UVB radiation induced effects on cells studied by FTIR spectroscopy

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm²) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spetroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698–705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.     

Agreement Between Deoxyribonucleic Acid Base Composition and Taxometric Classification of Gram-Positive Cocci.

Silvestri, L. G. (Università Statale, Milan, Italy), and L. R. Hill. Agreement between deoxyribonucleic acid base composition and taxometric classification of gram-positive cocci. J. Bacteriol. 90:136-140. 1965.-It had been previously proposed, from taxometric analyses, that gram-positive, catalase-positive cocci be divided into two subgroups. Thirteen strains, representative of both subgroups, were examined for deoxyribonucleic acid (DNA) base composition, determined from melting temperatures. Per cent GC (guanine + cytosine/total bases) values fell into two groups: 30.8 to 36.5% GC and 69 to 75% GC. Strains with low per cent GC values belonged to the Staphylococcus aureus-S. saprophyticus-S. lactis taxometric subgroups, and those with high per cent GC values belonged to the S. roseus-S. afermentans subgroup. The hypothetical nature of any classification is emphasized, and, in the present work, the hypothesis derived from taxometric analyses of division into two subgroups is confirmed by the study of DNA base ratios. The two subgroups correspond, respectively, to the genera Staphylococcus and Micrococcus.     

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.     

Effect of Kainic Acid, Glutamate, and Aspartate on CO2 Production by Goldfish Tectal Slices

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.     

Recognition of human minor alloantigen(s) by cytotoxic lymphocytes in vitro

Lymphocytes from an extensively transfused patient with aplastic anemia were induced to cytotoxicity against target cells from several HLA-matched siblings by in vitro stimulation with allogeneic cells. Effective stimulating cells shared HLA-B7 with the patient, but not all B7 individuals were effective. An additional factor, which was found to segregate in both the patient's and an unrelated sibship, was also necessary. Segregation of this minor alloantigen, W, was also revealed among the patient's HLA-matched sibs by differential susceptibility to lysis by effectors from the patient. The ratio of six positive to four negative siblings suggests that the antigen difference might be coded by a single locus. Lymphocytes from a normal sib, who like the patient is lacking the minor antigen, could not be induced to cytotoxicity against positive targets. Thus in vivo sensitization of the donor of the responding cells appears to be necessary for the demonstration of the cytotoxic response to the minor antigen in vitro. No correlation was observed between the segregation pattern of W and of known blood group antigens, and no cytotoxic antibody to W was detected in the patient's serum in several trials.Abbreviations used in this paper MHC major histocompatibility complex - Tc thymus dependent cells capable of mediating cytotoxicity in the absence of Immoral antibody - GVHD graft versus host disease - CML cell mediated lympholysis - MLC unidirectional mixed lymphocyte culture - ADCC antibody-dependent cell mediated cytotoxicity     

The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin

The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation, and is downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2(MASK)), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2(MASK) shows no cleavage activity and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2(MASK). The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.     

Exploring encapsulation strategies as a protective mechanism to avoid amensalism in mixed populations of Pseudomonas taetrolens and Lactobacillus casei

Bioprocess and Biosystems Engineering - Pseudomonas taetrolens constitutes an efficient platform for the biosynthesis of lactobionic acid, a potentially prebiotic compound. Unfortunately, an...     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.