A population-based study of the biochemical and clinical expression of the H63D hemochromatosis mutation

Background & Aims: Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y mutation have been well characterized, the effects of the H63D mutation remain unclear. We accessed a well-defined population in Busselton, Australia, and determined the frequency of the H63D mutation and its influence on total body iron stores. Methods: Serum transferrin saturation and ferritin levels were correlated with the H63D mutation in 2531 unrelated white subjects who did not possess the C282Y mutation. Results: Sixty-two subjects (2.1%) were homozygous for the H63D mutation, 711 (23.6%) were heterozygous, and 1758 (58.4%) were wild-type for the H63D mutation. Serum transferrin saturation was significantly increased in male and female H63D homozygotes and heterozygotes compared with wild-types. Serum ferritin levels within each gender were not influenced by H63D genotypes. Elevated transferrin saturation greater than or equal to45% was observed in a greater proportion of male H63D carriers than male wild-types. Male H63D homozygotes (9%) and heterozygotes (3%) were more likely to have both elevated transferrin saturation and elevated ferritin greater than or equal to300 ng/mL than male wild-types (0.7%). Homozygosity for H63D was not associated with the development of clinically significant iron overload. Conclusions: Presence of the H63D mutation results in a significant increase in serum transferrin saturation but does hot result in significant iron overload. In the absence of the C282Y mutation, the H63D mutation is not clinically significant.

The 2000 Ogura Lecture: Olfactory neural cells: An untapped diagnostic and therapeutic resource

Objective. This is an over-view of the cellular biology of upper nasal mucosal cells that have special characteristics that enable them to be used to diagnose and study congenital neurological diseases and to aid neural repair. Study Design: After mapping the distribution of neural cells in the upper nose, the authors' investigations moved to the use of olfactory neurones to diagnose neurological diseases of development, especially schizophrenia. Olfactory-ensheating glial cells (OEGs) from the cranial cavity promote axonal penetration of the central nervous system and aid spinal cord repair in rodents. The authors sought to isolate these cells from the more accessible upper nasal cavity in rats and in humans and prove they could likewise promote neural regeneration, making these cells suitable for human spinal repair investigations. Methods: The schizophrenia-diagnosis aspect of the study entailed the biopsy of the olfactory areas of 10 schizophrenic patients and 10 control subjects. The tissue samples were sliced and grown in culture medium. The ease of cell attachment to fibronectin (artificial epithelial basement membrane), as well as the mitotic and apoptotic indices, was studied in the presence and absence of dopamine in those cell cultures. The neural repair part of the study entailed a harvesting and insertion of first rat olfactory lamina propria rich in OEGs between cut ends of the spinal cords and then later the microinjection of an OEG-rich suspension into rat spinal cords previously transected by open laminectomy. Further studies were done in which OEG insertion was performed up to 1 month after rat cord transection and also in monkeys. Results: Schizophrenic patients' olfactory tissues do not easily attach to basement membrane compared with control subjects, adding evidence to the theory that cell wall anomalies are part of the schizophrenic lesion of neurones. Schizophrenic patient cell cultures had higher mitotic and apoptotic indices compared with control subjects. The addition of dopamine altered these indices enough to allow accurate differentiation of schizophrenics from control patients, leading to, possibly for the first time, an early objective diagnosis of schizophrenia and possible assessment of preventive strategies. OEGs from the nose were shown to be as effective as those from the olfactory bulb in promoting axonal growth across transected spinal cords even when added I month after injury in the rat. These otherwise paraplegic rats grew motor and proprioceptive and fine touch fibers with corresponding behavioral improvement. Conclusions. The tissues of the olfactory mucosa are readily available to the otolaryngologist. Being surface cells, they must regenerate (called neurogenesis). Biopsy of this area and amplification of cells in culture gives the scientist a window to the developing brain, including early diagnosis of schizophrenia. The Holy Grail of neurological disease is the cure of traumatic paraplegia and OEGs from the nose promote that repair. The otolaryngologist may become the necessary partner of the neurophysiologist and spinal surgeon to take the laboratory potential of paraplegic cure into the day-to-day realm of clinical reality.

Audit of maternal and fetal outcomes in women treated for glucose intolerance during pregnancy

Objective To determine whether one should aim for glycaemia that is statistically 'normal' or for levels of glycaemia low enough to prevent macrosomia (if such a threshold exists) when glucose intolerance is detected during pregnancy Design An audit of pregnancy outcomes in women with impaired glucose tolerance in pregnancy as compared to a local age-matched reference group with normal glucose tolerance. Results Our study suggests that for most patients, more intensive therapy would not have been justified. Maternal smoking appeared to convey some 'advantages' in terms of neonatal outcomes, with reduction in large-for-gestational-age (LGA) infants and jaundice in babies of impaired glucose tolerance (IGT) mothers. Conclusions These observations demonstrate the importance of considering risk factors other than GTT results in analysing pregnancy outcomes, while emphasising that 'normalisation' of fetal size should not be our only therapeutic endpoint. Our detailed outcome review allows us to reassure patients with GDM that with current treatment protocols, they should have every expectation of a positive pregnancy outcome.

Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M + H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M + H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M + H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1 a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels

Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor

Psychological stressors trigger the activation of medullary noradrenergic cells, an effect that has been shown to depend upon yet-to-be-identified structures located higher in the brain. To test whether the amygdala is important in this regard, we examined the effects of amygdala lesions on noradrenergic cell responses to restraint, and also looked at whether any amygdala cells that respond to restraint project directly to the medulla. Ibotenic acid lesions of the medial amygdala completely abolished restraint-induced Fos expression in A1 and A2 noradrenergic cells. In contrast, lesions of the central amygdala actually facilitated noradrenergic cell responses to restraint. Tracer deposits in the dorsomedial (but not ventrolateral) medulla retrogradely labelled many cells in the central nucleus of the amygdala, but none of these cells expressed Fos in response to restraint. These data suggest for the first time that the medial amygdala is critical to the activation of medullary noradrenergic cells by a psychological stressor whereas the central nucleus exerts an opposing, inhibitory influence upon noradrenergic cell recruitment. The initiation of noradrenergic cell responses by the medial amygdala does not involve a direct projection to the medulla. Accordingly, a relay through some other structure, such as the hypothalamic paraventricular nucleus, warrants careful consideration.

Present and future pharmacotherapy for benign prostatic hyperplasia

Around 50% of men 51-60 years of age have pathological benign prostatic hyperplasia (BPH). Pharmacotherapy for BPH includes the 5alpha-reductase inhibitor finasteride, and alpha(1)-adrenoceptor antagonists. Finasteride reduces prostate volume and symptom scores, while increasing peak urinary flow rates. The main problem with finasteride treatment is that it increases the incidence of ejaculation disorders. All of the alpha(1)-adrenoceptor antagonists have been shown to reduce symptom scores and increase peak urinary flow rates in BPH. The nonselective alpha(1)-adrenoceptor antagonists (prazosin, terazosin and doxazosin) were developed as antihypertensives, and hypotensive-related side effects are the main problem with these agents in BPH. These side effects can be diminished by reducing peak concentrations of the drugs, as with once-daily alfuzosin, or by using the uroselective antagonist tamsulosin. Phytopharmaceuticals are commonly used in the treatment of BPH, such as saw palmetto berry which has been shown to improve the symptoms and peak urinary flow rate. Androgen receptor antagonists are not used in BPH because of their adverse effects. Newer drugs under development for the treatment of BPH include alpha(1)-adrenoceptor antagonists that show more selectivity for alpha(1A)-adrenoceptors than tamsulosin, combined 5alpha-reductase/alpha(1)-adrenoceptor inhibitors and combined type 1/type 2 5alpha-reductase inhibitors. New targets for the drug treatment of BPH include indothelin, growth factors, estrogens and the phosphodiesterase isoenzymes.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.

Amino Acids involved in differences in the pharmacological profiles of the rat and human noradrenaline transporters

It has been suggested from a previous study in our laboratory that differences in the pharmacology of the species variants of the noradrenaline transporter (NET) are the result of four non-conservative amino acid exchanges from the total of 26 amino acids that are divergent between the rat NET (rNET) and human NET (hNET). The aim of this study was to examine the effects of changing the rNET at each of these four amino acid residues, which markedly alter local charge distribution, to the amino acid found in hNET. Site-directed mutagenesis was used to create mutant cDNAs from rNET cDNA. The mutant NETs (rK71), rE62K, rK375N and rR612Q), rNET and hNET were expressed in transiently transfected COS-7 cells to determine the effects of the mutations on the differing pharmacological properties of the species variants. The ratios of V-max for noradrenaline uptake and B-max for nisoxetine binding (which are a measure of the turnover number of the transporter, i.e. the number of transport cycles per min) were greater for rNET and rR612Q than for hNET, rK71), rE62K and rK375N. The K-m of noradrenaline was lower for hNET, rK713, rE62K and rK375N than for rNET or rR612Q. There were no differences between the K-i values for inhibition of noradrenaline uptake by nisoxetine for rNET, hNET or the mutants, but the K-i values of cocaine were lower for hNET, rE62K and rR612Q than rNET or rK375N. Hence, the study showed that: (1) the aspartate 7. lysine 62 and asparagine 375 amino acid residues are important in determining the lower substrate translocation by hNET than rNET; (2) the aspartate 7 and lysine 62 residues in the N-terminus of hNET determine the higher affinities of substrates for the hNET than the rNET; and (3) the lysine 62 and glutamine 612 residues in the N- and C-termini, respectively, of hNET Lire determinants of the higher cocaine affinity for the hNET than rNET.

The role of conserved CXXXRXG motif in the expression and function of the human norepinephrine transporter

Highly conserved motifs in the monoamine transporters, e.g. the human norepinephrine transporter (hNET) GXXXRXG motif which was the focus of the present study, are likely to be important structural features in determining function. This motif was investigated by mutating the glycines to glutamate (causing loss of function) and alanine, and the arginine to glycine. The effects of hG117A, hR121G and hG123A mutations on function were examined in COS-7 cells and compared to hNET. Substrate K-m values were decreased for hG117A and hG123A, and their K values for inhibition of [3 H]nisoxetine binding were decreased 3-4-fold and 4-6-fold, respectively. Transporter turnover was reduced to 65% of hNET for hG117A and hR121G and to 28% for hG123A, suggesting that substrate translocation is impaired. K values of nisoxetine and desipramine for inhibition of [H-3]norepinephrine uptake were increased by 5-fold for hG117A, with no change for cocaine. The K-i value of cocaine was increased by 3-fold for hG123A, with no change for nisoxetine and desipramine. However, there were no effects of the mutations on the K-d of [H-3]nisoxetine binding or K-i values of desipramine or cocaine for inhibition of [H-3]nisoxetine binding. Hence, glycine residues of the GXXXRXG motif are important determinants of NET expression and function, while the arginine residue does not have a major role. This study also showed that antidepressants and psychostimulants have different NET binding sites and provided the first evidence that different sites on the NET are involved in the binding of inhibitors and their competitive inhibition of substrate uptake. (C) 2002 Elsevier Science B.V. All rights reserved.

Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Role of PGF(2 alpha) and oxytocin in parturition in the brushtail possum (Trichosurus vulpecula)

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F-2alpha (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.