Overexpression of cytosolic sialidase Neu2 induces myoblast differentiation in C2C12 cells

Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady-state levels and in the cytosolic sialidase enzymatic activity. To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin-positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was on the contrary observed in parental and mock-transfected cells under the same experimental conditions. The results indicate that Neu2 upregulation per se is sufficient to trigger myoblast differentiation in C2C12 cells.     

The polytetrafluoroethylene-covered stent: a device with multiple potential advantages

The polytetrafluoroethylene (PTFE)-covered stent has emerged in the past year as a device with multiple potential advantages. Its structure (a sandwich composed of a layer of PTFE membrane between two stents) makes this the ideal tool for treating coronary ruptures, and for excluding coronary aneurysms. Furthermore, this device may be useful in the treatment of aortocoronary vein graft stenosis. In the present review, the authors summarize experiences with covered stents, and focus attention on available data on the implantation of PTFE-covered stents in human beings to treat coronary ruptures, aneurysms and aortocoronary vein graft stenosis.     

Evaluation of chromogranin A expression in serum and tissues of breast cancer patients

Human chromogranin A (CgA) is a member of the granin family and is widely distributed in large dense core granules of endocrine and neuroendocrine cells. A variety of non-neuroendocrine carcinomas arising in various tissues show patterns of neuroendocrine differentiation. Expression of CgA has been documented in epithelial cells of normal mammary gland as well as in breast cancers, and elevation of serum CgA has been detected in patients with breast cancer. Our study was undertaken to evaluate the relationship between serum CgA levels and neuroendocrine features in breast cancer. In addition, we evaluated the expression of serum CgA in patients affected by breast cancer compared to controls and the relationship between serum CgA and tumor histology, extent of disease, lymph node status, tumor stage and serum CA 15.3 levels. We enrolled 266 patients with infiltrating ductal or lobular breast carcinoma and a group of 100 age-matched healthy women serving as controls. Serum CgA and CA 15.3 were assayed by specific immunoradiometric methods. The overall sensitivity of CgA and CA 15.3 was 0.06 and 0.34, respectively (chi2 19.1, p<0.0005). No relationship was found between serum levels of CgA and tumor histology, extent of disease, lymph node status or tumor stage while serum levels of CA 15.3 were strongly correlated with all these variables but tumor histology. No relationship was found between serum levels of CgA and CA 15.3. Immunostaining against CgA, CgB, NSE and synaptophysin was performed on primary tumor tissue of 14 serum CgA-positive and 24 serum CgA-negative patients and was negative in all cases. We also evaluated eight cases of pathologically-proven neuroendocrine breast cancer: only four and two of these showed positive CgA immunostaining and increased serum CgA concentration, respectively. In conclusion, serum CgA assay offers no additional information regarding the presence, the extent and the histology of breast cancer compared to the CA 15.3 assay. Moreover, serum CgA was not an accurate marker to identify or exclude the rare neuroendocrine differentiation of breast cancer. We therefore conclude that CgA is not useful as a serum marker in breast cancer.     

Prevention by delay: nonspecific immunity elicited by IL-12 hinders Her-2/neu mammary carcinogenesis in transgenic mice

As a natural consequence of the expression of the activated transforming rat Her-2/neu oncogene all mammary glands of female transgenic BALB/c (BALB-neuT) mice develop atypical epithelial hyperplasia which progresses to invasive carcinoma. A lobular carcinoma is palpable in all mammary glands of 33-week-old BALB-neuT mice. This progression is markedly delayed by systemic administration of IL-12. In a series of studies the best administration schedule, the lowest dose and the most effective administration time have been defined. The cellular and molecular mechanisms resulting in the delay of carcinogenesis have been established. By means of a series of downstream mediators IL-12 inhibits the angiogenic burst that goes along with the passage from preneoplastic to neoplastic and invasive lesions; it also recruits lymphoid cells in the mammary pad and activates their cytotoxicity towards neoplastic cells and newly formed vessels; and furthermore, it induces lymphoid cells to trigger antiangiogenic activities in neoplastic epithelial cells. Effective, low-dose and non-toxic IL-12 treatments may thus be envisaged as a possible option in the management of preneoplastic mammary lesions and in mammary cancer prevention.     

Absence of the CD1 molecule up-regulates antitumor activity induced by CpG oligodeoxynucleotides in mice

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene(-/-)/transgenic V(alpha)14/Vbeta8.2 mice that generate NKT cells; J(alpha)281(-/-) mice and CD1(-/-) mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in J(alpha)281(-/-) mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1(-/-) mice the inhibition was dramatic. The increased tumor inhibition in CD1(-/-) correlated with a significantly higher ratio of IFN-gamma-IL-4 production in response to CpG as compared with C57BL/6 and J(alpha)281(-/-) mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-gamma- and IL-4-producing cells revealed that the differences between CD1(-/-) and C57BL/6 in the production of these two cytokines were mainly due to CD3(+) T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of V(alpha)14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.     

Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

MADS-box protein complexes control carpel and ovule development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.