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The La-like S2 state of 4-(1-piperidino)-benzonitrile and the corresponding 3,5-dimethyl-piperidino derivative was excited at 270 nm in the gas phase at 130 °C. Nonresonant multiphoton ionization at infrared wavelengths (810 and 1300 nm) with mass-selective detection of the ion yields then probed the subsequent processes. Two groups of time constants were found: in the range

Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.

In utero, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in C57BL/6J mice. Androgens, mesenchymal-epithelial interactions, and growth factor expression all have roles in initiating and regulating development and growth of the prostate. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), both of which bind the EGF receptor (EGFR), are expressed in human and rodent developing prostate. This study examines the influence of null expression of EGF and/or TGF-alpha on prostatic bud development and on the ability of TCDD to inhibit prostatic budding. Growth factor knockout (-/-) and wild-type (WT) mice were exposed either to vehicle or to TCDD (0, 0.2, 1, 5, 10, 50, 100, or 150 microg/kg) on gestation day (GD) 12. The number of anterior, dorsal, and lateral prostatic buds (ADLB) and ventral buds (VB) were counted on GD 17.5. Control WT and EGF (-/-) fetuses had similar numbers of ADLB and VB. In control TGF-alpha (-/-) fetuses, the number of ADLBs was higher relative to the C57BL/6J. Control EGF + TGF-alpha (-/-) had poor bud outgrowth, especially in the ADL region. TCDD induced a dose-related decrease in bud formation in all strains with the formation of VBs being more sensitive than ADLBs. The severity of the response depended on growth factor expression, with the most severe effects on VBs in the EGF (-/-) and on ADLBs in the EGF + TGF-alpha (-/-) fetuses. TGF-alpha (-/-) and C57BL/6J fetuses responded to TCDD similarly. In conclusion, EGF and TGF-alpha expression are important for the formation of ADLBs and VBs, and expression of EGF and TGF-alpha affects the ability of TCDD to inhibit prostatic bud formation in a region-specific manner.

Cholesterol is necessary for the proper growth and development of the fetus. Consequently, disruptions in cholesterol biosynthesis lead to abnormal fetal development. It has been shown that in cells exposed to polyunsaturated fatty acids (PUFA), the expressions of genes and activities of enzymes involved in cholesterol synthesis are reduced. Similarly, we found that adult male hamsters fed PUFA-enriched diets had an ≈60% reduction in in vivo hepatic sterol synthesis rates. If fetal tissues respond to PUFA in the same manner as do adult livers, then maternal dietary PUFA could lead to a reduction in fetal sterol synthesis rates and possibly abnormal development. To investigate the impact of maternal dietary fatty acids on fetal sterol synthesis rates, female hamsters were fed diets enriched in various fatty acids before and throughout gestation. In vivo sterol synthesis rates were measured in fetuses at mid- and late gestation. At both gestational stages, dietary PUFA had no effect on fetal sterol synthesis rates. This lack of effect was not a consequence of a lack of PUFA enrichment in fetal fatty acids or the lack of PUFA receptor expression in the fetus. We hypothesize that the fetus may experience a dysregulation of sterol synthesis as the result of the fetus being in a negative sterol balance; the PUFA-induced suppression of sterol synthesis in the adult male hamster liver was ablated by creating a net negative sterol balance across the adult hepatocyte.

The placental transport of various compounds, such as glucose and fatty acids, has been well studied. However, the transport of cholesterol, a sterol essential for proper fetal development, remains undefined in the placenta. Therefore, the purpose of these studies was to examine the transport of cholesterol across a placental monolayer and its uptake by various cholesterol acceptors. BeWo cells, which originated from a human choriocarcinoma, were grown on transwells for 3 days to form a confluent monolayer. The apical side of the cells was radiolabeled with either free cholesterol or LDL cholesteryl ester. After 24 h, the radiolabel was removed and cholesterol acceptors were added to the basolateral chamber. Cholesterol was found to be taken up by the apical surface of the placental monolayer, transported to the basolateral surface of the cell, and effluxed to fetal human serum, fetal HDL, or phospholipid vesicles, but not to apolipoprotein A-I. In addition, increasing the cellular cholesterol concentration further increased the amount of cholesterol transported to the basolateral acceptors. These are the first studies to demonstrate the movement of cholesterol across a placental cell from the maternal circulation (apical side) to the fetal circulation (basolateral side).

4-Dimethylamino-benzonitrile was excited in the gas phase at 270 nm into the S 2 (L a ) state and probed by femtosecond time-resolved photoionization at 2 μm. Coherent oscillations were detected in the transient ion signal. We claim that from S 2 , the molecule relaxes through a conical intersection and, going backwards along the charge-transfer (CT) reaction coordinate, enters into the S 1 (L b ) well, where it vibrates along the amino-group twist and inversion. Probably mainly the last slope stimulates these oscillations. We conclude that the conical intersection – and hence also the CT state – is displaced from the L b minimum in a direction containing the twist and inversion as components and that inversion is part of the nonadiabatic coupling vector.

Within the reproductive system, oestrogenic stimulation of uterine and pituitary tissue typically causes a proliferative response accompanied by an angiogenic induction of new blood vessels from existing ones, thereby providing nutrients and oxygen to the growing tissue. The pro-oestrogenic pesticide methoxychlor (MXC), however, has shown a differential effect on proliferative activity. An increase in uterine growth is present, while the pituitary undergoes a decrease in size, even though the effect is accompanied by a characteristic oestrogen-induced elevation in pituitary prolactin concentration. The focus of the current study was whether the observed differences in tissue growth between uterus and pituitary in response to MXC administration were paralleled by a corresponding disparity in the expression of those growth factors (members of the vascular endothelial growth factor (VEGF) and angiopoietin families and their receptors) that are involved in the angiogenic cascade. Ovariectomized adult Sprague-Dawley female rats were administered MXC (0-200 mg/kg, oral) for 1 or 3 weeks. Immunohistochemical staining of uteri and pituitaries was performed under strictly controlled conditions for VEGF and its receptor VEGFR2, Angiopoietin-1 (Ang1) and angiopoietin-2 and their tyrosine kinase receptor Tie2, and platelet endothelial adhesion factor (as an index of vascularity). Image acquisition and densitometric assessments of staining intensity were conducted under blind conditions. The results showed uterine MXC-induced increases in the expression of VEGFR2 and Ang1, changes consistent with a normal proliferative response to oestrogenic stimulation. For VEGF, staining tended to be most pronounced in the stromal region, although there did not appear to be a progressive increase with dose. VEGFR2 expression showed significant dose-related trends in luminal and glandular epithelia by 1 week. Similar effects at 1 week were evident for Ang1 in glandular epithelium. In the anterior pituitary, a dose-related increase in VEGF was present for the 1 and 3 week treatments, and the number of pituitary vessels per unit area was also increased after 3 weeks. The effects indicate that even though the insecticide has not been found to cause an augmentation in pituitary growth, a dose-related rise in the expression of at least one principal angiogenic factor is present that may be associated with an increase in vascular density.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate (CP) and hydronephrosis (HN) in mice. The etiology of these defects involves hyperproliferation of epithelial cells of the secondary palatal shelf and ureter, respectively. These effects correlate with altered expression of the epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). In this study, the developmental toxicity of TCDD was examined in EGF, TGF-alpha, and double EGF + TGF-alpha knockout (-/-) and wild type (WT) mice. The influence of background genetics in responsiveness to TCDD was examined using liver 7-ethoxyresorufin-O-deethylase (EROD) activity. Animals were dosed by gavage with 0, 0.2, 1, 5, 24, 50, 100, or 150 micro g TCDD/kg (5 ml/kg) body weight on gestation day 12. The mixed genetic background of WT, EGF (-/-), and EGF + TGF-alpha (-/-) made these mice less responsive to TCDD relative to C57BL/6J and TGF-alpha (-/-), which have a C57BL background. These results show that EGF and TGF-alpha are not required for response to TCDD; however, the specific ligand available to bind EGFR affects the responsiveness to TCDD. EGF (-/-) mice are less responsive for CP, but more sensitive to HN. TGF-alpha (-/-) mice were similar to WT in sensitivity for induction of CP and HN. The responses of EGF + TGF-alpha (-/-) mice were like the WT except at higher doses where sensitivity to CP increased, suggesting that the responses may be mediated by alternative ligands for EGFR that are not functional equivalents of EGF or TGF-alpha. In conclusion, the EGFR pathway is mechanistically important in responses of the embryo to TCDD. Specific ligands confer sensitivity or resistance that are target tissue-dependent.

We propose that gene expression changes in accessible tissues such as blood often reflect those in inaccessible tissues, thus offering a convenient biomonitoring method to provide insight into the effects of environmental toxicants on such tissues. In this pilot study, gene expression changes in peripheral blood leukocytes (PBL) were compared to those in the uteri of adult rats to identify genes that were altered in both tissues following estradiol treatment. Ovariectomized rats were treated with either 17beta-estradiol or vehicle control (corn oil) for 3 days. PBL and uterine RNAs were hybridized to arrays containing 1185 genes. One hundred and ninety three genes were expressed in common between the PBL and uterus. Eighteen were changed significantly in both tissues, 9 of which were treatment- but not tissue-specific (e.g., jun-D, phospholipase A2, thymidine kinase). These results demonstrate that many genes are coexpressed between PBL and uterus, and that some are coregulated by estradiol. Given the limited number of genes examined in this study and the estimated size of other mammalian genomes, we conclude that many more genes will also be coregulated and suggest that accessible tissues such as PBL can serve as surrogate tissues for observing gene expression changes in inaccessible target tissues.

We have isolated and functionally characterized an additional murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane transport proteins from mouse liver. The 3.6 kb cDNA insert contains an open reading frame of 2010 bp coding for a 670 amino acid protein. Based on its amino acid identity of 88% to the rat Oatp2, it is considered the mouse Oatp2 orthologue. Functional expression in Xenopus laevis oocytes demonstrated that mouse Oatp2 transports several general Oatp substrates such as estrone-3-sulfate, dehydroepiandrosterone sulfate (DHEAS), ouabain and BQ-123 but hardly any taurocholate nor rocuronium or deltorphin II. The high-affinity rat Oatp2 substrate digoxin is transported with a rather low affinity with an apparent Km value of 5.7 μM. Bromosulfophthalein (BSP), a substrate not transported by the rat Oatp2, is transported very well by mouse Oatp2. Northern blot analysis demonstrated a predominant expression in the liver with additional signals in kidney and brain. Using fluorescence in situ hybridization, the Oatp2 gene (gene symbol Slc21a5) was mapped to chromosome 6G1–G3.

The L(a)-like S2 state (2A) of 4-(dimethylamino)benzonitrile was pumped at 267 nm in the gas phase at 130 degrees C. Nonresonant multiphoton ionization at 800 nm with mass-selective detection then probed the subsequent processes. Whereas ionization at the Franck-Condon geometry only gave rise to the parent ion, fragmentation increased on motion towards the charge-transfer (CT) state. This useful difference is ascribed to a geometry-dependent resonance in the ion. The time constants found are interpreted by ultrafast (approximately 68 fs) relaxation through a conical intersection to both the CT and the L(b)-type S1 state (1B). Then the population equilibrates between these two states within 1 ps. From there the molecule relaxes within 90 ps to a lower excited state which can only be a triplet state (T(n)) and then decomposes within 300 ps. Previous experiments either investigated only 1B --CT relaxation-which does not take place in the gas phase or nonpolar solvents for energetic reasons--or, starting from S2 excitation, typically had insufficient time resolution (1 ps) to detect the temporary charge transfer. Only recently temporary population of the CT state was found in a nonpolar solvent (Kwok et al., J. Phys. Chem. A. 2000, 104, 4188), a result fully consistent with our mechanism. We also show that S2 --S1 relaxation does not occur vertically but involves an intermediate strong geometrical distortion, passing through a conical intersection.

In female rats, in uteroexposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during critical periods of organogenesis causes a permanent thread of tissue, consisting of a core of mesenchyme surrounded by keratinized epithelia, across the vaginal opening. The objective of the current study was to determine the earliest time after exposure to TCDD during fetal development that morphological changes in the development of the lower reproductive tract could be detected. In addition, the spatio-temporal expression of several growth factors within the developing reproductive tract was investigated to provide insight into the mechanism of action involved in TCDD-induced vaginal thread formation. Pregnant rats received a single oral dose of 1.0 microg TCDD/kg on gestation day (GD) 15. Dams were sacrificed on GD 17, 18, 19, and 21 and individual reproductive tracts were isolated from female fetuses. As early as GD 18, TCDD produced distinct abnormalities in the female reproductive tract. The width of mesenchyme separating the Mullerian ducts was significantly greater in TCDD-exposed female GD 18 and 19 fetuses and the zone of unfused Mullerian ducts was substantially increased on GD 19 and 21. TCDD induced alterations within the developing reproductive tract in the subcellular and temporal expression of transforming growth factor-beta3 (TGF-beta3) and epidermal growth factor receptor (EGFR). DNA array analysis suggested effects on several genes expressed on GD 18 and 19.

Excitation of norbornadiene (bicyclo[2.2.1]hepta-2,5-diene) at 200 nm populates two states in parallel, the second pi(pi*) state and a Rydberg state. We monitored both populations by transient nonresonant ionization. From the pi(pi*) state the molecule relaxes in consecutive steps with time constants 5, 31 and 55 fs down to the ground-state surface, whereas the Rydberg population merges to the other path on the pi(pi*) surface within 420 fs. The relaxation steps are discussed in terms of conical intersections (CoIns) between different surfaces Information on them is inferred from known spectroscopy and, for the last CoIn, from published calculations on Dewar benzene--prismane conversion and on ethylene photodimerization for which norbornadiene with its two nonconjugated double bonds is a model. The calculation predicts symmetry breaking for this CoIn, the two ethylenes forming a rhombus Although this distortion is hindered in norbornadiene by ring strain, this CoIn seems easily accessible as indicated by the short time (55 fs) found for passing through it.

To perform an interlaboratory comparison of FISH chromosome painting and to study the time-course of translocations and dicentrics in three accident victims exposed to radiation. Also, to use the data in the validation of the FISH technique as a retrospective dosimeter.Twelve blood samples were collected during 4 years from three subjects exposed to radiation in an accident in Estonia in 1994 involving gamma-radiation from a 137Cs source. Two of the subjects were exposed during approximately 7 h, both receiving a protracted dose of about 1 Gy and also localized exposure. The third subject received a protracted whole-body dose of 2.7 Gy during 4 weeks as well as a short-term partial-body dose. Preparations from 48-h metaphase cultures were painted by the FISH technique using routine methods and probe cocktails in four laboratories. Samples from each subject were analysed in two different laboratories that used different combinations of whole chromosome probes. The PAINT nomenclature was applied when recording chromosome aberrations.The intercomparison of FISH analysis data showed reasonable similarities between laboratories, the largest discrepancy being 21% in the frequency of two-way translocations in subject 3. Half-time calculations, based on combined data sets from two laboratories, showed that dicentrics decreased rapidly with half-times of approximately 2 years. In all cases, the initial dicentric yields were lower than the initial translocation yields. During the 4-year follow-up, the frequencies of all translocations in cells containing only simple rearrangements fell on average to about 65% of their initial value. Two-way translocations were slightly more persistent than all translocations. The average half-time was about 8 years for two-way translocations and around 6 years for all translocations. Cells containing complex rearrangements were few in number and they disappeared with time. In general, the inclusion of complex cells caused a more rapid fall in aberration yield.In general, the results imply that relatively consistent scoring data were obtained with different chromosome painting protocols. They also support the idea that the reduction of translocations with time is associated with partial-body irradiation.

The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 × 180 µm(2) and two Gaussian-type dose distributions of σ = 260 µm and σ = 520 µm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.

Sudden cardiac death (SCD) in young athletes during physical stress is a rare event with an incidence of 1-3 deaths per 100,000 athletes per year. A coronary anomaly is the second most common cause of death following hypertrophic cardiomyopathy. Symptomatic prodromes occur in 20% of cases prior to the SCD event. This case report describes a 35-year-old male who collapsed near the finishing line of a half marathon run. Despite immediate resuscitation attempts and initial return of spontaneous circulation (ROSC), a pulseless electrical activity (PEA) followed and the patient died 1 h after arrival in the resuscitation unit. The autopsy revealed an anomalous left coronary artery (ALCA), which can lead to ischemia of the respective heart muscles under severe stress.

The aim of the study was to examine risks, implications and outcomes of coronary sinus (CS) lead extraction in patients with infections of cardiac resynchronization therapy (CRT) systems.The study included 40 (65.5 ± 11.1 years; 80% male) transvenous CS lead extraction procedures performed between 2000-2011. Nine (22.5%) patients suffered from infection and included one sepsis (11.1%), two (22.2%) of lead and valve endocarditis, and four (44.4%) cases of pocket infection. CS lead extraction in the infection subgroup was performed between 14 days and more than five years after the last CIED-related surgical procedure.Totally 42 CS and 35 non-CS leads were extracted. Leads extracted in the infection subgroup were significantly longer in situ (49.7 ± 30.7 months) compared to the non-infection subgroup (19.2 ± 28.6 months). Extraction in infected patients required more aggressive methods and longer exposure to radiation than non-infected. Procedural success without major complications was achieved in all patients. Minor post-procedural complications occurred in four (44.4%) of the infected and one (3.2%) of the non-infected patients and were surgical-related in three cases. Overall hospitalization times were significantly longer for the infection than for the non-infection subgroup (21.4 ± 15 versus 9.6 ± 6.9 days).Our results support the concept of complete CIED-system removal in CIED-associated infection, regardless of whether or not infection appears to be limited to the generator pocket site, despite risk of heart failure, patient frailty and a high level of comorbidity. An interdisciplinary approach encompassing appropriate diagnostic, procedural and safety standards allows CS lead extraction in this high-risk subpopulation to be performed with excellent outcomes and low complication rates.

Magnetic nanoparticles are highly desirable for biomedical research and treatment of cancer especially when combined with hyperthermia. The efficacy of nanoparticle-based therapies could be improved by generating radioactive nanoparticles with a convenient decay time and which simultaneously have the capability to be used for locally confined heating. The core–shell morphology of such novel nanoparticles presented in this work involves a polysilico-tungstate molecule of the polyoxometalate family as a precursor coating material, which transforms into an amorphous tungsten oxide coating upon annealing of the FePt core–shell nanoparticles. The content of tungsten atoms in the nanoparticle shell is neutron activated using cold neutrons at the Heinz Maier-Leibnitz (FRMII) neutron facility and thereby transformed into the radioisotope W-187. The sizeable natural abundance of 28% for the W-186 precursor isotope, a radiopharmaceutically advantageous gamma–beta ratio of γ β ≈ 30% and a range of approximately 1 mm in biological tissue for the 1.3 MeV β-radiation are promising features of the nanoparticles' potential for cancer therapy. Moreover, a high temperature annealing treatment enhances the magnetic moment of nanoparticles in such a way that a magnetic heating effect of several degrees Celsius in liquid suspension – a prerequisite for hyperthermia treatment of cancer – was observed. A rise in temperature of approximately 3 °C in aqueous suspension is shown for a moderate nanoparticle concentration of 0.5 mg/ml after 15 min in an 831 kHz high-frequency alternating magnetic field of 250 Gauss field strength (25 mT). The biocompatibility based on a low cytotoxicity in the non-neutron-activated state in combination with the hydrophilic nature of the tungsten oxide shell makes the coated magnetic FePt nanoparticles ideal candidates for advanced radiopharmaceutical applications.

The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (∼100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons (P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

By exciting norbornene (bicyclo[2,2,1]heptene) in the gas phase at 200 nm and probing it by nonresonant multiphoton ionization with mass-selective detection of the ion yields, we found five time constants τi (30, 60, 52, 800 fs, and 92 ps). τ1−τ3 represent traveling times through observation windows on excited surfaces, whereas τ4 and τ5 reflect processes in the hot ground state. We assign τ1 to departure from the Franck−Condon region of the ππ* state populated at 200 nm, and τ2 to traveling along the ππ* surface. To explain the subsequent window (τ3), we suggest that the ππ* surface is crossed by the zwitterionic state Z and that the two known carbene products form from this state. The carbenes then stabilize on the ground-state surface within τ4 and τ5 to form the two known photoproducts. − The fragmentation pattern showed that cyclopentadiene was not detected until at least 600 ps. Hence the retro-Diels−Alder reaction, known to be allowed in the ground state, probably takes longer.

Flooding of soil occurs all over the world in areas of crop production. The resulting lack of available oxygen in the soil can hinder the establishment of field crops such as wheat (Triticum aestivum L.) and thus lead to a decrease in yield. The current study compares the tolerance of spelt (Triticum spelta L.) and wheat to wet and cold stress during germination and early growth. Two specific traits seem to be related to the superior early flooding tolerance of spelt: (1) fast growth of the coleoptile, which reaches the soil surface rapidly, thus reducing the time span of hypoxia sensitivity, independent of temperatures between 10 and 20 °C; (2) the improved physiological adaptation for coleoptile growth under hypoxia is probably due to lower oxygen consumption between germination and emergence. Because of this high level of flooding tolerance, spelt would also be a good source for breeding for stress tolerance in wheat. These findings will help to detect quantitative trait loci (QTL) for characters such as growth rate, flooding tolerance and oxygen consumption in future studies. A marker-assisted introgression of spelt into wheat is necessary to avoid combination with undesirable agronomic traits of spelt. Agronomische und physiologische Untersuchungen zur Kalte- und Ueberflutungstoleranz von Dinkel (Triticum spelta L.) und Weizen (Triticum aestivum L.) Uberflutung kommt praktisch uberall vor, wo Pflanzen angebaut werden. Der Mangel an Sauerstoff im Boden kann den Feldaufgang von Kulturen wie Weizen verhindern und dessen Ertrag reduzieren. Diese Studie vergleicht die Toleranzreaktionen von Dinkel und Weizen bezuglich Kalte und Nasse wahrend Keimung und Jungendentwicklung. Zwei spezifische Eigenschaften scheinen fur die Ueberflutungstoleranz in fruhen Stadien verantwortlich zu sein: (1) schnelles Wachstum der Koleoptilen, schnellere Erreichung der Bodenoberflache, welche die Zeit der hohen Sensibilitat gegenuber Hypoxia, unabhangig von Temperaturen zwischen 10 und 20 °C, verkurzt; (2) Die verbesserte physiologische Anpassung des Koleoptilenwachstums unter Hypoxia ist wahrscheinlich vom geringeren Sauerstoffkonsum zwischen Keimung und Aufgang verursacht. Wegen seiner besseren Ueberflutungstoleranz ist Dinkel eine interessante genetische Ressource fur die Stresstoleranzzuchtung von Weizen. Diese Resultate bilden die Grundlage fur die Ermittlung von `quantitative trait loci' (QTL) fur Wachstumsrate, Ueberflutungstoleranz oder Sauerstoffkonsum. Ein markergestutzter Transfer von Dinkeleigengeschaften in Weizen ist notwendig, um ungunstige Kombinationen mit unerwunschten Dinkeleigengeschaften zu vermeiden.

By exciting 1,3-butadiene in the gas phase at 200 nm and probing it by nonresonant multiphoton ionization, we found an internal-conversion time of about 110 fs, within which one can distinguish five consecutive phases and time constants. The first two times (10 and 44 fs) are assigned to two phases on the initially excited 1Bu surface and departure from it to the `dark' 2Ag state. The latter is then left in a remarkably short time (18 fs). Obviously the wave packet is very early (already in the 1Bu state) accelerated towards the 2Ag/1Ag conical intersection (CI). There follow two more short processes (both 18 fs) on the ground-state surface and then single-bond isomerization, taking 270 fs at the given excess energy (6.2 eV).

Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20--24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.

We investigated M(CO)6 (M = Cr, Mo, W), Fe(CO)5, Ni(CO)4 and M2(CO)10(M = Mn, Re) by femtosecond spectroscopy. The molecules were pumped by one photon at 267 nm and then probed by multiphoton ionization at 800 nm and mass selective detection of the resulting parent and fragment ions. Whereas it was previously believed that such metal carbonyls are excited to a repulsive potential, leading to elimination of one or several CO, we find that only one CO is photochemically split off in times typically below 100 fs and that this is already a multistep process involving relaxation between excited-state surfaces. The second elimination takes place in the S0 of the unsaturated carbonyl and requires much longer time (>1 ps with our pump wavelength of 267 nm). The unsaturated carbonyl is initially generated in its first excited singlet state S1. If this molecule has four- or fivefold coordination, it can relax from this S1 state to S0 within about 50 fs, the pathway leading through a symmetry-induced conical intersection involving pseudorotation of the ligands. Coherent oscillations along such coordinates were observed in several molecules. In the case of threefold coordination (Ni(CO)3), however, there is no such relaxation pathway. Therefore, this photofragment shows a beautiful luminescence with >10 μs lifetime. All processes only involve the singlet manifold. Intersystem crossing takes at least 500 ps.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are corre- lated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor- alpha (TGF-a), and altered epithelial cell proliferation and differen- tiation. This research examined the role of these growth factors in TCDD-induced teratogenicity by using wild type (WT) and knockout (-/-) mice that do not express EGF, TGF-a, or both EGF and TGF-a. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 mg/kg, 5 ml/kg. On GD 17.5, the maternal parameters evaluated included body weight, body weight gain, liver weight (absolute and adjusted for body weight). The number of implantations, live and dead fetuses, early or late resorp- tions, the proportion of males, fetal body weight, fetal absolute and relative liver weight, placenta weight, incidence of cleft palate, and the severity and incidence of hydronephrosis were recorded. TCDD did not affect maternal weight gain, fetal weight, or survival, but maternal and fetal liver weights and liver-to-body weight ratios were increased in all genotypes. The WT and TGF-a (-/-), but not the EGF (-/-) and EGF 1 TGF-a (-/-) fetuses, developed cleft palate after exposure to 24 mg TCDD/kg. Hydronephrosis was induced by TCDD in all genotypes, with the incidence in EGF 1 TGF-a (-/-) fetuses comparable to that of the WT. The incidence and severity of this defect was substantially increased in EGF (-/-) and TGF-a (-/-). In conclusion, this study demonstrated that expression of EGF influ- ences the induction of cleft palate by TCDD. Also, EGF and TGF-a The environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, dioxin) is the most potent and extensively studied member of the polychlorinated aromatic hydrocarbon (PAH) family of compounds. Members of this family of com- pounds produce similar patterns of toxicity and appear to have a common mechanism of action (Birnbaum et al., 1985; Mc- Connell and Moore, 1979; Poland and Knutson, 1982). TCDD is produced during industrial and combustion processes such as chemical pulp bleaching and waste incineration (Abbott et al., 1987; Firestone et al., 1972). The widespread distribution and stability of TCDD have resulted in bioaccumulation of the compound throughout the environment and food chain (Mc- Connell and Moore, 1979). TCDD has been shown to induce a variety of toxic responses in laboratory animals such as carci- nogenicity, hepatotoxicity, fetotoxicity, reproductive toxicity, and teratogenicity (McConnell et al., 1978; Poland and Knut- son, 1982; Schwetz et al., 1973). Human exposure to TCDD currently occurs primarily via dietary consumption, although there have been several accidental and occupational exposures (Fingerhut et al., 1991; Mastroiacovo et al., 1988; Reggiani, 1989). Because of its relative resistance to metabolism, TCDD tends to persist in the body and its half life in humans is estimated at 7 to 10 years (Pirkle et al., 1989). TCDD is a reproductive and developmental toxicant in wild- life and laboratory animals, including rats, hamsters, and mice. The primary teratogenic abnormalities produced in mice are hydronephrosis and cleft palate (Moore, 1973). In the C57BL/6N mouse, these responses are seen at doses that do not result in maternal or fetal mortality (Birnbaum et al., 1985).

By exciting cyclohexene in the gas phase at 200 nm and probing it by nonresonant multiphoton ionization with mass-selective detection of the ion yields, we found four time constants tau(i) (20, 47, 43, 350 fs). Whereas deuteration lengthens tau2 by a factor of 1.4, the other constants do not change. Tau1-tau3 represent traveling times through observation windows on excited surfaces, whereas tau4 reflects a process in the hot ground state. We assign tau1 (20 fs) to departure from the Franck-Condon regions of the Rydberg and pipi* states, which are both populated at 200 nm, and tau2 (47 fs) to traveling along the pipi* surface and suggest that a [1,3]-sigmatropic H shift begins in this state. This rationalizes the deuterium effect on tau2. To explain why this window is followed by a process not subject to a D effect, we postulate that the pipi surface is crossed late (i.e., at low energy) by the zwitterionic state Z and that formation of a carbene (the known photochemical product, cyclopentylcarbene) begins there. The corresponding 1,2-shift of a CC bond is then (within tau4 = 350 fs) largely reversed on the ground-state surface, while a smaller part of the carbene forms products such as methylenecyclopentane within the same time. Carbene formation is probably accompanied by some cis-trans isomerization. The wavelength dependence of carbene formation is attributed to a memory for the initially excited state, based on momentum conservation. The processes are most likely typical of simple olefins. The fragmentation pattern showed that butadiene is not formed until at least 500 ps. The retro-Diels-Alder reaction, known to take place in the ground state, thus only occurs later.

Ab initio multistate second-order perturbation theory (MS−CASPT2) calculations are used to map the reaction path for the ultrafast photochemical electrocyclic ring-opening of cyclohexa-1,3-diene (CHD). This path is characterized by evolution along a complex reaction coordinate extending over two barrierless excited state potential energy surfaces and ultimately leading to deactivation through a S1/S0 conical intersection. The observed excited-state dynamics involve three sequential phases with lifetimes (traveling times) of 10, 43, and 77 fs, respectively. In this work we associate each phase to the evolution of the CHD molecular structure along a different mode. In particular, we show that (a) the decay of CHD from its spectroscopic (1B2) state to a lower lying dark (2A1) excited state involves motion along a highly curved coordinate corresponding to a mixture of σ bond expansion and symmetry breaking skeletal bending, (b) the evolution on the 2A1 (S1) state and the final 2A1→1A1 (i.e., S1→S0) decay invo...

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

Background Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells. Materials and Methods Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted. Results In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data. Conclusions Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics. Cytometry 44:156–160, 2001. © 2001 Wiley-Liss, Inc.

In marginal, agroclimatic zones, yield is often affected by flooding, but the effect is much less for winter spelt (Triticum spelta L.) than for winter wheat (Triticum aestivum L.). This study evaluates the reaction of a wheat x spelt population (F5 RILs of Forno x Oberkulmer) to flooding stress in the early phase of germination. Lines with greater tolerance to 48 h flooding just after imbibition showed less electrolyte leakage (r = -0.79) indicating greater membrane integrity and better survival. Five QTL explaining 40.6% of the phenotypic variance for survival to flooding were found, and localized on the chromosomes 2B, 3B,5A, and 7S. The tolerance to 48 h flooding four days after sowing was best correlated with the mean germination time (r = 0.8), indicating that the plants with a fast coleoptile growth during flooding are less susceptible to flooding. Ten QTL were found for seedling growth index after flooding explaining 35.5% of the phenotypic variance. They were localized on chromosomes 2A, 2B, 2D, 3A, 4B, 5A, 5B, 6A, and 7S. Standard varieties of spelt and wheat showed the same tolerance characteristics. The possibility to use marker assisted selection for flooding tolerance is discussed.

High-resolution transmission electron microscopy (HRTEM) and selectedarea electron diffraction (SAED) were used to study the formation of 20 nm thick platinum silicide films in the presence of an interfacial native silicon oxide layer. Pt films 10 nm thick were sputtered on Si[001] substrates covered by a native oxide layer 0–2.2 nm thick and annealed between 165 and 800°C. HRTEM observations on cross-sections show that, when an interfacial oxide layer is present, the reactants interdiffuse through the oxide pinholes. The pinholes influence the Pt–Si reaction over all the annealing temperature range examined. Up to 250°C their influence is observed by differences in the silicide phases formed and in the silicide–Si interface flatness. In the 350–550°C annealing temperature range. films with or without an interfacial oxide layer are continuous, polycrystalline and quite homogeneous in thickness, being equivalent for electrical uses. Silicide films grown through an interfacial oxide layer consist o...

Aneuploidy induction in male germ cells of mice and men after chronic exposure to diazepam (DZ; CAS 439-14-5; Valium was assessed by multicolor fluorescence in situ hybridization (FISH). DZ, a widely administered sedative and muscle relaxant, was proposed to act as an aneugen by disturbing spindle function in various assay systems. Male mice were treated by oral intubation with 3mg/kg DZ once or daily for 14 consecutive days. At 22 days after the last treatment, epididymal sperm were collected from the caudae epididymes. Evaluation of aneuploid and diploid sperm (10,000 sperm per animal) was performed by multicolor FISH employing DNA probes specific for chromosomes X, Y, and 8 simultaneously. We found a significant increase in the frequency of disomy 8 in subchronically DZ-treated mice when compared to the concurrent solvent control group (2.4-fold; P0.01), while no increase was detected for sex-chromosome hyperhaploidies. No effect was seen when mice were treated with a single dose (3mg/kg DZ). In a parallel human approach, two men were evaluated who chronically ingested0.3mg/kg/d DZ for more than 6 months. Multicolor FISH was applied to human sperm probing for chromosomes X, Y, and 13. Frequencies for sperm with disomy 13, disomy X, and total sex-chromosomal disomies were found to be elevated among the two subjects after chronic DZ-exposure compared to control subjects. In conclusion, the results indicate that diazepam acts as an aneugen during meiosis in male spermatogenesis, both in mice and humans. The quantitative comparison indicates that humans may be at least 10 times more sensitive than mice for aneuploidy induction by DZ during male meiosis.

By time-resolved nonresonant (800 nm) multiphoton ionization we found six consecutive exponential processes after excitation of Ni(CO)4 at 267 nm in the gas phase. Up to four steps (time constants 22 to 70 fs) probably correspond to relaxation and internal conversion between metal-to-ligand charge-transfer states in the intact molecule. Only the next step (600 fs) represents elimination of a CO group. This is an order of magnitude slower than in most other metal carbonyls investigated so far. The dissociation product is Ni(CO)3 in its S1 state. It does not relax to the ground state but luminesces (probably after intersystem crossing). This implies the absence of an easily accessible S1/S0 conical intersection. Such an intersection is induced by the Jahn−Teller effect in other carbonyls (which therefore do not luminesce), but not in Ni(CO)3. To explain a pump−wavelength-dependent time constant (42, 55, and 113 ps at 260, 267, and 276 nm, respectively), we assume that part of Ni(CO)3 dissociates to electron...

By time-resolved nonresonant (800 nm) multiphoton ionization we found five consecutive processes and pronounced coherent oscillations after excitation of M(CO) 6 , M=Cr, Mo and W at 267 nm in the gas phase. We suggest that the first two steps correspond to relaxation along a Jahn–Teller (JT) active coordinate and internal conversion between metal-to-ligand charge-transfer states via a JT induced conical intersection, whereas in the third step the molecules change over to a repulsive ligand-field surface and dissociate. The primary product is M(CO) 5 in its S 1 state which can again relax in an ultrashort time through a JT induced conical intersection to S 0 ; the JT active coordinate (relaxation pathway) corresponds to pseudorotation. The total time to reach S 0 takes 110, 165 and 195 fs for the three carbonyls (calculated from the measured first four time constants). After arrival at S 0 , M(CO) 5 oscillates coherently along a pseudorotation coordinate. In S 0 , M(CO) 5 eliminates a second CO in about 1 ps owing to its vibrational excess energy, a step which is suppressed in solution by cooling. All processes take place in the singlet manifold of states.

The concerted photochemical ring opening of 1,3-cyclohexadiene was investigated in the gas phase by low-intensity pumping at 267 nm and subsequent probing by high-intensity photoionization at 800 nm and mass-selective detection of the ion yields. We found five different time constants which can be assigned to traveling times along consecutive parts of the potential energy surfaces. The molecule is first accelerated in the spectroscopic state 1B along Franck–Condon active coordinates, then alters direction before changing over to the dark state 2A. All constants including that for leaving the 2A surface are below 100 fs. These times are shorter than appropriate vibrational periods. Such a maximum speed is evidence that the pathway is continuous leading from surface to surface via real crossings (conical intersections) and that the molecule is accelerated right into the outlet of the 2A/1A funnel. On the ground state it arrives as a compact wave packet, indicating a certain degree of coherence. The experimental method promises a high potential for investigating dynamics, since many consecutive phases of the process can be detected. This is because the fragmentation pattern depends on the location on the potential energy surface, so that monitoring several different ions permits to conclude on the population flow through these locations. Ionization at the intensities used is normally considered to be an effect of the electric field of the radiation. But in our case it is enhanced by resonances in the neutral molecule and in particular in the singly positive ion, and it is not sensitive for the length of the molecule (different conformers of the product hexatriene). The ionic resonances explain why hexatriene has a much richer fragmentation pattern than cyclohexadiene. Coulomb explosion is observed from an excited state of a doubly positive ion. Its mechanism is discussed.

Summary 1 This paper summarises the most important contributions on trophic relationships of lotic meiofauna. In contrast to marine research, the few quantitative studies of the freshwater meiobenthos have shown that these invertebrates not only take up particulate/fine organic matter, but also dissolved organic substances attached to organic particles. In lotic ecosystems, further estimates of grazing rate and bacterial/algal ingestion rate are needed, particularly in situ measurements. 2 The effects of macroinvertebrate predators upon meiofauna are still under debate. Depending on the type of experiments (laboratory vs. field) it seems that macrofauna may or may not affect meiofauna. Field samples and analyses of gut contents of larval tanypod chironomids have shown that the impact upon meiofauna was low and larvae were nonselective predators. Predation amounted to 2.2% of the combined prey density and prey consumption averaged 1.3 individuals per predator individual per year. 3 Adding taxonomic resolution by including the meiofaunal component within lotic food webs distinctly increases the number of total species and, as a consequence, changes food web statistics. Webs that included meiofauna revealed that these metazoans contributed substantially to the percentage of intermediate species (species with predators and prey). The resolution of dietary analyses of major consumers of macro- and meiobenthos showed that many stream invertebrates feed on meiofauna.

We found five consecutive processes with time constants 21, 15, 30, 47, and 3300 fs in Fe(CO)5 after excitation at 267 nm in the gas phase. The first four represent a continuous pathway of the molecule from the Franck−Condon region down to the lowest singlet state (S0) of Fe(CO)4 through a chain of Jahn−Teller-induced conical intersections. The motion before dissociation initially involves more than one of the equatorial ligands, but then eliminates only one CO. The product Fe(CO)4 is initially generated in its first excited singlet state S1, then it relaxes to S0 in 47 fs via a triply degenerate conical intersection at tetrahedral geometry. The pathway for this process involves pseudorotation of the ligands. The fifth step is assigned to thermal elimination of a second CO. Intersystem crossing to the triplet ground states of Fe(CO)4 and Fe(CO)3 takes more than 500 ps.

Summary In this study we present an efficient protocol for the production of embryos in isolated wheat microspore culture and discuss parameters determining the efficiency of embryo induction and green plant regeneration. Microspores were isolated from the spring wheat genotypes DH83Z118.32 and DHBW3 showing high and low androgenetic response, respectively. The washing (WM) and induction/culture medium (AMC) were optimised and led to an average embryo yield of 1350 embryos per 10 5 microspores with DH83Z118.32. One single culture dish yielding 7250 embryos per 10 5 microspores demonstrated that the androgenetic potential of this genotype is even higher. With DHBW3 an average of 82 embryos per 10 5 microspores was achieved. We developed a non-destructive UV fluorescence image processing system that revealed a correlation between size parameters of microspore populations and their embryo yield. For DH83Z118.32 we found the ability of embryos to regenerate green plants (embryo quality) to be strongly determined by embryo age and size. The highest yield of regenerated green plants was obtained when large embryos (\s>4 mm) were transferred to regeneration medium 25 days after microspore isolation. The formation of albinos was negatively correlated with the embryo size. The early assessment of microspore size and embryo quality combined with optimised culture methods demonstrated that the potential of wheat haploid induction by isolated microspore culture is not yet fully exploited.

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l−1 for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l−1 colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33).

In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40-120 microg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 microg/ml trichlorfon, whereas at 120 microg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.

Capsular block syndrome (CBS) is a complication of uncomplicated cataract surgery. Depending on etiology and the time of onset CBS can be subdivide into three types. The cases of 4 patients with CBS type III who presented with a milky fluid in the space between the posterior surface of the implanted intraocular lens (IOL) and the anterior surface of the distended posterior capsular bag are described. All patients complained of reduced vision. The therapy of choice is Nd:YAG laser posterior capsulotomy but with suspected low-grade endophthalmitis surgical intervention can also be carried out and submitting the plug for bacterial culture.

We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent Km values of 12 μM and 5 μM respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.