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The behavioral analysis of laboratory mice plays a key role in several medical and scientific research areas, such as biology, toxicology, pharmacology, and so on. Important information on mice behavior and their reaction to a particular stimulus is deduced from a careful analysis of their movements. Moreover, behavioral analysis of genetically modified mice allows obtaining important information about particular genes, phenotypes or drug effects. The techniques commonly adopted to support such analysis have many limitations, which make the related systems particularly ineffective. Currently, the engineering community is working to explore innovative identification and sensing technologies to develop new tracking systems able to guarantee benefits to animals' behavior analysis. This work presents a tracking solution based on passive Radio Frequency Identification Technology (RFID) in Ultra High Frequency (UHF) band. Much emphasis is given to the software component of the system, based on a Web-oriented solution, able to process the raw tracking data coming from a hardware system, and offer 2D and 3D tracking information as well as reports and dashboards about mice behavior. The system has been widely tested using laboratory mice and compared with an automated video-tracking software (i.e., EthoVision). The obtained results have demonstrated the effectiveness and reliability of the proposed solution, which is able to correctly detect the events occurring in the animals' cage, and to offer a complete and user-friendly tool to support researchers in behavioral analysis of laboratory mice.

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The makers have examined 455 fingerprint cards of accidents at biologic risk needed in a sanitary structure from the month of november 1995 to the month of december 1998. After they have described the protocol of the sanitary supervision applied, the procedure of the accidents, the qualifications, and the departments mainly interested by the subject of study event, they have pointed out the need of a greater vaccinable covering against the virus B, parvying attention to the "non responders" subjects, thinking also it's necessary bigger resources for the personnel, training and information.

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Alkaline (pI 8.6-7.5) and neutral (pI 7.0-6.0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values) = 0.25 and 0.29 nM respectively) compared with their neutral counterparts (ED50 values = 0.66 and 0.70 nM respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation. To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH. In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.

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To understand better why patients with TSH-secreting pituitary tumors exhibit variable degree of hyperthyroidism, we analyzed the various isoforms of TSH and alpha-subunit secreted by 4 TSH-secreting adenomas in primary culture. All patients had macrodenomas clinically associated with hyperthyroidism with normal to elevated TSH plasma levels. The in vivo molar alpha/TSH ratio ranged from 18.4 to 3.8. The hormone material secreted over 4 to 48 h in culture was separated by gel isoelectrofocusing, eluted and estimated by immunoassays. The release of free alpha-subunit was noticeably different among adenomas. Three tumors were found to release an homogeneous and acidic (pI = 5.4-4.5) species totally unrelated to the alpha-subunit dissociated from intrapituitary TSH (5 isoforms, pI = 8.8-5.8) while another was more heterogeneous (pI = 8.8, 8.4, 7.6, 6.8, 5.8, 5.4-4.5). Tumoral TSH exhibited at least six detectable isoforms (pI = 8.6, 8.3-8.0, 7.5, 7.0, 6.5, 6.0) very similar to those present in a purified intrapituitary hormone preparation. While intrapituitary TSH was composed of 70% of alkaline (pI = 8.6-7.5), 25% of neutral (pI = 7.0-6.0) and 5% (pI = 5.8-4.5) of acidic forms, these species were found to be more evenly distributed in adenomatous secretion (43%/42%/15%). The TSH-secreting tumors thus appeared to relase preferentially neutral and acidic forms of TSH than alkaline components but for one tumor, this ratio could be modified by chronic incubation with TRH. When assayed for their capacity to stimulate 3H-thymidine incorporation in FRTL-5 cells, neutral TSH appeared definitely less potent than the alkaline and acidic isohormones. Altogether, these data show that pituitary adenomas synthesize normal forms of TSH but release them in variable amount in the medium. When circulating in the blood, the ratio between active and inactive isoforms of TSH may thus be responsible for the variable stimulation of the thyroid gland observed in the patients.

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To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.

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Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.

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Isoforms of intrapituitary human TSH were separated by gel isoelectrofocusing, and their immunoreactivity analyzed by subsequent immunoblotting using polyclonal and monoclonal antibodies. Under these conditions, TSH polymorphism could be resolved as seven major isoforms (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) by both silver staining of the gels and binding to anti-TSH polyclonal antibodies. The distribution pattern of these forms appeared totally distinct from that of individual TSH alpha (pI 8.8, 8.4, 8.2, 7.6, 7.4, 6.8, 6.6, 5.8, and 5.4) and TSH beta (pI 8.7, 8.1, 7.2, 6.8, 6.2, and 5.8) subunits. While most anti-TSH polyclonal antibodies recognized neutral and alkaline isoforms of TSH (pI 8.6, 8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) through beta determinants, they displayed a variable potency to bind acidic forms of the hormone (pI 5.8, 5.5, 4.8, and 4.5), in contrast to anti-TSH alpha antisera, which enlighted the broadest spectrum of isoforms. Monoclonal antibodies of various specificities largely reproduced this distribution, indicating that at least five distinct epitopes are coexpressed in the neutral and alkaline forms of TSH, but only two are expressed in the acidic ones. All of the forms were found to induce cAMP production and stimulate growth of FRTL-5 rat thyroid cells, although neutral forms proved to be definitely less potent than the others. We therefore, conclude that TSH isoforms differ in the expression of both their immunoreactive and bioactive domains and that the bioactive/immunoreactive ratio is not an accurate index for the biopotency of the hormone.

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When wheat-germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220,000-Mr and 140,000-Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172,800, 155,000, 143,000, 133,800, 125,000 and 115,000. The use of affinity-purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220,000-Mr and 140,000-Mr subunits of wheat-germ RNA polymerase II and their breakdown products generated in the presence of trypsin. None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones. Furthermore, in transcription of a poly[d(A-T)] template, the catalytic activity of the proteolyzed form of wheat-germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template-directed catalysis of phosphodiester bond formation.

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To probe possible effects of carbohydrate chains in the conformation of pituitary glycoprotein hormones, two radiolabeled derivatives of human thyroid-stimulating hormone (hTSH), either partially deglycosylated in the beta-subunit or fully deglycosylated in both the alpha- and beta-subunits, were compared to the native hormone for binding to monoclonal as well as polyclonal antibodies. Monoclonal antibodies were screened for their ability to bind the intact hormone (anti-hTSH), hTSH and its free alpha-subunit (anti-alpha) or its free beta-subunit (anti-beta). A panel of 14 monoclonal antibodies directed against at least eight out of the 12 epitopes known to be present in the hormone was tested in solid-phase assays for their capacity to bind intact and deglycosylated forms of hTSH. All of them displayed identical recognition of native and partially deglycosylated 125I-hTSH. In contrast, binding of fully deglycosylated 125I-hTSH to anti-hTSH and anti-beta antibodies was dramatically lost while that of anti-alpha was preserved. This clearly indicates that most of the epitopes specific for subunit association as well as those present on the beta-subunit are glycosylation dependent. No alteration was found in antibody recognition following deglycosylation of free individual subunits, indicating that the carbohydrate effect can only occur in the combined dimer. Using polyclonal antisera raised against the International Reference Preparations, we found that the deglycosylated hormone could be bound by the anti-beta antiserum although at a much lower dilution than the native antigen, suggesting the presence of at least one glycosylation-independent epitope in the beta-subunit. Competitive binding assays revealed that deglycosylated hTSH is 5 times less immunoreactive toward the anti-beta compared to the anti-alpha antiserum. The current data thus demonstrate the presence of the glycosylation-independent epitopes in the alpha-subunit of hTSH and the localization of most of the glycosylation-dependent domains in the beta-subunit.

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