Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence.

STUDY QUESTION: Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days? SUMMARY ANSWER: Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h. WHAT IS KNOWN ALREADY: Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods. STUDY DESIGN, SIZE, DURATION: This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory's local manual method (Makler chamber) was used for comparison. MAIN RESULTS AND THE ROLE OF CHANCE: The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate. LIMITATIONS, REASONS FOR CAUTION: The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS: Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from 'Ferring Pharmaceuticals' to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Effects of the neonatal intensive care environment on circadian health and development of preterm infants.

The circadian system in mammals ensures adaptation to the light-dark cycle on Earth and imposes 24-h rhythmicity on metabolic, physiological and behavioral processes. The central circadian pacemaker is located in the brain and is entrained by environmental signals called Zeitgebers. From here, neural, humoral and systemic signals drive rhythms in peripheral clocks in nearly every mammalian tissue. During pregnancy, disruption of the complex interplay between the mother's rhythmic signals and the fetal developing circadian system can lead to long-term health consequences in the offspring. When an infant is born very preterm, it loses the temporal signals received from the mother prematurely and becomes totally dependent on 24/7 care in the Neonatal Intensive Care Unit (NICU), where day/night rhythmicity is usually blurred. In this literature review, we provide an overview of the fetal and neonatal development of the circadian system, and short-term consequences of disruption of this process as occurs in the NICU environment. Moreover, we provide a theoretical and molecular framework of how this disruption could lead to later-life disease. Finally, we discuss studies that aim to improve health outcomes after preterm birth by studying the effects of enhancing rhythmicity in light and noise exposure.

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 h) sexual abstinence periods.

OBJECTIVE: Metabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described. To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men. STUDY DESIGN: Semen samples collected after long (4-7 days) and short abstinence (2 h) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy. RESULTS: In total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume × concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12-3.87) vs. 1.29 (0.83-2.62) (P < 0.001) and taurine 0.58 (0.36-0.92) vs. 0.43 (0.28-0.95) (P < 0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence. CONCLUSION: The generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples. The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Morphometric dimensions of the human sperm head depend on the staining method used.

BACKGROUND: Assessment of sperm morphology (including morphometry) is extensively used to determine one of the qualities of a semen sample and depends on the differential staining of spermatozoa. A staining technique should cause as little change to sperm dimensions and form as possible in order to reliably evaluate the morphometric features of the sperm. Various staining techniques have been employed, but only a few have been recommended by the World Health Organization and are amenable to automated sperm morphometry analysis. Our study was aimed at comparing the effect of three staining techniques [Papanicolaou (PAP), Rapidiff (RD) and SpermBlue (SB)] on human sperm head dimensions and to compare these with the head dimensions in fresh semen. METHODS: Smears made from human semen samples (n = 24) were stained according to the three staining techniques and sperm head morphometry was assessed with the Sperm Class Analyzer. Head dimensions of fresh spermatozoa were measured with a digital calliper on a computer screen. The minimum number of spermatozoa to be analyzed to represent the sperm population and the degree of inter-laboratory variation were determined. Electron micrographs from the same semen samples were used to determine the actual acrosome coverage of the spermatozoa in the semen (n = 7) in order to verify the results of the automatic analyses. RESULTS: The osmolality of human semen differs from that of the RD and PAP fixatives and stains, but is more similar to the SB fixative and stain. At least 100 spermatozoa should be analyzed to include a representative sample of the sperm population. RD caused sperm heads to swell, PAP caused them to shrink and SB had no significant effect on sperm head dimensions when compared with spermatozoa in fresh semen. Very little inter-laboratory variations were found. The percentage acrosome coverage was significantly different between the three staining techniques, as well as between the RD and PAP stains and the manual measurements obtained using the electron micrographs. CONCLUSIONS: Different staining techniques change the morphometric dimensions of the human sperm head, probably due to the fact that either the fixatives or stains are not iso-osmotic in relation to human semen. Since these changes in sperm head dimensions are not uniform, care should be taken when selecting a staining technique. Ideally, stained spermatozoa should have dimensions as close to spermatozoa in fresh semen as possible, as was found with the SB staining method, resulting in accurate evaluations of sperm head morphometry.

DNA repair in mammalian cells: Direct DNA damage reversal: elegant solutions for nasty problems.

The genomic integrity of all living organisms is constantly jeopardized by physical [e.g. ultraviolet (UV) light, ionizing radiation] and chemical (e.g. environmental pollutants, endogenously produced reactive metabolites) agents that damage the DNA. To overcome the deleterious effects of DNA lesions, nature evolved a number of complex multi-protein repair processes with broad, partially overlapping substrate specificity. In marked contrast, cells may use very simple repair systems, referred to as direct DNA damage reversal, that rely on a single protein, remove lesions in a basically error-free manner, show high substrate specificity, and do not involve incision of the sugar-phosphate backbone or base excision. This concise review deals with two types of direct DNA damage reversal: (i) the repair of alkylating damage by alkyltransferases and dioxygenases, and (ii) the repair of UV-induced damage by spore photoproduct lyases and photolyases. (Part of a Multi-author Review).

Molecular mechanisms of the biological clock in cultured fibroblasts.

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. Here, using wild-type and cryptochrome (mCry)-deficient cell lines derived from mCry mutant mice, we show that the peripheral oscillator in cultured fibroblasts is identical to the oscillator in the SCN in (i) temporal expression profiles of all known clock genes, (ii) the phase of the various mRNA rhythms (i.e., antiphase oscillation of Bmal1 and mPer genes), (iii) the delay between maximum mRNA levels and appearance of nuclear mPER1 and mPER2 protein, (iv) the inability to produce oscillations in the absence of functional mCry genes, and (v) the control of period length by mCRY proteins.

Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock.

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.

Interacting molecular loops in the mammalian circadian clock.

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret.

In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months.

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry.

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.

Disruption of the Cockayne syndrome B gene impairs spontaneous tumorigenesis in cancer-predisposed Ink4a/ARF knockout mice.

Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage.

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues.

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.

A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response.

Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening.

Impact of global genome repair versus transcription-coupled repair on ultraviolet carcinogenesis in hairless mice.

The nucleotide excision repair (NER) system is comprised of two subpathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). To establish the relative importance of TCR and GGR for UV effects on the skin, we have used hairless knockout mouse strain lacking either TCR (CSB -/-) or GGR (XPC -/-). In single exposure experiments, we found that CSB -/- mice have a 7-16 times higher susceptibility to sunburn than XPC -/- mice and than heterozygous (+/-) and wild-type (+/+) controls. Exposure to 80 J/m2 UV radiation (i.e., suberythemogenic in CSB -/-) on 10 consecutive days gives rise to epidermal hyperplasia in CSB -/- and XPC -/-, whereas repair-proficient controls do not show epidermal hyperplasia from these exposures. In addition, CSB -/- mice develop marked parakeratosis, whereas XPC -/- mice and controls do not. Under continued exposure to this daily dose, squamous cell carcinomas appear in CSB -/-, XPC -/-, and in the control groups, whereas only in the CSB -/- animals is a fairly high number of benign papillomas also found. The median latency time of squamous cell carcinomas (diameters > or = 1 mm) is 84 days for the XPC -/- mice, 115 days for the CSB -/- mice, and 234-238 days for the heterozygous and wild-type control groups. These results indicate that GGR is more important than TCR in protection against UV-induced carcinomas of the skin but not against other UV effects such as sunburn, epidermal thickening, scaling of the stratum corneum, and development of papillomas. These results also indicate that GGR capacity may serve as a better predictor for skin cancer susceptibility than sensitivity to sunburn. The relative cancer susceptibilities of GGR- and TCR-deficient skin could well depend on the balance between an increased mutation rate and the presence (in CSB -/-) or lack (in XPC -/-) of a compensatory apoptotic response.

The relationship between benzo[a]pyrene-induced mutagenesis and carcinogenesis in repair-deficient Cockayne syndrome group B mice.

Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.

Mouse model for the DNA repair/basal transcription disorder trichothiodystrophy reveals cancer predisposition.

Patients with the nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) are highly predisposed to develop sunlight-induced skin cancer, in remarkable contrast to photosensitive NER-deficient trichothiodystrophy (TTD) patients carrying mutations in the same XPD gene. XPD encodes a helicase subunit of the dually functional DNA repair/basal transcription complex TFIIH. The pleiotropic disease phenotype is hypothesized to be, in part, derived from a repair defect causing UV sensitivity and, in part, from a subtle, viable basal transcription deficiency accounting for the cutaneous, developmental, and the typical brittle hair features of TTD. To understand the relationship between deficient NER and tumor susceptibility, we used a mouse model for TTD that mimics an XPD point mutation of a TTD patient in the mouse germline. Like the fibroblasts from the patient, mouse cells exhibit a partial NER defect, evident from the reduced UV-induced DNA repair synthesis (residual repair capacity approximately 25%), limited recovery of RNA synthesis after UV exposure, and a relatively mild hypersensitivity to cell killing by UV or 7,12-dimethylbenz[a]anthracene. In accordance with the cellular studies, TTD mice exhibit a modestly increased sensitivity to UV-induced inflammation and hyperplasia of the skin. In striking contrast to the human syndrome, TTD mice manifest a dear susceptibility to UV- and 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis, albeit not as pronounced as the totally NER-deficient XPA mice. These findings open up the possibility that TTD is associated with a so far unnoticed cancer predisposition and support the notion that a NER deficiency enhances cancer susceptibility. These findings have important implications for the etiology of the human disorder and for the impact of NER on carcinogenesis.

Standing sedation with medetomidine and butorphanol in captive African elephants (Loxodonta africana).

Doses for standing sedation allowing for various procedures in otherwise inaccessible, untrained captive African elephant bulls are presented. Thirty-three standing sedations were performed in 12 males aged 8-30 years (one to four sedations per animal). Each bull received a combination of 0.009 ± 0.002 mg/kg medetomidine and 0.03 ± 0.007 mg/kg butorphanol. Full sedation was reached on average 25.5 min after injection. The addition of hyaluronidase (1000-2000 IU) significantly reduced time to full sedation to 16.5 min (paired t test, P = 0.024). Reversal was induced with intramuscular atipamezole 0.008 (±0.002) and naltrexone 0.035 (±0.015) mg/kg. Recovery took on average 7 min (3-18 min). The medetomidine/butorphanol combination provided safe standing sedation for smaller procedures.

The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

Formation of Shc/Grb2- and Crk adaptor complexes containing tyrosine phosphorylated Cbl upon stimulation of the B-cell antigen receptor.

B-cell antigen receptor (BCR) stimulation induces tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2. The Shc/Grb2 complex may be involved in Ras activation, since Grb2 interacts with the guanine nucleotide exchange factor Sos. We reveal here an additional complexity of the BCR-induced Shc/Grb2 complex: it contains tyrosine phosphorylated proteins of 130, 110 and 75 kDa. The 130 kDa molecule inducibly associates with Shc, while the 75 kDa protein interacts with the carboxy-terminal SH3 domain of Grb2. The 110 kDa molecule is defined as Cbl, the product of the c-cbl oncogene, which is strongly phosphorylated on tyrosine upon BCR stimulation. Cbl constitutively interacts with the SH3 domains of Grb2, with a preference for the amino-terminal domain, and is in this way recruited to Shc upon BCR stimulation. Immunodepletion studies showed that Grb2-associated Cbl can be phosphorylated by BCR-induced tyrosine kinases independent of a Shc/Grb2 interaction. This indicates that the BCR can also couple to a Grb2 complex without the involvement of Shc. Cbl not only interacts with Grb2, but also with the adaptor protein Crk. In contrast to its constitutive interaction with Grb2, tyrosine-phosphorylated Cbl only associates with Crk after BCR stimulation. In summary, we observe that the BCR activates Shc/Grb2-, Grb2- and Crk adaptor complexes of distinct composition, which may allow selective coupling to different signal transduction cascades. Cbl participates in all three adaptor complexes and is tyrosine phosphorylated upon BCR stimulation, pointing to a central role for this molecule in the regulation of antigen receptor-induced B cell responses.