Assessment of the postoperative discomfort of intra-auricularly hypophysectomized rats

Rats subjected to hypophysectomy make up one of the largest groups of experimental animals in Europe, since there is a legal demand for batch testing of industrially produced growth hormones. To describe the clinical performance of rats having undergone hypophysectomy, animals were examined postoperatively by monitoring behaviour, body temperature and food intake. Behavioural changes were observed in rats that had only been anaesthetized, as well as in sham-operated rats, while no behavioural deviations could be shown in hypophysectomized rats. On the first day after surgery all rats had declining body temperature and food intake; and this change was not reversed by treatment with carprofen, buprenorphine or oxytetracycline. The mortality rate in rats treated with buprenorphine was increased, as was the mortality rate in rats hypophysectomized when weighing more than 100 g. As there seemed to be no differences whether methohexital or a combination of fentanyl, fluanison and midazolam was used, the latter anaesthesia is recommended due to its analgesic potential. For post-surgical analgesic treatment, carprofen is recommended rather than buprenorphine. At best, the use of hypophysectomized rats should be replaced in industrial batch testing by an existing in vitro method.     

Revision of<Emphasis Type="Italic">Cheliderpeton vranyi</Emphasis><Emphasis Type="SmallCaps">Fritsch</Emphasis>, 1877 (Amphibia,Temnospondyli) from the Lower Permian of Bohemia (Czech Republic)

The temnospondylCheliderpeton vranyi Fritsch, 1877 from the Lower Permian Ruprechtice horizon (Rotliegend) of the Intrasudetic Basin (Bohemia, Czech Republic) is redescribed. Many features of the skeleton permit a new understanding of the type species and consequently of the genus. Diagnostic characters are the narrow and round-tipped snout, straight to convex outline of the skull roof, narrow and long otic notch, posteriorly expanded quadratojugal, and the relatively wide and short rhombic interclavicle. The ilium with a short, expanded dorsal branch and the missing contact of nasal/maxilla are features shared with the related Upper PermianIntasuchus from Russia and the Eryopidae.Actinodon germanicus is a junior synonym ofCheliderpeton vranyi.      

Monthly patterns of testosterone and behavior in prospective fathers

The individual time patterns of salivary testosterone of adult healthy men, self-reported sexual behavior and their co-occurrence with regular weekly or monthly intervals were studied. Twenty-seven volunteer males (mean age 33 +/- 1 years) collected daily morning saliva over a period of 90 days. Evening questionnaires provided daily information on sexual activity. From the saliva, testosterone immunoreactive substances were determined using enzyme immunoassay. To detect events in which increases of testosterone were associated with sexual activity and at the same time controlling for regular internal patterns in men, data were analyzed using Theme software. First results indicated a varying number of complex nonrandom interaction patterns of testosterone with sexual activity, but also with weekly (i.e., Saturdays) and monthly intervals (i.e., 28-day full-moon intervals). The social context of the occurrence of specific pattern combinations was elaborated using parameters from the men's self-reported general life history profiles. Peak hormone levels occurred around weekends in the majority of the males. The 28-day monthly interval coincided with testosterone peaks only in those of the paired men who reported a current wish for children ("prospective fathers"), but not in unpaired men or in those who did not wish to have children with their current partner. Rather than representing a direct regular pattern of the male testosterone per se, the observed patterns suggest that men have the facultative potential to adjust their testosterone responses to their female partner's cycle. In line with the interactions between behavior and androgens observed in vertebrates in general, this study adds an example of the mutual character of hormone-behavior interactions and, thus, for the social context of testosterone patterns in human males.     

Hepatitis C virus nonstructural 5A protein and interferon resistance: a new model for testing the reliability of mutational analyses

Presumably due to the capability of the hepatitis C virus (HCV) to evade the antiviral effects of alpha interferon, treatment is ineffective in more than half of chronically genotype HCV type 1 (HCV-1)-infected patients. Previous approaches to correlate the number of amino acid mutations within regions of HCV nonstructural (NS)-5A protein with virologic treatment response provided conflicting results. In the present study, we developed a new mathematical model to investigate NS5A sequences of HCV-1-infected patients. The mean number of all mutations within the complete NS5A protein was significantly higher in virologic responders compared to nonresponders (P = 0.008 and P = 0.0001 for amino acid residues predicted on the surface of the NS5A protein). Differences did not achieve statistical significance for NS5A regions that are currently assumed to be functionally relevant (e.g., the interferon sensitivity-determining region, the RNA-activated protein kinase-binding domain, etc.). Analyses of smoothed mutational frequencies showed that the number of mutations in other NS5A regions correlated with virologic response. Such a correlation was observed for both genuine and randomly generated NS5A sequences. The existence of local accumulations of mutations within genuine NS5A isolates that truly correlated with treatment response was defined by a refined test procedure. Upon considering the predicted residue accessibility, we identified the main focus of mutations correlating with treatment response to be the sequence from amino acids 2350 to 2370. Thus, evaluation of NS5A mutations in correlation with treatment response is improved by consideration of functional and predicted conformational amino acid properties. As shown by simulations with randomly generated sequences, multiple analyses of simple counts of local NS5A amino acid mutations and correlation with treatment response are insufficient. For improvement of mutational analysis, a refined specific functional data test procedure is proposed.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

The importance of stereoselective determination of drugs in the clinical laboratory

About 56% of the drugs currently in use are chiral compounds, and 88% of these chiral synthetic drugs are used therapeutically as racemates. Only a few of these drugs qualify for a stereospecific determination in a clinical laboratory for therapeutic drug monitoring of patients. If the qualitative and quantitative pharmacokinetic and pharmacodynamic effects are similar, the enantiomers do not need to be separated. However, if the metabolism of the different stereoisomers is handled by different enzymes which are either polymorphic or can be induced or inhibited, and if their pharmacodynamic effects have differences either in strength or in quality, enantiospecific analysis is urgently needed. Unfortunately, there are many racemic drugs where the stereospecificity of the metabolism and/or the pharmacodynamic effects of the enantiomers is not known today. For these drugs, there is a great need for studies concentrating on these differences to improve treatment of the patients.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.     

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.