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A 6-week old infant who had been conceived through in-vitro fertilisation (IVF) presented with a skin lesion and enlarged lymph nodes, and developed severe respiratory distress. Mycobacterium tuberculosis was identified; his mother was the only potential source identified. To our knowledge, this is the first case of congenital tuberculosis after IVF reported in Australia and the second worldwide. It highlights the importance of adequate screening during investigation of infertility and the difficulties in diagnosing congenital tuberculosis. [ABSTRACT FROM AUTHOR]

Compared to the well-known conjugated (1,3-dipolar) and cross-conjugated (1,4-dipolar) heterocyclic mesomeric betaines (HMBs), semi-conjugated HMBs are unexplored and almost unknown. The three discrete classes of HMB are defined by the connectivity between their ring 2π heteroatoms and the odd-conjugated fragments that complete the ring. A single example of a stable, fully-characterized semi-conjugate HMB has been reported. This study employs the density functional theory (DFT) methodology to investigate the properties of a series of six-membered semi-conjugated HMBs. The electronic character of ring substituents is found to significantly influence the structure and electronic properties of the ring. The aromaticity measured by HOMA and NICS(1) zz indices is increased by π-electron-donating substituents whereas π-electron-withdrawing substituents decrease the calculated aromatic character and ultimately lead to non-planar boat or chair structures. A notable property of all derivatives is the small energy gap between their frontier orbitals.

Using a set of twelve selected substituents, the influence of substituent properties on the ease of deprotonation of imidazolium cations and mesoionic imidazolium-4-olates measured by the CREF index has been investigated. Significant correlations between CREF values and the Swain and Lupton field ( F ) and resonance ( R ) substituent constants have been found. In all cases the field effect has the greatest influence but resonance effects are also significant., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The index described previously (carbene relative energy of formation) has been extended to oxygen and sulfur heterocycles. This provides a quantitative overview of factors determining ease of formation of (i) neutral N-heterocyclic carbenes (NHCs) by deprotonation of heterocyclic salts and (ii) anionic NHCs by deprotonation of heterocyclic mesomeric betaines. The influence of the nature and ring position of oxygen and sulfur is discussed for a range of known and unknown systems. Attention is directed to unexplored systems of potential interest.

An energy-based index of the ease of N-heterocyclic carbene (NHC) formation either by deprotonation of precursor salts to give neutral NHCs or deprotonation of heterocyclic mesomeric betaines to give anionic NHCs is described. This index (CREF; Carbene Relative Energy of Formation), which is easily calculated using DFT methods, also gives a quantitative measure of the relative σ-donor strength of NHCs. CREF index values for a wide range of known and unknown NHC ring systems are reported and their significance discussed.

Contradictory reports on the behaviour of hydroquinone as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-quinone to tautomerise to the thermodynamically more stable para-quinone isomer. Oxidation of phenols by native tyrosinase requires activation by in situ formation of a catechol formed via an enzyme generated ortho-quinone. In the special case of hydroquinone, catechol formation is precluded by rapid tautomerisation of the ortho-quinone precursor to catechol formation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Tyrosinase is an enzyme widely distributed in the biosphere. It is one of a group of proteins with a strongly conserved bicopper active centre able to bind molecular oxygen. Tyrosinase manifests two catalytic properties; monooxygenase and oxidase activity. These actions reflect the oxidation states of the active centre. Tyrosinase has four possible oxidation states and the details of their interaction are shown to give rise to the unusual kinetic behaviour of the enzyme. The resting state of the enzyme is met-tyrosinase [Cu(II)2] and activation, associated with a 'lag period', involves reduction to deoxy-tyrosinase [Cu(I)2] which is capable of binding dioxygen to form oxy-tyrosinase [Cu(II)2·O2]. Initially the conversion of met- to deoxy-tyrosinase is brought about by a catechol that is indirectly formed from an ortho-quinone product of tyrosinase action. The primary function of the enzyme is monooxygenation of phenols to ortho-quinones by oxy-tyrosinase. Inactivation of the enzyme results from monooxygenase processing of catechols which can lead to reductive elimination of one of the active-site copper ions and conversion of oxy-tyrosinase to the inactive deact-tyrosinase [Cu(II)Cu(0)]. This review describes the tyrosinase pathways and the role of each oxidation state in the enzyme's oxidative transformations of phenols and catechols., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The inactivation of tyrosinase by resorcinol (1,3-dihydroxybenzene) and seventeen simple derivatives has been investigated using combined spectrophotometry and oximetry together with hplc/ms examination of the oxidation products. The results are consistent with a Quintox mechanism, analogous to that proposed for catechol inactivation of tyrosinase, in which the resorcinol substrate is oxidised via the monooxygenase route leading to a hydroxy intermediate that undergoes deprotonation and results in irreversible elimination of Cu(0) from the active site. Hplc/ms evidence for formation of the resorcinol monooxygenase product (3-hydroxy-ortho-quinone) is presented and the relationship between the ring position of simple resorcinol substituents (H, Me, F, Cl) and tyrosinase inactivation is rationalised., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: A haemodynamically significant ductus arteriosus (HSDA) is commonly associated with morbidity in preterm infants., Aim: To study the effect of the first dose of indomethacin on coronary blood flow in preterm neonates diagnosed with an HSDA., Method: A prospective observational echocardiographic study was performed on preterm infants. A single study dose of intravenous indomethacin (0.1 mg/kg) was administered over 1 h. Serial echocardiography was performed before and after indomethacin treatment to study the effect on coronary artery perfusion and cardiovascular performance., Results: Eighteen infants born at a median gestation of 25.8 (24.2, 28.1) weeks and a birth weight of 773 g (704, 1,002) were evaluated. The median age at indomethacin administration was 7.5 days (4, 17). There was no significant change in arterial pressure or ventilatory indices. Left anterior descending artery diastolic velocity and time integral declined from 0.3 ± 0.1 and 3.19 ± 1.2 m/s to 0.22 ± 0.08 and 2.01 ± 0.9 m/s, respectively, within 10 min of completion of infusion. These indices showed partial recovery when reassessed after 60 min. There were no changes in left ventricular output or transductal flow., Conclusions: Intravenous indomethacin was followed by a decline in coronary arterial diastolic blood flow., (Copyright © 2011 S. Karger AG, Basel.)

The reasons behind changes of aromaticity in 10 unsubstituted aza-azoles were analysed by employing the natural bond orbital (NBO) approach at the MP2/6-311+G(d,p) level of theory. Sum of occupations of p(z) orbitals at atoms in the ring correlates well with the magnetism based aromaticity index NICS as well as with the number of nitrogen atoms in the ring. Changes of NICS depend strongly in a linear way on the number of NN bonds. Classification of azoles based on the number of pyridine-type nitrogen atoms vicinal to NH is supported by plotting the relative occupation of π orbitals (π(occ)) against the relative occupation of σ orbitals (σ(occ)) for all individual atoms in rings.

4-Fluoro-1,2-benzoquinone, generated by tyrosinase oxidation of 4-fluorocatechol in aqueous buffer, rapidly undergoes substitution by O-nucleophiles (water or catechols) with release of fluoride. 4-Chloro- and 4-bromocatechol behave similarly. The reactions, which have toxicological implications, have been monitored by spectrophotometry and HPLC/MS, and intermediate and final products, including dibenzodioxins, identified.

Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder characterized by severe and rapidly progressive neurologic damage caused by the functional loss of sulfite oxidase, 1 of 4 molybdenum-dependent enzymes. To date, no effective therapy is available for MoCD, and death in early infancy has been the usual outcome. We report here the case of a patient who was diagnosed with MoCD at the age of 6 days. Substitution therapy with purified cyclic pyranopterin monophosphate (cPMP) was started on day 36 by daily intravenous administration of 80 to 160 microg of cPMP/kg of body weight. Within 1 to 2 weeks, all urinary markers of sulfite oxidase (sulfite, S-sulfocysteine, thiosulfate) and xanthine oxidase deficiency (xanthine, uric acid) returned to almost normal readings and stayed constant (>450 days of treatment). Clinically, the infant became more alert, convulsions and twitching disappeared within the first 2 weeks, and an electroencephalogram showed the return of rhythmic elements and markedly reduced epileptiform discharges. Substitution of cPMP represents the first causative therapy available for patients with MoCD. We demonstrate efficient uptake of cPMP and restoration of molybdenum cofactor-dependent enzyme activities. Further neurodegeneration by toxic metabolites was stopped in the reported patient. We also demonstrated the feasibility to detect MoCD in newborn-screening cards to enable early diagnosis.

3,6-Difluorocatechol, which cannot act as a monooxygenase tyrosinase substrate, is an oxidase substrate, and, in contrast to other catechols, oxidation does not lead to suicide-inactivation, providing experimental evidence for an inactivation mechanism involving reductive elimination of Cu(0) from the active site.

The influence of N-substituents on the mode of reaction of ortho-quinones generated by oxidation of N-substituted dopamine derivatives has been studied. Ortho-quinones with amide, urea or guanidine side chains are relatively stable, with evidence of rearrangement to para-quinomethanes. The N-methylthiourea derivative rapidly cyclises giving a bicyclic product . The trichloromethylamidine derivative also rapidly cyclises but in this case gives a spirocyclic derivative . In contrast to the transient formation of spirocyclic products by other ortho-quinones derived from dopamine derivatives, e.g., , the product is stable and has been isolated and fully characterised.

Tyrosinase is a mono-oxygenase with a dinuclear copper catalytic center which is able to catalyze both the ortho-hydroxylation of monophenols (cresolase activity) and the oxidation of catechols (catecholase activity) yielding ortho-quinone products. Tyrosinases appear to have arisen early in evolution and are widespread in living organisms where they are involved in several processes, including antibiosis, adhesion of molluscs, the hardening of the exoskeleton of insects, and pigmentation. Tyrosinase is the principal enzyme of melanin formation in vertebrates and is of clinical interest because of the possible utilization of its activity for targeted treatment of malignant melanoma. Tyrosinase is characterised by an irreversible inactivation that occurs during the oxidation of catechols. In a recent publication we proposed a mechanism to account for this feature based on the ortho-hydroxylation of catecholic substrates, during which process Cu(II) is reduced to Cu(0) which no longer binds to the enzyme and is eliminated (reductive elimination). Since this process is dependent on cresolase activity of tyrosinase, a strong prediction of the proposed inactivation mechanism is that it will not be exhibited by enzymes lacking cresolase activity. We show that the catechol oxidase readily extracted from bananas (Musa cavendishii) is devoid of cresolase activity and that the kinetics of catechol oxidation do not exhibit inactivation. We also show that a species with the molecular mass of the putative cresolase oxidation product is formed during tyrosinase oxidation of 4-methylcatechol. The results presented are entirely consistent with our proposed mechanism to account for suicide-inactivation of tyrosinase.

Tyrosinase is a copper-containing mono-oxygenase, widely distributed in nature, able to catalyze the oxidation of both phenols and catechols to the corresponding ortho-quinones. Tyrosinase is characterised by a hitherto unexplained irreversible inactivation which occurs during the oxidation of catechols. Although the corresponding catechols are formed during tyrosinase oxidation of monophenols, inactivation in the presence of monophenolic substrates is minimal. Previous studies have established the kinetic features of the inactivation reaction which is first-order in respect of the enzyme concentration. The inactivation reaction exhibits the same pH-profile and saturation properties as the oxidation reaction, classing the process as a mechanism-based suicide inactivation. The recent elucidation of the crystallographic structure of tyrosinase has stimulated a new approach to this long-standing enigma. Here we report the results of an investigation of the tyrosinase-catalysed oxidation of a range of hydroxybenzenes which establish the structural requirements associated with inactivation. We present evidence for an inactivation mechanism based on catechol hydroxylation, with loss of one of the copper atoms at the active site. The inactivation mechanism involves two linked processes occurring in situ: (a) catechol presentation resulting in alpha-oxidation, and (b) deprotonation of an adjacent group. On the basis of our experimental data we believe that a similar mechanism may account for the inhibitory action of resorcinols.

A pulse radiolytic investigation has been conducted to establish whether a redox reaction takes place between dopaquinone and 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) and to measure the rate constants of the interactions. To obviate possible confounding reactions, such as nucleophilic addition, the method employed to generate dopaquinone used the dibromide radical anion acting on dopa to form the semiquinone which rapidly disproportionates to dopaquinone. In the presence of DHI the corresponding indole-5,6-quinone (and/or tautomers) was also formed directly but, by judicious selection of suitable relative concentrations of initial reactants, we were able to detect the formation of additional indolequinone from the redox exchange reaction of DHI with dopaquinone which exhibited a linear dependency on the concentration of DHI. Computer simulation of the experimental time profiles of the absorption changes showed that, under the conditions chosen, redox exchange does proceed but not quite to completion, a forward rate constant of 1.4 x 10(6)/M/s being obtained. This is in the same range as the rate constants previously established for reactions of dopaquinone with cyclodopa and cysteinyldopa. In similar experiments carried out with DHICA, the reaction more obviously does not go to completion and is much slower, k (forward) =1.6 x 10(5)/M/s. We conclude that, in the eumelanogenic pathway, DHI oxidation may take place by redox exchange with dopaquinone, although such a reaction is likely to be less efficient for DHICA.

The influence of side-chain structure on the mode of reaction of ortho-quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N-substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase-oximetry. Ortho-quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para-quinomethanes was observed. A thiourea derivative cyclized fairly rapidly (k = 1.7/s) to a product containing a seven-membered ring, whereas a related amidine gave more rapidly (k approximately 2.5 x 10(2)/s) a stable spirocyclic product. The results suggest that cyclization of amides, ureas and carbamates (NHCO-X; X = R, NHR or OR) does not occur and is not, therefore, a viable approach to the formation of tyrosinase-activated antimelanoma prodrugs. It is also concluded that for N-acetyldopamine spontaneous ortho-quinone to para-quinomethane isomerization is slow.

Acts of dyadic orienting (responses to attention bids by a researcher) and acts of joint attention (e.g. pointing and showing behaviors) were observed in preschool children with autism and children with developmental delay. Children with autism responded to fewer adult vocal and non-vocal attention bids that were made singly and by combining modalities (e.g. name call plus touch). Sensitivity in dyadic orienting was significantly related to child-initiated acts of joint attention (IJA). Sensitivity to dyadic orienting was also significantly related to language and non-verbal ability. These findings indicate that dyadic orienting difficulties are found alongside triadic joint attention difficulties in children with autism.

Oxidation of amide, urea and guanidinium derivatives of dopamine gives relatively stable ortho-quinones whereas oxidation of corresponding thioamide and amidinium derivatives rapidly and quantitatively gives novel bicyclic and spirocyclic products formed via the corresponding ortho-quinone.

We measured the velocity and attenuation of audible sound in the isolated lung of the near-term fetal sheep to test the hypothesis that the acoustic properties of the lung provide a measure of the volume of gas it contains. We introduced pseudorandom noise (bandwidth 70 Hz-7 kHz) to one side of the lung and recorded the noise transmitted to the surface immediately opposite, starting with the lung containing only fetal lung liquid and making measurements after stepwise inflation with air until a leak developed. The velocity of sound in the lung fell rapidly from 187 +/- 28.2 to 87 +/- 3.7 m/s as lung density fell from 0.93 +/- 0.01 to 0.75 +/- 0.01 g/ml (lung density = lung weight/gas volume plus lung tissue volume). For technical reasons, no estimate of velocity could be made before the first air injection. Thereafter, as lung density fell to 0.35 +/- 0.01 g/ml, there was a further decline in velocity to 69.6 +/- 4.6 m/s. High-frequency sound was attenuated as lung density decreased from 1.0 to 0.5 g/ml, with little change thereafter down to a density of 0.35 +/- 0.01 g/ml. We conclude that both the velocity of audible sound through the lung and the degree to which high-frequency sound is attenuated in the lung provide information on the degree of inflation of the isolated fetal lung, particularly at high lung densities. If studies of sound transmission through the lung in the intact organism were to confirm these findings, the acoustic properties of the lung could provide a means for monitoring lung aeration during mechanical ventilation of newborn infants.

Enzymatic and chemical oxidation of 4-(4-N,N-dialkylaminobutyl)catechols leads to formation of 1,1-dialkyl-pyrrolidinium salts in good yield. It is proposed that these products are formed by tautomerism of the initially formed ortho-quinones to para-quinomethanes. The corresponding secondary amines do not form para-quinomethanes but cyclise giving tetrahydro-1H-benzo[b]azepine-7,8-diones. The failure of the dialkylaminobutyl derivatives to cyclise to bicyclic betaines, in a manner analogous to lower homologues and monoalkylaminobutyl derivatives, is attributed to steric hindrance. This proposal is supported by evidence that the sterically hindered N-tert-butylaminobutyl derivative, in contrast to other secondary amines, does not cyclise but gives a para-quinomethane-derived product. Based on pulse radiolysis and spectrophotometric evidence, para-quinomethane formation appears to be much slower than cyclisation and only occurs when cyclisation is unfavourable. The ortho-quinones formed from 5-aminopentylcatechols neither cyclise nor tautomerise suggesting that the chain length in these derivatives is too long for both cyclisation and intramolecular deprotonation.

The biological significance of the spontaneous cyclization and redox reactions of ortho-quinone amines is that these appear to be the mechanism of formation of the indolic components of melanin and are also involved in the autoactivation of tyrosinase. We have previously shown that activation of tyrosinase is prevented by the formation of a cyclic betaine from a tertiary amine analogue. Evidence is presented to show that cyclization of ortho-quinones by Michael addition also occurs in the oxidation of secondary catecholamines. Three varieties of cyclic product have been detected and their formation is influenced by the nature of the N-substituent. Five-membered betaine rings form directly and, although six- and seven-membered rings also form, a transient spiro isomer of the ortho-quinone was in some cases detected as an intermediate. The heterocyclic products formed as betaines undergo redox exchange with residual quinone to form the corresponding aminochromes. We have established the kinetic constants of these reactions, either directly by pulse radiolysis measurements or by inference using a computer model of the reaction pathway to fit the observed data. To investigate the potential biological applications of this chemistry the system was also examined by tyrosinase-catalysed oxidation of the catecholamine substrates in which there is re-oxidation of the catechol formed by the redox exchange reaction and enables measurement of oxygen utilization stoichiometry. We show that the redox exchange reaction is unaffected by side-chain modification whereas cyclization is dependent on both electronic and steric factors. In the light of these studies we conclude that the failure of tertiary amine-derived betaines to undergo redox exchange, and thus block in vitro activation of tyrosinase, is due to the absence of a second exchangeable proton.

Tyrosinase oxidizes tyrosine to dopaquinone, which undergoes nonenzymatic reactions leading to precursors of melanin pigments. Cyclization of dopaquinone gives cyclodopa, which participates in redox exchange with dopaquinone to give the eumelanin precursor dopachrome plus dopa. The indirect formation of the catechol (dopa) from the phenol (tyrosine) leads to unusual enzyme kinetics. Using a combination of enzyme oximetry, pulse radiolysis, and chemical oxidation, the study of structurally modified dopaquinones provides firm evidence of nonenzymatic catechol formation during tyrosinase oxidation of phenols and reveals significant differences in their modes of reaction.

The contributions of pulse radiolysis towards characterisation of unstable ortho-quinones relevant to melanogenesis are reviewed. The quinones discussed include dopaquinone, the precursor of both eumelanogenesis and phaeomelanogenesis, and 5-S-cysteinyldopaquinone, an early component of the phaeomelanogenic pathway. Redox exchange between dopaquinone and 5-S-cysteinyldopa is shown to be a determinant of the balance between eumelanogenesis and phaeomelanogenesis. Ortho-quinones resulting from the oxidation of tertiary N,N-dialkylcatecholamines cyclise to redox-inactive betaines which fail to autoactivate tyrosinase. This is consistent with the dopa detected during melanogenesis catalysed by tyrosinase being formed indirectly by a combination of dopaquinone intramolecular reductive addition to form leucodopachrome (cyclodopa), followed by redox exchange between remaining dopaquinone and leucodopachrome. Rapid tautomerism of the ortho-quinone of 4-cyanomethylcatechol to a redox-inactive quinomethane likewise inhibits tyrosinase autoactivation. The incorporation of trihydric phenol moieties in melanin is modelled by the reactions of several ortho-quinones with phloroglucinol, which itself is not directly oxidised by tyrosinase due to the meta-positioning of the hydroxyl groups. The importance of a susceptibility towards nucleophilic attack as well as a propensity to undergo redox-exchange, in the chemistry of melanogenic ortho-quinones, is emphasised.

This study aimed to examine the performance characteristics of four high-frequency oscillatory-type ventilators, using an in vitro model of the intubated neonatal respiratory system. Each ventilator was examined across its operative range of settings and at varying model lung compliance (C) and resistance. The oscillatory pressure waveform was measured at the airway opening (Pao). Tidal volume (VT) and flow were determined from pressure changes within the model lung (DeltaPA). The spectral content of the Pao waveform differed between ventilators. The maximum ventilator VT ranged from 3.7 to 11.1 ml at 15 Hz and a mean airway pressure (Paw) of 12 cm H(2)O to oscillate a model lung (C = 0.4 ml/cm H(2)O) through a 3.0-mm internal diameter (i.d.) endotracheal tube (ETT). A small drop in C was associated with a decrease in VT and marked increase in DeltaPA from 0.1 to 0.8 ml/cm H(2)O. The influence of C on VT and DeltaPA and the pressure cost of ventilation (DeltaPA/f.VT(2)) was dependent on the oscillatory frequency, ETT inner diameter, and the specific ventilator used. Substantive differences exist between oscillatory ventilators that need to be considered in their clinical application. The rapid establishment of optimal lung volume and oscillatory frequency is important in minimizing barotrauma during high-frequency oscillatory ventilation.

1. Most of the liquid that fills the lung of the fetal sheep in late gestation is cleared by the end of labour. Clearance of this liquid has a beneficial effect on postnatal gas exchange and therefore represents an important adaptation for postnatal life. Despite its importance, there is disagreement about whether clearance begins prior to labour, or occurs entirely within labour. 2. To address this issue, we made serial determinations of lung liquid volume by indicator dilution during late gestation and labour in the fetal sheep. 3. Regression analysis demonstrated that lung liquid volume exhibited a plateau level in the near-term fetus before it began to decline. Two models provided a fit to the decline in volume. In one, lung liquid clearance occurred in two linear phases, the first beginning 70 h before the study was terminated when the ewe was in advanced labour, the second occupying the last 8 h of the study period. In the initial phase, average lung liquid volume fell from 38.3 to 26.4 ml x kg(-1) before a rapid decline in the second phase reduced the volume to 13.8 ml x kg(-1). An exponential decay model was also found to fit the data; this showed a gradual decline in lung liquid volume in the 2 days preceding onset of labour, followed by a much more rapid decline within labour. 4. The rate of lung liquid secretion also declined in two linear phases, both of which commenced earlier than the changes in lung liquid volume. An exponential decay model also gave a significant fit to the data, but the fit was significantly weaker than that achieved with the two-slope model. 5. We conclude that clearance of lung liquid begins well before commencement of labour in the full term fetal sheep, and then accelerates once labour is established. In our study, lung liquid volume fell even in the absence of reabsorption of liquid across the pulmonary epithelium, indicating that outflow of liquid through the trachea must have occurred at a rate in excess of the secretion rate.

The efficacy of pulmonary gas exchange immediately after delivery is inversely related to the volume of liquid in the lung at birth, but aspiration of as much liquid as possible from the lung before Caesarean delivery fails to improve postnatal oxygenation (Pa,O2) to the level achieved after spontaneous term delivery. We hypothesised that the differing respiratory benefit of aspiration and vaginal delivery results from the differing volume of lung liquid remaining after aspiration (17 ml (kg body weight)-1) and labour (7 ml kg-1). We addressed this hypothesis by reducing lung liquid volume to an estimated 7 ml kg-1 by infusing adrenaline to seven fetal lambs at 140 days gestation (term is 147 days) before performing Caesarean delivery and obtaining postnatal blood gases for comparison with samples from lambs delivered vaginally. Infusion of adrenaline to fetuses caused a progressive decline in arterial O2 saturation (Sa, O2), pH and base excess, but no change in arterial partial pressure of O2 (Pa,O2) or CO2 (Pa,CO2). After birth, Pa,O2 rapidly rose to the same level in adrenaline-treated and vaginal-delivery groups. A severe acidosis occurred in the adrenaline-treated group and this appeared to be related to a higher Pa,CO2 and a transiently lower Sa, O2 in this group. We conclude that adrenaline infusion can enhance postnatal Pa,O2 levels in the newborn lamb, but this beneficial effect may be outweighed by the severe acidosis that develops after prolonged prenatal adrenaline treatment.

Objective: To describe and characterize a new disorder of hyaluronan metabolism associated with marked abnormalities of cutaneous tissue and to determine whether a relationship with a phenotypically similar disorder in the shar-pei dog exists., Methods: Biopsy specimens of the skin of a child with extreme cutaneous thickening and folding were examined by light and electron microscopy. The concentration of hyaluronan and the activity of hyaluronidase were measured in the patient's serum and plasma, respectively, and the activity of hyaluronan synthase was examined in cultured dermal fibroblasts. Hyaluronan concentration was also measured in the plasma of 23 shar-pei and 34 control dogs., Results: The patient's skin displayed gross accumulation of hyaluronan, and the serum concentration of hyaluronan was markedly elevated (up to 3100 microg/L) during infancy. Hyaluronan synthase activity of cultured dermal fibroblasts was increased, whereas hyaluronidase activity in plasma was normal (5.5 +/- 0.08 IU/L). Plasma hyaluronan concentration was higher in the shar-pei dogs than in control dogs (median, 378 microg/L vs 73 microg/L, respectively)., Conclusion: The child we describe has a novel disorder of hyaluronan metabolism, which appears to result from abnormal control of hyaluronan synthesis. An analogous disorder may be present in the shar-pei dog.

Fetal lung liquid volume is usually determined by using radio-iodinated serum albumin (RISA) or blue dextran (BD) as volume tracers. We tested the reliability of both tracers at 124 (G124) and 142 days of gestation (G142; term = G147) when the labels were employed simultaneously. We measured the proportion of label bound reversibly to the lung, or apparently lost from the lung compartment, by washing out the lung with saline and 5% albumin. At G124, volume estimates with the two labels were similar. At G142, the volume estimate with BD (36.3 +/- 8.7 ml/kg of body wt) was higher (P < 0. 05) than with RISA (22.3 +/- 3.5 ml/kg). This difference resulted from reversible binding of BD, because 5% albumin washout released 38.5 +/- 4.0% of the BD added at the start of the experiment but a lesser amount of RISA (9.8 +/- 0.7%; P < 0.05). At G142, when RISA was used alone, its reversible binding was 1.3 +/- 0.2%. Background absorbance increased during experiments, giving rise to an apparent increase in BD concentration. We conclude that RISA is an effective tracer for lung liquid volume determination in the fetal lamb, whereas our findings of substantial epithelial binding of BD and large changes in background absorbance demonstrate that, under the conditions of our experiments, BD is a poor tracer close to term.

This study investigated factors contributing to differences between mean alveolar pressure (PA) and mean pressure at the airway opening (Pao) during high-frequency oscillatory ventilation (HFOV). The effect of the inspiratory-to-expiratory time (I/E) ratio and amplitude of oscillation on the magnitude of - Pao (Pdiff) was examined by using the alveolar capsule technique in normal rabbit lungs (n = 4) and an in vitro lung model. The effect of ventilator frequency and endotracheal tube (ETT) diameter on Pdiff was further examined in the in vitro lung model at an I/E ratio of 1:2. In both lung models, fell below Pao during HFOV when inspiratory time was shorter than expiratory time. Under these conditions, differences between inspiratory and expiratory flows, combined with the nonlinear relationship between resistive pressure drop and flow in the ETT, are the principal determinants of Pdiff. In our experiments, the magnitude of Pdiff at each combination of I/E, frequency, lung compliance, and ETT resistance could be predicted from the difference between the mean squared inspiratory and expiratory velocities in the ETT. These observations provide an explanation for the measured differences in mean pressure between the airway opening and the alveoli during HFOV and will assist in the development of optimal strategies for the clinical application of this technique.

A range of imidazoline derivatives are known to be effective stimulators of insulin secretion, and this response correlates with closure of ATP-sensitive potassium channels in the pancreatic beta-cell. However, mounting evidence indicates that potassium channel blockade may form only part of the mechanism by which imidazolines exert their effects on insulin secretion. Additionally, it remains unclear whether members of this class of drugs can bind directly to potassium channel components and whether occupation of a single binding site accounts for their functional activity. This review considers recent developments in the field and highlights evidence that does not fit readily with the concept that a single mechanism of action is sufficient to mediate the effects of imidazolines on pancreatic hormone secretion.

We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP-sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino-efaroxan blocks ATP-sensitive potassium channels in human pancreatic beta-cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.

The insulin secretagogue activity of certain imidazoline compounds is mediated by a binding site associated with ATP-sensitive K+ (K(ATP)) channels in the pancreatic beta-cell. We describe the effects of a series of structural modifications to efaroxan on its activity at this site. Substitution of amino-, nitro- or azide- groups onto the 5-position of the benzene ring of efaroxan did not significantly affect the functional interaction of the ligand with the islet imidazoline binding site. Modification of the imidazoline ring to an imidazole to generate 2-(2-ethyl-2,3-dihydrobenzo[b]furan-2-yl)-1H-imidazole (KU14R) resulted in loss of secretagogue activity. Indeed, this reagent appeared to act as an imidazoline antagonist since it blocked the secretory responses to imidazoline compounds and also inhibited the blockade of beta-cell K(ATP) channels by efaroxan in patch clamp experiments. Application of KU14R alone resulted in a modest reduction in K(ATP) channel opening, suggesting that it may display weak partial agonism, at least in patch-clamp experiments.

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1.cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol.min-1 and the apparent Km was 374 microM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol.min-1 and Km 309 microM. The quinomethane was found to decay with a rate constant of 2k 38 M-1.s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.

Objective: Our aim was to determine the volume of liquid remaining in the lungs of the fetal lamb just before a normal vaginal delivery at term to assess the extent to which an excess of liquid in the airspaces might contribute to the respiratory morbidity that accompanies elective cesarean delivery., Study Design: The volume of liquid in the future airspace of the lungs was determined at the end of labor in eight fetal lambs at term from the dilution of an impermeable tracer (125I-labeled human serum albumin) mixed into the liquid. This volume was compared with that measured in a second group of 10 fetal lambs studied 7 days before the expected date of delivery (term = 147 days)., Results: The volume of lung liquid present at the end of labor was 6.8 +/- 1.0 ml x kg(-1) (n = 8) compared with 28.2 +/- 1.8 ml x kg(-1) (n = 10) in the second group of lambs studied before the onset of labor at 140 days of gestation., Conclusion: Our results indicate that the bulk (>75%) of the liquid that fills the lungs of the fetal lamb at 140 days of gestation is cleared at some time before normal term birth, suggesting that the adverse respiratory impact of elective cesarean delivery may be largely explained by denying the fetus this important adaptive mechanism.

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s-1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N, N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

Recent studies have suggested that a variety of ion channels possess a binding site for ligands such as phencyclidine (PCP), dizocilpine and certain sigma ligands and that some imidazoline compounds can also bind to this site. We have investigated whether interaction with this binding site could account for the ability of imidazolines to stimulate insulin secretion from rat islets. Neither PCP nor dizocilpine shared the insulin secretory activity of the imidazoline efaroxan in rat islets suggesting that they do not have similar actions in the pancreatic B-cell. Further, we were able to define a new antagonist, KU14R (2(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole), which selectively blocks the insulin secretory response to imidazolines. The results suggest that imidazolines do not stimulate insulin secretion by causing physical blockade of the K(+)-ATP channel in pancreatic B-cells and show that their effects are not reproduced by PCP or sigma receptor ligands.

A set of 26 substituted phenols, 10 of which were synthesised in our laboratories, were tested for their rate of oxidation by mushroom tyrosinase in vitro as determined by oximetry and spectrophotometry and for their cytotoxic action in a model system. With one exception (4-hydroxybenzoic acid) all the agents tested were oxidised to the corresponding ortho-quinones. The maximum rates of oxidation varied between 15.1 +/- 0.59 nmoles oxygen consumed per minute (4-(2-thioethylthio)phenol) and 372.9 +/- 5.61 nmoles O2/ min. (4-(2-Hydroxyethylthio)phenol) in a reaction system comprising 300 units tyrosinase and 200 microM substrate. The rates of generation of quinone were in close agreement with these oximetric data. Some anomalies in oxygen stoichiometry were observed due to reoxidation of reaction products. Four categories of compounds were tested: those known to undergo side-chain cyclisation (such as tyrosine) (Group A), alkylphenols of increasing chain length with or without terminal hydroxyl groups (Group B), compounds with charged or bulky side-chains (Group C) and agents with oxy-, thio- and selenyl-ether side-chains (Groups D, E and F). In the majority of cases, the cytotoxicity, measured by the reduction of thymidine incorporation in cells exposed for 30 min to the agent in the presence of tyrosinase, reflected the rate of oxidation and is ascribed to the toxic action of the derived ortho-quinone. Tyrosinase-dependent cytotoxicity was absent in cyclising (Group A) and in Group C compounds. Toxicity, expressed by comparison with 4-hydroxyanisole (4HA) (IC50 = 11.7 microM), ranged between 0.36 (4-hydroxybenzyl alcohol) and 1.07 (3-(4-hydroxyphenyl)propanol) for Group B compounds, and be-tween 0.83 (4-ethoxyphenol) and 2.08 (4-(2-hydroxyethylthio)phenol) for groups D, E and F. Addition of glutathione to the toxicity assay system abrogated the cytotoxic action and, on the basis of spectrophotometric data, this is ascribed to the prevention of cellular thiol depletion by the ortho-quinone products of tyrosinase oxidation of the phenolic substrates. The lack of toxicity of the group C compounds may be due to the inability of their derived quinones to gain access to the cells. Addition of catalase or deferoxamine to the incubation medium was without effect on tyrosinase-dependent toxicity.

1. The volume of liquid in the lungs of the fetal lamb is reported to fall in the final days of gestation and during labour itself. We aimed to test the hypothesis that this fall in liquid volume adapts the lungs for air breathing and pulmonary gas exchange. 2. In twelve chronically catheterized fetal lambs we measured lung liquid volume at 140 days gestation (term is 147 days) and then delivered the fetuses by Caesarean section under maternal spinal anaesthesia. In five fetuses we removed approximately half the liquid contained in the lungs just before delivery (experimental group) while the remaining seven fetuses were delivered without change to their lung liquid (control group). 3. Lambs born with reduced lung liquid volume improved their arterial blood gas and acid-base status more quickly than lambs born without alteration to lung liquid. 4. Carotid arterial blood gas values in the first 60 min of postnatal life were significantly related to the volume of liquid present in the lungs at birth, with higher arterial partial pressure of oxygen (Pa,02) and arterial oxygen saturation (Sa,02) and lower arterial partial pressure of carbon dioxide (Pa,CO2) levels being associated with lower lung liquid volumes. 5. We conclude that postnatal gas exchange is enhanced by a reduction in the volume of liquid remaining in the lungs when breathing starts.

Many women attend the Accident and Emergency department with vaginal bleeding as the first signs of a possible miscarriage. This study was undertaken to identify the views of Accident and Emergency nurses with regard to counselling these women in the Accident and Emergency department, and who should be undertaking the counselling. 50 questionnaires were distributed to 10 Accident and Emergency departments within the Yorkshire region, to qualified nurses of various grades and experience. 50% of the respondents said that counselling should be undertaken during the couple's stay in the department. This paper discusses where and when counselling should be undertaken, by whom and also focuses on the feelings, thoughts and needs of the couple.

Rate constants have been determined for reactions between biologically significant thiols, represented by cysteine and glutathione, and a series of 10 4-substituted o-quinones, and unsubstituted o-quinone itself, generated by rapid disproportionation of the semiquinones formed from the corresponding catechols by pulse radiolysis. The quantitative structure-reactivity relationships were investigated by examining the correlation between the rate constants and various Hammett and other parameters characterizing the electronic nature of the substituents. From these relationships, it can be concluded that the o-quinone reactivity with thiols increases with the electron-withdrawing capacity of the substituent groups and that this effect is principally due to resonance effects. Such relationships allow the prediction of likely reactivities with cellular thiols of further o-quinones whose 4-substituents have known electronic parameters. These reactivities are likely to be one of the critical factors determining overall cytotoxicity, assisting in the choice of improved melanogenesis-targeted anti-melanoma drugs.