PhSOD) was purified to homogeneity from cells grown at 4°C by two chromatographic steps on a DEAE-Sepharose and HTP. The relative molecular mass of the purified enzyme, estimated by SDS-PAGE is 22,000. As SsSOD, also PhSOD shows a homotetrameric structure, as determined by gel filtration. PhSOD has a unusual thermal stability for a psychrophilic enzyme, as evaluated by its half-life of 10 min at 52°C. Similar results were obtained by UV-melting curves. The heat stability seems to be a feature possessed by a number of SODs; for instance, rat mitochondrial Mn-SOD has a a melting temperature of 87°C. Enzymatic assays showed that PhSOD has a specific activity of 6500 U/mg, The enzyme is inactivated by hydrogen peroxide and it is inhibited by sodium azide, whereas PMSF, a specific SsSOD inactivator, has no effect. According to this preliminary characterization PhSOD could be classified as a Fe-SOD. Future research plan includes the determination of the metal content and the cloning of the gene encoding PhSOD from a genomic DNA library. To this aim, a molecular probe will be designed on the basis of the amino acid sequence of some tryptic fragments of the purified protein.

CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBP beta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBP beta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBP beta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBP beta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBP beta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBP beta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBP beta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBP beta as a critical signaling molecule in BM B lymphopoiesis.

A thermostable thioredoxin reductase isolated from S. solfataricus (SsTrxR) was successfully crystd. in the absence and in the presence of NADP. Two different crystal forms were obtained. Crystals of the form that yielded higher resoln. data (1.8 .ANG.) belonged to space group P212121, with unit-cell parameters a = 76.77, b = 120.68, and c = 126.85 .ANG.. The structure of the enzyme was solved by MAD methods using the anomalous signal from the Se atoms of selenomethionine-labeled SsTrxR.