Detection of synthetic corticosteroids in bovine urine by chemiluminescence high-performance liquid chromatography.

The development of a black market of chemical cocktails for illegal growth promotion in food-producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v/v) and centrifuged. The upper layer was collected and Strata X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra- and inter-day assay, respectively, in all cases.     

On the uncertainty beneath the name Oithona similis Claus, 1866 (Copepoda,Cyclopoida)

The marine cyclopoid Oithona similis sensu lato Claus, 1866, is considered to be one of the most abundant and ubiquitous copepods in the world. However, its minimal original diagnosis and the unclear connection with its (subjective) senior synonym Oithona helgolandica Claus, 1863, may have caused frequent misidentification of the species. Consequently, it seems possible that several closely related but distinct forms are being named Oithona similis or Oithona helgolandica without explicit and accurate discrimination. Here the current situation concerning the correct assignment of the two species is revised, the morphological characters commonly used to identify and distinguish each species are summarized, and the nomenclatural implications of indiscriminately using these names in current taxonomic and ecological practice is considered. It is not intended to upset a long-accepted name in its accustomed meaning but certainly the opposite. “In pursuit of the maximum stability compatible with taxonomic freedom” (International Commission of Zoological Nomenclature), we consider that reassessment of the diagnostic characters of Oithona similis sensu stricto cannot be postponed much longer. While a consensus on taxonomy and nomenclatural matters can be attained, we strongly recommend specifically reporting the authority upon which the identification of either Oithona similis s.l. or Oithona helgolandica s.l. has been accomplished.     

Deoxyribonucleic Acid Repair in Bacillus subtilis: Development of Competent Cells into a Tester for Carcinogens

The development of competent transformed Bacillus subtilis into a tester system for carcinogens is described. Precocious or noninduced activation of SOS functions occur in competent cells. Thus, lower doses or concentrations of SOS inducing agents are needed to cause cell death due to indigenous prophage activation and lysis of bacteria. The two known defective prophages in B. subtilis enhance the sensitivity of competent cells to the carcinogens ultraviolet light, mitomycin C, and methyl methanesulfonate. However, these same cells have no enhanced sensitivity for the non-carcinogenic ethyl methanesulfonate or for nalidixic acid. Therefore, competent B. subtilis appear to be a sensitive tester for carcinogens.     

A vector representation for amino acid sequences

A scheme for representing amino acids as vectors in the plane is presented and justified. The two dimensions of the plane are size and hydrophobicity. The vector representation is then applied to generate a consensus sequence for some sets of homologous proteins. A figure of merit for the degree of homology of a set of sequences results from the analysis. Some other applications of the scheme are considered also. This work grew from ideaseeds planted by Margaret Dayhoff's work in theAtlas of Protein Structure and Sequence. It is with gratitude that I dedicate this paper to her memory.     

Requirement of chelating compounds for the growth of Corynebacterium glutamicum in synthetic media

Summary The effects of various iron-complexing substances on the growth of Corynebacterium glutamicum in synthetic medium were investigated. The data obtained indicate that C. glutamicum has an absolute requirement for the presence of an iron-complexing compound as a growth factor for rapid and abundabnt growth in synthetic media. This requirement can be met by adding low concentrations (10^–5 M) of certain dihydroxyphenols (catechol, protocatechuate) or relatively high concentratuions (0.1%) of citrate to the medium or by autoclaving a small amount of glucose together with other media components. The addition of catechol or protocatechuate to synthetic media has advantages over media preparation with citrate or autoclaved glucose. In the described synthetic broth supplemented with catechol or protocatechuate growth is largely independent of the iron content of the medium in a range between 0.037 and 0.37 mM.Offprint requests to: W. Liebl     

Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.     

Tim-3+ T-bet+ tumor-specific Th1 cells colocalize with and inhibit development and growth of murine neoplasms

Although T cells infiltrate many types of murine and human neoplasms, in many instances tumor-specific cytotoxicity is not observed. Strategies to stimulate CTL-mediated antitumor immunity have included in vitro stimulation and/or genetic engineering of T cells, followed by adoptive transfer into tumor-bearing hosts. In this model of B cell lymphoma in SJL/J mice, we used Tim-3(+) T-bet(+) Th1 cells to facilitate the development of tumor-specific CTL. Tumor-specific Th1 cell lines were polarized with IL-12 during in vitro stimulation and long term maintenance. As few as 5 million Tim-3(+) T-bet(+) Th1 cells enabled recipients to resist growth of malignant transplantable cells. In addition, similar numbers of Th1 cells injected into 2- to 3-mo-old mice inhibited development of the spontaneous primary lymphomas, which normally arise in 90% of aging mice. CFSE(+) Th1 cells colocalized with injected tumor cells in vivo and formed conjugates with the tumor cells within follicles, whereas in nontumor-challenged recipients the CFSE(+) Th1 cells localized only within the T cell zones of the spleen. These results provide evidence that adoptive immunotherapy with Tim-3(+) T-bet(+) tumor-specific Th1 cells can be used to induce host cytotoxic responses that inhibit the development and growth of neoplastic cells.