Reduction of urinary 8-epi-prostaglandin F2α during cyclo-oxygenase inhibition in rats but not in man

1. 8-epi-prostaglandin (PG) F_2α, a major F₂ isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF_2α as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF_2α in vivo is mostly COX-independent and urinary 8-epi-PGF_2α is therefore an accurate marker of ‘basal'' oxidative stress in vivo.
2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF_2α vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg⁻¹ day⁻¹; indomethacin: 4 mg kg⁻¹ day⁻¹) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF_2α was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB₁ and 2,3-dinor-6-keto-PGF_1α). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry.
3. Naproxen reduced urinary excretion of 2,3-dinor-TXB₁ and 2,3-dinor-6-keto-PGF_1α but, unexpectedly, also that of 8-epi-PGF_2α (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF_2α in liver and plasma remained unchanged after indomethacin.
4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF_2α in man. We gave naproxen (1 g day⁻¹) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF_2α remained unchanged in the two groups (9.63±0.99 before vs 10.24±1.01 after and 20.14±3.00 vs 19.03±2.45 ng h⁻¹ 1.73 m⁻²), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB₂ and 2,3-dinor-TXB₂; >50% for 2,3-dinor-6-keto-PGF_1α).
5. To investigate whether rat COX-1 produces 8-epi-PGF_2α more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF_2α and TXB₂ simultaneously in whole clotting blood. Serum levels of 8-epi-PGF_2α and TXB₂ were similar in rats and man.
6. We conclude that a significant amount of COX-dependent 8-epi-PGF_2α is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF_2α cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.

     

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca²⁺]_i, in a population of rat striatal astrocytes loaded with the fluorescent Ca²⁺ probe Fura2, by means of fluorescence spectrometry.
2. ATP triggered a fast and transient elevation of [Ca²⁺]_i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5′-O-(2-thiodiphosphate) (ADPβS), as well as uridine-5′-triphosphate (UTP) resembled that of ATP, while α,β-methylene-ATP (α,β-meATP) and β,γ-methylene-ATP (β,γ-meATP) were totally ineffective.
3. Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response.
4. Extracellular Ca²⁺ did not contribute to the observed [Ca²⁺]_i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni²⁺ (1 mM) or Mn²⁺ (1 mM) did not modify the nucleotide responses.
5. Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide-evoked [Ca²⁺]_i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved.
6. Both application of the G-protein blocker guanosine-5′-O-(2-thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml⁻¹) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 μM), significantly reduced the ATP-evoked [Ca²⁺]_i rise.
7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca²⁺]_i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

     

Prevention by morphine of apomorphine- and oxytocin-induced penile erection and yawning: involvement of nitric oxide

The possible involvement of nitric oxide in the prevention by morphine of apomorphine- and oxytocin-induced penile erection and yawning was investigated by measuring the concentration of NO₂- and NO₃- in the dialysate obtained with a vertical microdialysis probe implanted in the paraventricular nucleus of the hypothalamus of male rats. Either apomorphine (80 μg/kgs.c.) or oxytocin (30 ng i.c.v.) increased significantly basal NO₂- and NO₃- concentration in the paraventricular dialysate, penile erection and yawning. Morphine (1, 5 and 10mg/kg i.p.) prevented dose-dependently either apomorphine or oxytocin responses when given 15min before apomorphine or oxytocin. Prevention by morphine of apomorphine and oxytocin responses was abolished by naloxone (3mg/kg i.p.) given 15min before morphine. Morphine prevented apomorphine and oxytocin responses also when given in the lateral ventricles or directly in the paraventricular nucleus. In contrast, the selective agonist of the kappa opioid receptor subtype U-69,593 was found to be ineffective. The present results confirm previous findings showing that morphine acts through μ receptors in the paraventricular nucleus to prevent apomorphine and oxytocin-induced penile erection and yawning and suggest that this morphine effect is mediated by a decreased activity of nitric oxide in the paraventricular nucleus of the hypothalamus. Received: 30 September 1996 / Accepted: 24 January 1997     

Factors Influencing the Growth in Knowledge of Trainees in General Practice

Objectives: The relation between the content of postgraduate training for general practice and the outcome in terms of the growth in knowledge of trainees was investigated. The training variables included were: (1) the number of patients seen per day, (2) the trainer, (3) the practice and (4) the theoretical curriculum.Methods: Subjects were 58 trainee-trainer pairs. Growth in knowledge was assessed by two written tests administered with eight months interval. Training variables were evaluated by means of questionnaires and logbook-registration. The correlation was explored between each of the training variables and the knowledge tests scores. To correct for interactional effects, a step-wise multiple regression analysis was performed with the second test as dependent variable and the first test as well as the training variables as independent variables.Results: Significant growth in knowledge was demonstrated. Non of all training variables investigated explained the variance in scores on the second test equally good or better than the scores on the entry test.Conclusions: The impact of the separate training-components on the growth in knowledge, remains unclear. We may speculate, that the sort of knowledge assessed with the written literature based true/false test is different from the sort of knowledge transferred during every day training: evidence based knowledge versus experience based knowledge. Equally valid is the conclusion that these findings fit into the theory that in adult learning the outcome is more learner than teacher dependent.This revised version was published online in September 2005 with corrections to the Cover Date.     

Formation of extra digits in the interdigital spaces of the chick leg bud is not preceded by changes in the expression of the Msx and Hoxd genes

By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msx1 and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles for Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissue involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.     

The structure of human S-phase chromosome fibres

Recentin situ hybridization studies suggested that within the range of 0.1–1.0 Mb, human interphase chromosomes follow a random walk model (i.e. they behave as flexible polymers without major constraints). However, chromosome structure may differ in the G1, S, and G2 phases, and phase-specific constraints may be masked if the chromosome analysis does not discriminate between the phases. Therefore, using confocal microscopy, we examined the structure of S-phase chromosomes labelled with 5-iododeoxyuridine after prolonged treatment with 5-fluorodeoxyuridine. In the S-phase, labelled 0.32 µ chromosome fibres mostly appear as semi-circles with an average diameter of 0.83 ±0.03 µ. These semi-circles are joined together to form different 3D structures, and two semicircles frequently adopt s- or-like conformations involving about 2.5 µ of the chromosome contour length (L). Morphometric analysis of the S-phase fibres suggests that our data fit both the random flexible polymer model and also a model in which two constrained semi-circles are attached to each other by a flexible joint, thus eliminating constraints at long distances (L more than 2 µ).     

“Nowadays it isn't easy to advise the young”: Grandmothers and granddaughters among Abaluyia of Kenya

Among Abaluyia of Kenya relative age (seniority) structures relationships hierarchically among co-wives, siblings and females of different generations. Ambiguous equality and affectionate informality in grandmother-granddaughter relationships mute the hierarchical implications of their different age and generation statuses. This facilitates grandmothers' educational roles although, as grandmothers say, Nowadays it isn't easy to advise the young. Increased physical, cognitive and experiential distances between these generations resulted from radical changes in the female lifecourse associated with modernization and delocalization. Nevertheless, reciprocal exchanges continue. Granddaughters are also intermediaries in exchanges between older women and their adult daughters. Some grandmothers are denied the companionship and assistance of granddaughters caught in the middle of mother-in-law/daughter-in-law conflicts. Many grandmothers assume parental responsibilities as they deal with the modern problem of daughters' premarital pregnancies. All in all, these intergenerational relationships reveal both cultural persistence and the effects of social change, and continue to have instrumental and expressive value for both grandmothers and granddaughters.     

Sex selection may be inadvertently performed in in-vitro fertilization--embryo transfer programmes

The present study aims to ascertain whether sex selection maybe inadvertently performed in human in-vitro fertilization (IVF)and embryo transfer (IVF-embryo transfer) programmes when selectingfor high quality embryos (those with the fastest cleaving ratesand/or the best morphology) at the fresh transfer cycle. Allpatients entering into the study were treated with gonadotrophinsafter pituitary suppression with gonadotrophin-releasing hormoneagonists (GnRHa) and had intrauterine embryo transfer on day2 post-insemination. These patients were retrospectively dividedinto three groups according to whether the difference in meannumber of cells between embryos transferred and all embryosavailable for transfer in a given cycle was less than (negativeselection), equal to (no selection) or greater (positive selection)than zero. In cycles resulting in singleton births, the sexratio of the resulting babies was significantly (P 0.005) shiftedtoward the female (88.8%) and to the male (90.0%) in the negativeand positive selection groups respectively. No shift in sexratio was observed in cycles resulting in multiple births. Maternalage was another independent factor affecting sex ratio at birth.Sex ratio was significantly (P 0.05) skewed in favour of males(62.7%) and females (71.4%) in women <35 and 235 years ofage respectively. Maternal age, number of embryos transferredand the event of selecting or not selecting the slowest cleavingembryos for transfer were entered automatically in a three-groupdiscriminant model for distinguishing cycles resulting in onlyboys, both boys and girls, and only girls. These data suggestthat (i) sex selection may be inadvertently performed in IVF-embryotransfer programmes when selecting for high quality embryosat the fresh transfer cycles; (ii) human endometria may be favourable,indifferent or hostile to either fast cleaving or slow cleavingembryos depending on maternal age; and (iii) ‘natural’sex selection may be performed for social, psychological ormedical reasons.     

Child neurology

Poster Session 1

Child neurology