Browsing by Author "Mueller, C."

The murine calgizzarin like gene (Cal) encodes for a calcium binding protein, which belongs to the S100 family of EF-hand proteins. It is specifically expressed in Sertoli cells in the testis and its expression is down-regulated by unknown factor(s) from spermatocytes/spermatids. In this paper, we show by transfection of a fusion protein of green fluorescent protein and Cal protein into NIH3T3 cells, that the expression of Cal is restricted only in the cytoplasm of the cell. A differentially regulated cytoplasmic expression of the Cal in Sertoli cells during mouse development suggests that Cal might play an important role during spermatogenesis. In order to elucidate the function of the Cal protein in the spermatogenesis, we disrupted the Cal locus in mouse by homologous recombination. In our knockout mouse, we deleted exon 2 and exon 3 of the Cal gene and replaced them with a neomycin cassette, which resulted in a complete loss of the Cal transcript. Male and female Cal4(+/-) and Cal4(-/-) mice from genetic backgrounds C57BL/6J x 129X1/SvJ hybrid and 129X1/SvJ inbred exhibited normal phenotype and were fertile. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. The lack of the Cal protein also does not affect the parameters of sperm, as they are able to fertilize the oocytes in a competent manner, which is comparable to wild-type sperm. Collectively our results demonstrate that Cal is a nonessential protein and it does not play an important role in mouse spermatogenesis or in process of fertilization. (C) 2003 Wiley-Liss, Inc.

We report the cloning and characterization of the spermatogenesis associated 6 gene (Spata6) encoding a predicted protein of 488 amino acids. It exhibits similarity with the motor domain of kinesin related proteins and with the Caenorhabditis elegans neural calcium sensor protein (NCS-2). The gene encodes three mRNAs of similar to2.6, similar to1.8 and similar to1.2 kb. The expression of the 2.6 kb mRNA is detected at low levels in testis, ovary, thymus and placenta, while the 1.8 and 1.2 kb transcripts are exclusively expressed in testis. The 1.8 and 1.2 kb transcripts are specifically expressed in haploid germ cells. Data from in situ hybridization experiments suggested that mRNA expression of Spata6 in spermatids is higher than in spermatocytes and spermatogonia. RT-PCR analysis and whole mount in situ hybridization demonstrate that the Spata6 transcript is expressed during embryonic development and is localized in neural tube, somites and limb buds of mouse embryo. The Spata6 gene consists of 15 exons ranging in size between 40 and 596 bp. The 2.6 and 1.8 kb transcripts have different 5' untranslated sequences but have the same translational initiation site and therefore may encode the same protein with a predicted molecular weight of 49.7 kDa. The 1.2 kb transcript is derived from a proximal promoter between exons; 7 and 8, and contains a translation initiation codon AUG, which is in frame with initiator AUG codon of the 2.6 and 1.8 kb transcripts. Therefore, the 1.2 kb transcript may code for a truncated protein of 32 kDa. Western blot analysis with the antiserum raised against a synthetic peptide from the C-terminal of the deduced Spata6 protein detects only a single protein of 53 kDa in all tissues studied. The Spata6 gene was localized to chromosome 5, region q34-35 in the rat and to chromosome 1, region p32-35 in the human. In an effort to determine the function of Spata6, we inactivated the mouse gene in embryonic stem cells through homologous recombination. Although the heterozygous mutant cells were able to generate low coat colour chimeric mice, all chimeras did not transmit the targeted allele to their progeny suggesting that a high contribution of Spata6(+/-) cells lead to the lethality of the chimeric embryos.

The aim of this study was to compare the inheritance of the chromosomal SSLP markers with the inheritance of sperm movement parameters in order to map genes responsible for these quantitative traits (QTs). Chromosome 7 and 14 SSLP markers were tested to obtain the strain distribution pattern (SDP) for recombinant inbred (RI) strains developed from two progenitors, KE and CBA/Kw, which differ significantly in gamete quality. Sperm motility characteristics were determined using the computer assisted semen analysis (CASA) system. The Map manager software was used in order to assess linkage between the analyzed motility parameters and chromosome regions. The marker regression, interval mapping and permutation tests matched the QT loci of BCF with chromosome 7q11. The likelihood ratio statistic for this association was 18.1 with 79% of the total trait variance explained by QTL at this locus. These mapping results suggest that the BCF trait depends on the genetic factor(s) located in this region.

Animal and environmental care. health, product safety and consumer acceptance are factors that are becoming increasingly important for the assessment of pig housing. It can be foreseen that housing conditions will undergo a documentation and certification process as part of a quality assurance scheme according to international standards [ISO-9000 Series, Quality Management and Quality Assurance Standards, 1994]. As already implemented for the food processing industry, critical control points (CCP. based on the Hazard Analysis of Critical Control Point concept) have to be defined in order to objectively assess animal housing based on sound scientific data. The German working group "Animal Husbandry and Animal Welfare" of the German Society of Animal Production (DGFZ) has proposed a concept for the "Assessment of Animal Housing and Management according to Welfare and Environmental Criteria". Based on this concept, CCP and critical management points (CMP) have been developed for the categories health, behaviour, management and environmental impact. These criteria are intended to be used primarily by the farmer as an internal on farm assessment scheme. In the long run this concept of housing assessment through critical control and management points, measurable parameters and tolerance limits can be further developed and utilised by government agencies, consumer organisations and commodity groups that have an interest to evaluate, monitor and licence housing systems. (C) 2001 Elsevier Science B.V. All rights reserved.

Aim: To determine the value of multi-slice CT for the diagnosis of acute pulmonary embolism and an underlying venous thrombosis. Methods: 70 patients with clinically suspected acute pulmonary embolism were examined. Using multi-slice CT a combined examination of the pulmonary arteries and the veins of the lower limb, pe(vis and abdomen was performed. Only one single bo[us of 150 ml iopromid 300 was injected into a cubital vein with a flow of 4 ml/s. First, the pulmonary arteries were scanned with a slice thickness of 2.5 mm and a pitch of 1.5. On arrival of the contrast medium at the popliteal veins, indicated by bolus trakking, the veins of the lower limbs up to the end of the inferior vena cava were imaged using a slice thickness of 3.75 mm and a pitch of 1.5. The results could be compared with a ventilation-perfusion scan in 48 cases, with a Doppler ultrasound examination in 46 cases, and with a venography in 70 cases. Furthermore, the image quality of all arterial and Venous regions was subjectively assessed. Results: in all patients who underwent multi-slice CT the pulmonary arteries as well as the veins of the lower half of the body could be recorded completely. Regarding the pulmonary arteries the image quality showed excellent results for the central and segmental arteries. The region up to the 3rd division in subsegmental branches could be sufficiently judged. More peripherally, a diagnostic assessment was not possible. The image quality of the veins was excellent in all sections, except the calf, where a reliable diagnosis could not be made. The comparison with the other techniques confirmed the superiority of multi-slice CT concerning the central and segmental pulmonary arteries and the veins from the popliteal vein to the inferior vena cava. In contrast, peripheral pulmonary emboli can be detected more certainly in ventilation/perfusion scans. The veins of the calf can be evaluated more reliably with venography. Conclusion: Multi-slice CT proved to be an outstanding tool in the diagnosis of acute pulmonary embolism. The clinically suspected disease and a causing venous thrombosis can be detected in a fast and reliable way. At present, multi-slice CT is not suitable for the recognition of peripheral emboli. However, expected technical developments hold promise for future improvements.

Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD), In most cases, axonemal defects are present, i.e, PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD, However, to date it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals. To elucidate the role of the mouse dynein heavy chain 7 (MDHC7) gene, which encodes a component of the inner dynein arm, we have generated mice lacking this dynein heavy chain isoform, Both MDHC7(+/-) and MDHC7(-/-) mice are viable and show no malformations; however, homozygous males produce no offspring. In comparison to MDHC7(+/-) and wild-type mice the spermatozoa of MDHC7(-/-) mice revealed a dramatic reduced straight line velocity and progressive movement, resulting in the inability of MDHC7-deficient sperm to move from the uterus into the oviduct, Additionally, we measured the beat frequency of tracheal cilia and observed a decrease in the beat frequency of similar to 50% in MDHC7(-/-) mice. The reduction in both ciliary and flagellar motility is not correlated with any gross defects in the axonemal structure, The phenotype of MDHC7(-/-) mice is similar to that observed in some patients suffering from PCD, and our data strongly suggest that in some patients this disease could be due to mutations in the homologous human gene DNAH1 (HDHC7),

Background: Patients with systemic sclerosis (SSc) are endagered by tissue fibrosis and by microvasculopathy, with the latter caused by endothelial cell expansion/proliferation. SSc-associated fibrosis potentially results from mesenchymal transdifferentiation of endothelial cells. Early Endothelial Progenitor Cells (eEPCs) act proangiogenic under diverse conditions. Aim of the study was to analyze eEPC regeneration and mesenchymal transdifferentiation in patients with limited and diffuse SSs (lSSc and dSSc). Methods: Patients with both, lSSc and dSSc were included into the study. The following parameters were evaluated: eEPC numbers and regeneration, concentrations of vasomodulatory mediators, mesenchymal properties of blood-derived eEPC. Serum samples of healthy subjects and SS patients were used for stimulation of cultured human eEPC, subsequently followed by analysis of mesenchymal cell characteristics and mobility. Results: Twenty-nine patients were included into the study. Regenerative activity of blood-derived eEPCs did not differ between Controls and patients. Circulating eEPC were significantly lower in all patients with SSc, and in limited and diffuse SSc (lSSc/dSSc). Serum concentrations of promesenchymal TGF-b was elevated in all patients with SSc. Cultured mononuclear cells from SS patients displayed higher abundances of CD31 and of CD31 and aSMA combined. Finally, serum from SSc patients inhibited migration of cultured eEPCs and the cells showed lower sensitivity towards the endothelin antagonist Bosentan. Conclusions: The eEPC system, which represents an essential element of the endogenous vascular repair machinery is affected in SSc. The increased appearance of mesenchymal properties in eEPC may indicate that alterations of the cells potentially contribute to the accumulation of connective tissue and to vascular malfunction.

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants in the environment. They are present in body fluids associated with reproduction such as follicular fluid, seminal fluid and cervical mucus. Most PCB effects are mediated through the aryl hydrocarbon receptor, which is present in human spermatozoa. Additionally, PCBs may alter various biochemical reactions, such as calcium homeostasis. Therefore we investigated the effects of single non-ortho PCB 126, mono-ortho PCB 118, and di-ortho PCB 153 on human sperm motility, vitality, and calcium-dependent acrosome reaction (AR) in vitro. Human spermatozoa were either treated with different single PCB congeners or their combinations for 5 h at 37 degrees C (spontaneous AR), or for 16 h at room temperature and 4 degrees C (induced AR). Motility was measured after 5 h of incubation. Compared with the controls, PCB exposure had no effects on the percentage of living acrosome reacted spermatozoa, vitality, and motility. There was no difference in the inducibility of the AR between treatment groups and the respective controls after long term incubation. The PCB concentrations used were far higher than those found in cervical mucus or seminal fluid. In vivo effects of PCB congeners on human ejaculated spermatozoa seem to be unlikely. However these results cannot be easily transferred to the in vivo situation, because individual susceptibility has to be considered, and there is no information about synergistic or additive effects with other chemicals present in the male and female reproductive tract. (c) 2005 Elsevier Inc. All rights reserved.

Perioperative hypothermia is a common complication of general anesthesia and occurs in up to 50 % of patients during ear, nose and throat (ENT) surgery. In this prospective, randomized controlled study the hypothesis that a new conductive warming blanket (BarrierA (R) EasyWarmA (R), Molnlycke Health Care Erkrath, Germany) is better in reducing the incidence of perioperative hypothermia in ENT surgery than insulation with a conventional hospital duvet alone was tested. After approval of the local ethics committee and written informed consent 80 patients with a planned procedure time between 1 and 3 h were recruited. Anesthesia was induced and maintained using propofol, remifentanil and rocuronium and the core temperature was measured using an esophageal temperature probe. Patients in the study group were warmed at least 30 min prior to induction of anesthesia using the novel warming blanket (BarrierA (R) EasyWarmA (R)) and patients in the control group were insulated with a standard hospital duvet. Data were tested using Fisher's exact test, Student's t-test or the Mann-Whitney U-test as appropriate. Time-dependent changes in core temperature were evaluated using repeated measures analysis of variance (ANOVA) and post hoc Scheff,'s test. Results are expressed as mean +/- SD or as median and interquartile range (IQR) as appropriate. A p < 0.05 was considered to be statistically significant. The ANOVA did not identify a significantly higher core temperature in the study group at any time point. Furthermore, Fisher's exact test showed no differences in the incidence of intraoperative (12 out of 29 versus 10 out of 32 patients, p = 0.44) or postoperative hypothermia (12 out of 29 versus 9 out of 32 patients, p = 0.30) between the groups. No adverse effects were observed. In the studied patient group the new conductive warming blanket (BarrierA (R) EasyWarmA (R)) showed no superiority compared to conventional thermal insulation alone.

Objective is the use of phenoloxidases like laccases to activate the surfaces of fibres for making fibreboards without any adhesive. The concept of using lignin-oxidising enzymes for bonding applications is based on the reactivity of phenoxy radicals in the plant cell wall. A problem is that laccase can only oxidise the phenolic constituents of lignin, due to its lower oxidation potential, Therefore the use of appropriate low molecular-mass compounds (so-called mediators), in combination with laccase, makes this enzyme competent for the oxidation of nonphenolic substrates. The oxidised mediator can rely on an oxidation mechanism that is not available to the enzyme.

Purpose: To evaluate the detection rate of vascular stenosis in contrast-enhanced 3D MR angiography using a flow phantom. Material and methods: The examinations were performed on a 1.5 T whole body imaging system (Magnetom Symphony/Quantum) with 30 mT/m gradient field strength using a body-phased-array coil. Different 3D sequences (TR/TE/FA < 5 ms/< 2 ms/25 degrees) with slice thicknesses ranging from 0.67 to 1.25 mm were applied. A gelantine-filled plastic cylinder with PVC tubes of 8 mm diameter was used as a vascular phantom. The tubes had concentric and excentric stenoses (50-90%) of different lengths. For the detection of different vessel diameters another phantom with 0.3 - 8 mm silicon tubes was used. Both systems were flushed with a solution of Gd-DTPA (0.15 mmol/l) and saline at flow rates from 50 to 200 cm/s. The phantoms were positioned 0 degrees, 45 degrees, and 90 degrees towards the z-axis. Results: The degree of stenosis was under- and overestimated in less than 10 %. The sequence with the highest spatial resolution provided the best results. Detection and evaluation of tubes greater than or equal to 2 mm proved to be reliable. Conclusion: Contrast-enhanced 3D MR angiography provides an almost exact evaluation of the degree of stenosis in the phantom study. Evaluation of vessel diameters < 2 mm is not possible.

We investigated the spectroscopy of the first excited singlet electronic state S, of 2-phenylindene using both fluorescence excitation spectroscopy and resonantly enhanced multiphoton ionization spectroscopy. Moreover, we investigated the dynamics of the S, state by determining state-selective fluorescence lifetimes up to an excess energy of similar to 3400 cm(-1). Ab initio calculations were performed on the torsional potential energy curve and the equilibrium and transition state geometries and normal-mode frequencies of the first excited singlet state (S)1 on the CIS level of theory. Numerous vibronic transitions were assigned, especially those involving the torsional normal mode. The torsional potentials of the ground and first excited electronic states were simulated by matching the observed and calculated torsional frequency spacings in a least-squares fitting procedure. The simulated S, potential showed very good agreement with the ab initio potential calculated on the CIS/6-31G(d,p) level of theory. TDDFT energy corrections improved the match with the simulated S-1 torsional potential. The latter calculation yielded a torsional barrier of V-2 = 6708 cm(-1), and the simulation a barrier of V-2 = 6245 cm(-1). Ground-state normal-mode frequencies were calculated on the B3LYP/6-31G(d,p) level of theory, which were used to interpret the infrared spectrum, the FDS spectrum of the 0(0)(0) transition and hot bands of the FES spectrum. The fluorescence intensities of the v(49) overtone progression could reasonably be reproduced by considering the geometry changes upon electronic excitation predicted by the ab initio calculations. On the basis of the torsional potential calculations, it could be ruled Out that the uniform excess energy dependence of the fluorescence lifetimes is linked to the torsional barrier in the excited state. The rotational band contour simulation of the 0(0)(0) transition yielded rotational constants in close agreement to the ab initio values for both electronic states. Rotational coherence signals were obtained by polarization-analyzed, time-resolved measurements of the fluorescence decay of the 0(0)(0) transition. The simulation of these signals yielded corroborating evidence as to the quality of the ab initio calculated rotational constants of both states. The origin of the anomalous intensity discrepancy between the fluorescence excitation spectrum and the REMPI spectrum is discussed.

The testicular haploid expressed gene (Theg) encodes for a novel similar to42.0-kDa nuclear protein, which is specifically expressed in spermatid cells. Its expression is upregulated by some unknown factor(s) from Sertoli cells. To elucidate the function of Theg protein and its role in spermatogenesis, we disrupted the Theg locus in mouse by homologous recombination. For functional dissection of the domain structure of the Theg protein, two different knockout approaches were undertaken. In the first knockout mouse (Th14), the C-terminal region of the Theg protein (amino acids 137-376) was deleted. Both Th14(+/-) and Th14(-/-) mice from genetic backgrounds of C57BL/6J X 129X1/ SvJ hybrid and 129X1/SvJ inbred exhibited a normal phenotype and were fertile. The testes of Th14(-/-) mice were smaller than those of Th14(+/-) and Th14(+/+) mice; however, the testicular morphology and the properties of sperm, including morphology and motility, from Th14(-/-) mice were similar to those of Th14(+/-) and Th14(+/+) mice. These results demonstrate that the C-terminal region of Theg (amino acids 137-376) does not play an important role in progression of spermatogenesis. In the second knockout mouse (Th15), we deleted the N-terminal domain of the Theg protein, which resulted in complete loss of Theg transcripts. Both Th15(+/-) and Th15(-/-) mice from genetic backgrounds C57BL/6J x 129X1/SvJ hybrid, C3H/J congenic, and 129X1/SvJ inbred appeared normal and were fertile, with no gross abnormalities detected in testicular morphology or sperm properties. Our results from both knockout mouse model systems clearly illustrate that Theg is not essential for spermatogenesis in the mouse.