Gestational chronodisruption leads to persistent changes in the rat fetal and adult adrenal clock and function.

KEY POINTS: Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Here we explored the developmental impact of gestational chronodisruption (chronic photoperiod shift, CPS) on adult and fetal adrenal biorhythms and function. We found that gestational chronodisruption altered fetal and adult adrenal function, at the molecular, morphological and physiological levels. The differences between control and CPS offspring suggest desynchronization of the adrenal circadian clock and steroidogenic pathway, leading to abnormal stress responses and metabolic adaptation, potentially increasing the risk of developing chronic diseases. ABSTRACT: Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long-term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down-regulated and 437 up-regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48-20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long-term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases.

Puberty arises with testicular alterations and defective AMH expression in rams prenatally exposed to testosterone.

The male gonadal tissue can be a sensitive target to the reprogramming effects of testosterone (T) during prenatal development. We have demonstrated that male lambs born to dams receiving T during pregnancy-a model system to the polycystic ovary syndrome (PCOS)-show a decreased number of germ cells early in life, and when adult, a reduced amount of sperm and ejaculate volume. These findings are a key to put attention to the male offspring of women bearing PCOS, as they are exposed to increased levels of androgen during pregnancy which can reprogram their reproductive outcome. A possible origin of these defects can be a disruption in the expression of the anti-Müllerian hormone (AMH), due to its critical role in gonadal function at many postnatal stages. Therefore, we addressed the impact of prenatal T excess on the expression of AMH and factors related to its expression like AP2, SOX9, FSHR, and AR in the testicular tissue through real-time PCR during the peripubertal age. We also analyzed the testicular morphology and quantified the number of Sertoli cells and germ cells to evaluate any further defect in the testicle. Experiments were performed in rams at 24 wk of age, hence, prior puberty. The experimental animals (T-males) consisted of rams born to mothers receiving 30 mg testosterone twice a wk from Day 30 to 90 of pregnancy and then increased to 40 mg until Day 120 of pregnancy. The control males (C-males) were born to mothers receiving the vehicle of the hormone. We found a significant increase in the expression of the mRNA of AMH and SOX9, but not of the AP2, FHSR nor AR, in the T-males. Moreover, T-males showed a dramatic decrease in the number of germ cells, together with a decrease in the weight of their testicles. The findings of the present study show that before puberty, T-males are manifesting clear signs of disruption in the gonadal functions probably due to an alteration in the expression pattern of the AMH gene. The precise way by which T reprograms the expression of AMH gene remains to be established.

Environmental control of biological rhythms: effects on development, fertility and metabolism.

Internal temporal organisation properly synchronised to the environment is crucial for health maintenance. This organisation is provided at the cellular level by the molecular clock, a macromolecular transcription-based oscillator formed by the clock and the clock-controlled genes that is present in both central and peripheral tissues. In mammals, melanopsin in light-sensitive retinal ganglion cells plays a considerable role in the synchronisation of the circadian timing system to the daily light/dark cycle. Melatonin, a hormone synthesised in the pineal gland exclusively at night and an output of the central clock, has a fundamental role in regulating/timing several physiological functions, including glucose homeostasis, insulin secretion and energy metabolism. As such, metabolism is severely impaired after a reduction in melatonin production. Furthermore, light pollution during the night and shift work schedules can abrogate melatonin synthesis and impair homeostasis. Chronodisruption during pregnancy has deleterious effects on the health of progeny, including metabolic, cardiovascular and cognitive dysfunction. Developmental programming by steroids or steroid-mimetic compounds also produces internal circadian disorganisation that may be a significant factor in the aetiology of fertility disorders such as polycystic ovary syndrome. Thus, both early and late in life, pernicious alterations of the endogenous temporal order by environmental factors can disrupt the homeostatic function of the circadian timing system, leading to pathophysiology and/or disease.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

This study isolated the effects of maternal hypoxia independent of changes in maternal nutrition on maternal circulatory and placental molecular indices of oxidative stress and determined whether maternal antioxidant treatment conferred protection. Pregnant rats were subjected to normoxic pregnancy or 13% O2 chronic hypoxia for most of gestation with and without maternal treatment with vitamin C in the drinking water. Maternal hypoxia with and without vitamin C did not affect maternal food or water intake and led to a significant increase in maternal and fetal haematocrit. At gestational day 20, maternal plasma urate and L-cysteine concentrations, and placental levels of 4-hydroxynonenal and heat shock protein 70 were increased while placental heat shock protein 90 levels were decreased in hypoxic pregnancy. The induction of maternal circulatory and placental molecular indices of oxidative stress in hypoxic pregnancies was prevented by maternal treatment with vitamin C. Maternal hypoxia during pregnancy with or without vitamin C increased placental weight, but not total or compartmental volumes. Maternal treatment with vitamin C increased birth weight in both hypoxic and normoxic pregnancies. The data show that maternal hypoxia independent of maternal undernutrition promotes maternal and placental indices of oxidative stress, effects that can be prevented by maternal treatment with vitamin C in hypoxic pregnancy. While vitamin C may not be the ideal candidate of choice for therapy in pregnant women, and taking into consideration differences in ascorbic acid metabolism between rats and humans, the data do underlie that antioxidant treatment may provide a useful intervention to improve placental function and protect fetal growth in pregnancy complicated by fetal hypoxia.

Redox modulation of the fetal cardiovascular defence to hypoxaemia.

Episodes of hypoxia in utero present a potentially serious challenge to the fetus, but are counteracted by defence responses including marked redistribution of blood flow from peripheral circulations to the brain. Here, we report the novel observation that the oxidant tone is an important modulator of this cardiovascular defence. Using pregnant Welsh Mountain sheep surgically prepared for long-term recording, we investigated in vivo the effects on the fetal cardiovascular defence to acute hypoxaemia of fetal treatment with the antioxidant vitamin C. The mechanisms via which vitamin C may affect the vascular oxidant tone were investigated by monitoring fetal plasma concentrations of nitrates and nitrites, by determining changes in the activity of superoxide dismutase (SOD) in fetal plasma, and by investigating the effect of vitamin C treatment on the fetal cardiovascular defence to hypoxaemia following nitric oxide (NO) synthase blockade. Fetal treatment with vitamin C markedly depressed the normal femoral constrictor response to acute hypoxaemia in the fetus (5.2 ± 1.0 vs. 1.1 ± 0.3 mmHg (ml min(-1))(-1), mean ± s.e.m.; P < 0.05) an effect which was completely restored following NO synthase blockade (6.2 ± 1.3 mmHg (ml min(-1))(-1)). Compared to saline infusion, fetal treatment with vitamin C during acute hypoxaemia also significantly increased fetal plasma SOD activity from normoxic baseline (-8.9 ± 6.5 vs. 15.0 ± 6.6% inhibition, P < 0.05) and decreased the plasma concentration ratio of nitrate:nitrite from normoxic baseline (ΔNO3(-):NO2(-); 0.15 ± 0.30 vs. -0.29 ± 0.11, P < 0.05). The data provide in vivo evidence of redox modulation of redistribution of blood flow in the fetus, part of the fetal brain sparing during acute hypoxaemic stress.

Discovery of potent and selective histamine H3 receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold.

A novel series of potent histamine H(3) receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold has been discovered. Several compounds display high selectivity over other histamine receptor subtypes and have favorable physicochemical properties, low potential for CYP450 enzyme inhibition and high metabolic stability in microsomal preparations. (R)-2-Cyclopropylmethyl-8-(1-isopropyl-piperidin-4-yloxy)-3-methyl-3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one (8t) showed good in vivo efficacy after per os application in an acute rat dipsogenia model of water intake.

Cryptochrome 2 expression level is critical for adrenocorticotropin stimulation of cortisol production in the capuchin monkey adrenal.

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Clock gene expression in adult primate suprachiasmatic nuclei and adrenal: is the adrenal a peripheral clock responsive to melatonin?

The circadian production of glucocorticoids involves the concerted action of several factors that eventually allow an adequate adaptation to the environment. Circadian rhythms are controlled by the circadian timing system that comprises peripheral oscillators and a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, driven by the self-regulatory interaction of a set of proteins encoded by genes named clock genes. Here we describe the phase relationship between the SCN and adrenal gland for the expression of selected core clock transcripts (Per-2, Bmal-1) in the adult capuchin monkey, a New World, diurnal nonhuman primate. In the SCN we found a higher expression of Bmal-1 during the h of darkness (2000-0200 h) and Per-2 during daytime h (1400 h). The adrenal gland expressed clock genes in oscillatory fashion, with higher values for Bmal-1 during the day (1400-2000 h), whereas Per-2 was higher at nighttime (about 0200 h), resulting in a 9- to 12-h antiphase pattern. In the adrenal gland, the oscillation of clock genes was accompanied by rhythmic expression of a functional output, the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase. Furthermore, we show that adrenal explants maintained oscillatory expression of Per-2 and Bmal-1 for at least 36 h in culture. The acrophase of both transcripts, but not its overall expression along the incubation, was blunted by 100 nm melatonin. Altogether, these results demonstrate oscillation of clock genes in the SCN and adrenal gland of a diurnal primate and support an oscillation of clock genes in the adrenal gland that may be modulated by the neurohormone melatonin.

On the fracture of a zirconia ball head.

Y-TZP ball heads were introduced into the market in 1985. Since then these components have had wide diffusion in hip replacements, due to their good mechanical performance and reliability. Namely, only a few papers were published up to year 2000 reporting failures of Y-TZP ball heads. The worldwide recall in August 2001 of some Y-TZP batches changed this situation. This paper analyse the material of a ceramic ball head that fractured in vivo 34 months after surgery. The retrieved fragments were submitted for visual inspection, fractographic analysis by optical microscopy, X-ray diffractometry, and FEG-SEM ceramography analysis. The results obtained show that the hydrothermal stability of the material had only a secondary role in the fracture. The literature reporting on failures of THR making use of Y-TZP ball heads is discussed.

The floor plate cells from bovines express the mRNA encoding for SCO-spondin and its translation products.

The floor plate (FP) is a transient structure of the embryonic central nervous system (CNS) which plays a key role in development driving cell differentiation and patterning in the ventral neural tube. The fact that antisera raised against subcommissural organ (SCO) secretion immunostain FP cells and react with high-molecular-mass proteins in FP extracts, prompted us to investigate the expression of a SCO-related polypeptide in FP cells. RNA from bovine FP was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR), using primers derived from the 3' end of SCO-spondin which revealed products of 233, 237, 519 and 783 bp. Sequence analysis of the 233 bp PCR fragment confirmed the identity between this FP product and SCO-spondin. FP-translation of the SCO-spondin encoded polypeptide(s) was demonstrated by Western blot analysis and immunocytochemistry, using antisera raised against (i) the glycoproteins secreted by the bovine SCO, and (ii) a peptide derived from the open reading frame of the major SCO secretory protein, SCO-spondin, respectively. Additional evidence pointing to active transcription and translation of a SCO-spondin related gene was obtained in long term FP organ cultures. On the basis of partial sequence homologies of SCO-spondin with protein domains implicated in cell-cell contacts, cell-matrix interactions and neurite outgrowth it is possible to suggest that the SCO-spondin secreted by the FP is involved in CNS development.

Y-TZP ceramics for artificial joint replacements.

Due to their excellent mechanical properties, Yttria-stabilized Tetragonal Zirconia Polycrystal ceramics (Y-TZP) are used in ball heads for Total Hip Replacements. It is known that Y-TZP materials may show strength degradation due to ageing or to hydrothermal treatment. Also high wear of UHMWPE sockets coupled to steam sterilized Y-TZP ball heads after a short implantation period was recently reported. This effect may be related to ball head surface phase transformation, due to corrosive attack. The aim of this study is the evaluation of Y-TZP ceramics stability. Y-TZP made out of Yttria coated powders were aged at 140 degrees C under 0.2 MPa water pressure, in Ringer's solution at 37 degrees C, in NZW rabbits. Samples made out Yttria coated powders show lower strength degradation than samples made out coprecipitated powders, and UHMWPE discs coupled to Y-TZP rings made out coated powders do not show increase in wear after repeated sterilization cycles of the ceramic rings.

New Y-TZP powders for medical grade zirconia.

There is interest in using zirconia for biomedical applications as ball heads for total hip prostheses. Two potential types are under discussion: partially stabilized zirconia (PSZ) and tetragonal zirconia polycrystals (TZP) materials. Because of its enhanced material properties, TZP stabilized with yttria is favourable. To eliminate high amounts of natural radioactive impurities, the precursors are purified. The kind of precursor and purification method determine the powder impurity level. The disadvantage of Y-TZP is that the hydrothermal decomposition reaction method is that it depends very strongly on the grain size and the distribution of the stabilizing yttria within the zirconia grains. Thermodynamical and kinetic investigations on high purity coprecipitated and yttria-coated zirconia powders show different behaviours. Y-TZP materials based on yttria-coated zirconia powders show excellent mechanical strength of more than 1000 MPa, a Weibull modulus of up to 20 a! nd a fracture toughness of 9 MPa radical m. The material properties of Y-TZP ceramics based on coprecipitated powders and prepared under the same conditions are less attractive. It is expected that materials based on yttria-coated zirconia will show enhanced properties compared to materials derived from coprecipitated powders. Therefore Y-TZP materials derived from yttria-coated powders are very attractive as medical grade zirconia.

Design, synthesis, and evaluation of 2 beta-alkenyl penam sulfone acids as inhibitors of beta-lactamases.

A general method for synthesis of 2 beta-alkenyl penam sulfones has been developed. The new compounds inhibited most of the common types of beta-lactamase. The level of activity depended very strongly on the nature of the substituent in the 2 beta-alkenyl group. The inhibited species formed with the beta-lactamase from Citrobacter freundii 1205 was sufficiently stable for X-ray crystallographic studies. These, together with UV absorption spectroscopy and studies of chemical degradation, suggested a novel reaction mechanism for the new inhibitors that might account for their broad spectrum of action. The (Z)-2 beta-acrylonitrile penam sulfone Ro 48-1220 was the most active inhibitor from this class of compound. The inhibitor enhanced the action of, for example, ceftriaxone against a broad selection of organisms producing beta-lactamases. The organisms included strains of Enterobacteriaceae that produce cephalosporinases, which is an exceptional activity for penam sulfones.

Synthesis and structure-activity relationship of (lactamylvinyl)cephalosporins exhibiting activity against staphylococci, pneumococci, and enterococci.

The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone.

[Increasing the safety of ceramic femoral heads for hip prostheses]. / Steigerung der Sicherheit von keramischen Kugelköpfen für Hüftendoprothesen.

Since 1974 Biolox ceramic femoral ball heads have been used successfully for artificial modular hip joints. The revision rate due to ball head fracture is lower than 0.02%. This is an extremely low value. In this article it is shown how the safety of a ceramic ball head can be improved using the procedures of HIPing, engraving by laser technique, and 100% proof testing. By applying these means the materials properties density, grain size, grain size distribution, and the strength of the ball head, i.e. the fracture load can be improved significantly.

[Fracture load of ceramic ball heads of hip joint prostheses]. / Bruchlast keramischer Kugelköpfe von Hüftgelenkprothesen.

To ensure the stability of ceramic ball heads of hip joint prostheses over the long term, both standards for the material and FDA-regulations for the components have been established. In this paper the philosophy underlying design and reliability is discussed. On the basis of fracture loads determined for Biolox ball heads the high level of stability and reliability that has been achieved and can now be guaranteed, is demonstrated.

[Type A behavior--a uniform construct?]. / Das Typ-A-Verhalten--ein einheitliches Konstrukt?

Single dimensions of the Type-A-behaviour pattern and their contributions to the etiology and pathogenesis of cardiovascular and psychosomatic diseases are very often discussed. Our studies are concerned with the analysis of 40 blue collar workers and patients (40 patients with hyperuricemia, 36 psychotherapy patients) in definite groups of age. By item analysis and multivariate statistical procedures (variance, factor and discriminant analysis) we found an overlapping effect of psychosocial factors of work and on the other hand the influence of neurotic symptoms and behaviour patterns.

Pathophysiology of subglottic tracheal stenosis in childhood.

Two particularly problematic clinical entities are chosen from the many possible causes of the pathophysiology of subglottic tracheal stenoses in childhood: malformations of the "hard" and "soft" types of stenosis occurring typically as primary lesions; and stenoses caused by trauma (following intubation or faulty tracheostomy) as the most common secondary lesions. It is shown with reference to experimental investigations that subepithelial layers play the leading role in the origin of stenoses, in contrast to hitherto existing ideas based on clinical observations. Traumatic stenoses caused by unphysiological mechanical stressing show striking parallels to those in other age groups, suggesting the conclusion that a reaction of the mesenchymal tissue layers of the airway regions in question is a typical response to mechanical forces.

[A scanning electron microscopic study of the tracheal mucosa damage after long-term translaryngeal intubation (author's transl)]. / Das rasterelektronen-mikroskopische Bild der Trachealschleimhaut nach translaryngealer intratrachealer Langzeitintubation.

Tracheal mucosa tissue obtained by tracheotomy from 23 patients who had been intubated between 4 to 20 days was examined in the scanning electron microscope. In spite of different intubation periods almost identical tissue defects with extensive areas of epithelial degeneration reaching as far as the basal lamina were found. In contrast, patients with short-term intubation, i.e. during intubation anaesthesia, showed no mucosal changes in the areas studied. The expected damage in the lower trachea caused by the cuff of the intubation tube is described and the dangers of prolonged intubation are discussed.