Natural polysaccharide-based gels for dairy food preservation.

The innovative packaging systems described in the present work, based on natural gels, have been shown to increase the shelf life of the Mozzarella cheese, without adding any chemical substance and without thermal procedures. Physical, physicochemical, microbiological, analytical, and mechanical analyses were used to monitor the quality of the cheese as a function of storage type and storage time. In particular, microbiological analysis confirmed that the characteristics of the Mozzarella cheese stored at 4 degrees C in gel are maintained for more than 15 d, whereas samples stored in the mother solution lost important characteristics after 5 d. A penetration test (texture) confirmed that the Mozzarella cheese preserved in the gel maintained mechanical properties similar to those of the fresh product, even after storage for 30 d at 4 degrees C.

Transferrin-like autocrine growth factor, derived from T-lymphoma cells, that inhibits normal T-cell proliferation.

Transferrin, the major iron-binding protein in the plasma of vertebrate species, is an essential growth factor for cells in serum free medium. We have established a cell line, Fr, from peripheral blood mononuclear cells of a patient affected by Sézary syndrome. Fr cells show a very immature antigenic phenotype, while constitutively bearing transferrin receptor on their surface. Furthermore the Fr line does not produce or respond to interleukin 2. Finally its conditioned medium contains both a growth stimulating activity for the Fr cell line and a factor which inhibits T-lymphocyte proliferation. We have identified a protein, produced in large amounts by Fr cells, which shares the immunological properties of human transferrin. Our data suggest that this transferrin-like factor can act as an autocrine growth factor for the producer cells and as an inhibitory factor for normal lymphocytes.

Behavior of some enzymatic activities in the serum of rabbits after tissue damage by CO2 laser radiation.

A comparative evaluation of the biochemical damage observed after tissue injury by high-power CO2 laser, scalpel, and electrical diathermy has been made in rabbits. The tissue injury consisted of surgery performed by making two perpendicular linear cuts on the muscle of the posterior thigh. At different times after surgery, enzymatic levels of creatine kinase, aspartate amino transferase, and lactate dehydrogenase were determined in the serum. Only creatine kinase showed an increase, and it was lower in both extent and dispersion in the animals treated with CO2 laser. The results suggest that CO2 laser radiation caused damage to muscular tissue which was moderately lower compared to that from scalpel or electrical diathermy.

Immunochemical evaluation of bovine beta-casein and its 1-28 phosphopeptide in cheese during ripening.

Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.

Detection of pH 4.6 insoluble beta-lactoglobulin in heat-treated milk and Mozzarella cheese.

Different protein aggregates including beta-lactoglobulin (beta lg) were detected in the pH 4.6 insoluble fraction recovered from actual heat-treated milk samples by gel electrophoresis and immunoblotting. A competitive enzyme-linked immunosorbent assay (ELISA) using anti-beta lg polyclonal antibodies was developed to analyze the beta lg partition in the protein fractions obtained upon acidification of both milk and Mozzarella cheese at pH 4.6. According to ELISA determinations, nearly 90% of the pH 4.6 soluble beta lg included in raw milk was found in the pH 4.6 insoluble fraction of ultrahigh temperature (UHT)-treated milk. As concerns Mozzarella cheese analysis, ELISA results indicated that about 36% of the total beta lg milk content was transferred from pasteurized milk to Mozzarella cheese, whereas less than 0.5% was transferred from raw milk. The pH 4.6 insoluble beta lg proved to be a suitable indicator of the intensity of the heat treatment applied to milk. The ELISA-based detection of this parameter was suggested for quality control of both drinking milk and raw milk cheese.

Expression of interleukins in L cells transfected with human DNA.

We have obtained, by transfection of mouse L cells with total human DNA, clones that constitutively secreted human interleukins IL1, IL2 and B-cell grown-factor activities, as assessed by specific biological assays. Southern analysis with IL2 and IL1 beta cDNAs confirmed the integration of the corresponding human genes in the genome of recipient mouse cells and showed their amplification and rearrangement. All the four IL2-secreting clones integrated in the mouse genome the human IL2 gene. Three out of the sixteen IL1-producing clones contained the IL1 beta gene. The IL1 activity secreted by the remaining clones exhibited a molecular mass of 17 kDa similar to that reported for mature human macrophage IL1. Our results demonstrate that DNA-mediated gene transfer may represent a suitable tool for the production of human growth and differentiation factors and the cloning of their genes.

Antipeptide Antibodies as Analytical Tools To Discriminate among Bovine alpha(s1)-Casein Components.

Polyclonal antibodies raised against synthetic peptides reproducing sequence stretches of bovine alpha(s1)-casein were used as probes to discriminate within the alpha(s1)-casein fraction of bovine milk and cheese. A minor alpha(s1)-casein component, selectively recognized by an antisera directed against the bovine 139-149 alpha(s1)-casein sequence, was found to be a C-terminally truncated alpha(s1)-casein form. This component coeluted with the main alpha(s1)- and alpha(s2)-casein by anion-exchange chromatography of whole casein, whereas by RP-HPLC it eluted with alpha(s2)-casein only. Similarly to the main alpha(s1)-casein, the C-terminally truncated form was hydrolyzed in vitro by chymosin and early in the cheese-making.

Mass spectrometry-based procedure for the identification of ovine casein heterogeneity.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HTPC into four major peaks. With ESI-MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography-ESI-MS allowed us to determine each fraction's composition by detecting thirteen alphas1-, eleven alphas2-, seven beta-, and three kappa-casein (CN) components. The alphas1-CN and alphas2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as kappa-CN and beta-CN. By CE at pH 2.5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLC-derived fractions. The separation of alphas1-CN and alphas2-CN proved to be excellent, with the exception of a co-migration of kappa0-CN with a minor alphas1-CN component and of a glycosylated kappa-CN for with low-phosphorylated = alphas1-CN and beta-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI-MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative alphas1-CN variants, the non-allelic alphas1-CN and alphas2-CN forms, dimeric kappa-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

Primary structure of ovine alpha s1-caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D.

The primary structures of ovine alpha s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141-148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for approximately 80% of the entire translated alpha s1-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89% degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in alpha s1-casein A, whereas Ser115 and Ser41 were phosphorylated by approximately 50 and approximately 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13-->Pro13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP12-->Ser12. A further substitution, SerP68-->Asn68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser64 and Ser66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.

Identification and characterization of a T cell growth inhibitory factor produced by K562 erythromyeloid cells.

Cells of the human erythroleukemia cell line K562 constitutively secrete a factor that inhibits human T lymphocyte proliferation induced via CD3/Ti. The factor, termed K-TIF (K562-derived T cell inhibitory factor) is produced in either the presence or absence of fetal calf serum in cultures of K562 cells and can be precipitated by 70% NH4SO4. Gel filtration chromatography on Superose 12 resin by FPLC showed that the inhibitory factor has a molecular weight of approximately 30-35 kDa. A protein of this size, metabolically labeled with [35S]methionine, specifically bound human peripheral blood mononuclear cells. Chromatofocusing with Mono P by FPLC (pH gradient 7.2-5) indicates that the inhibitory factor has an isoelectric point of 6.0-6.4.

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation.

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.