Investigation of the magnetic ground state of GaV4S8using powder neutron diffraction.

The magnetic ground state of polycrystalline Néel skyrmion hosting material GaV4S8has been investigated usingacsusceptibility and powder neutron diffraction. In the absence of an applied magnetic field GaV4S8undergoes a transition from a paramagnetic to a cycloidal state below 13 K and then to a ferromagnetic-like state below 6 K. With evidence fromacsusceptibility and powder neutron diffraction, we have identified the commensurate magnetic structure at 1.5 K, with ordered magnetic moments of 0.23(2) µBon the V1 sites and 0.22(1) µBon the V2 sites. These moments have ferromagnetic-like alignment but with a 39(8)° canting of the magnetic moments on the V2 sites away from the V4cluster. In the incommensurate magnetic phase that exists between 6 and 13 K, we provide a thorough and careful analysis of the cycloidal magnetic structure exhibited by this material using powder neutron diffraction.

Real-space observation of ferroelectrically induced magnetic spin crystal in SrRuO3.

Unusual features in the Hall Resistivity of thin film systems are frequently associated with whirling spin textures such as Skyrmions. A host of recent investigations of Hall Hysteresis loops in SrRuO3 heterostructures have provided conflicting evidence for different causes for such features. We have constructed an SrRuO3-PbTiO3 (Ferromagnetic - Ferroelectric) bilayer that exhibits features in the Hall Hysteresis previously attributed to a Topological Hall Effect, and Skyrmions. Here we show field dependent Magnetic Force Microscopy measurements throughout the key fields where the 'THE' presents, revealing the emergence to two periodic, chiral spin textures. The zero-field cycloidal phase, which then transforms into a 'double-q' incommensurate spin crystal appears over the appearance of the 'Topological-like' Hall effect region, and develop into a ferromagnetic switching regime as the sample reaches saturation, and the 'Topological-like' response diminishes. Scanning Tunnelling Electron Microscopy and Density Functional Theory is used to observe and analyse surface inversion symmetry breaking and confirm the role of an interfacial Dzyaloshinskii-Moriya interaction at the heart of the system.

Ultrathin frozen sections. II. Demonstration of enzymic activity.

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35 degrees C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70 degrees C and the knife at -23 degrees C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.

Management of multispecies fisheries.

With the overexploitation of many conventional fish stcocks, and growing interest in harvesting new kinds of food from the sea, there is increasing need for managers of fisheries to take account of interactions among species. In particular, as Antarctic krill-fishing industries grow, there is a need to agree upon sound principles for managing the Southern Ocean ecosystem. Using simple models, we discuss the way multispecies food webs respond to the harvesting of species at differrent trophic levels. These biological and economic insights are applied to a discussion of fisheries in the Southern Otean and the North Sea and to enunciate some for harvesting in multispecies systems.

Hypertrophy and hyperplasia of the neonatal rat exocrine pancreas induced by orally administered soybean trypsin inhibitor.

Measurement of RNA, DNA, protein and phospholipid synthesis in the neonatal rat pancreas following the oral administration of partially purified soybean trypsin inhibitor demonstrates an enhanced synthesis of all these constituents. Evidence of true hyperplasia in addition to this cellular hypertrophy is provided by an increased mitotic activity in the exocrine pancreas following a wave of enhanced [3H] thymidine incorporation into DNA. Complete inhibition of the stimulated RNA synthesis by low doses of actinomycin D indicates the importance of transcription as a regulatory step in the response of the exocrine pancreas to trophic stimulation by this means. Collateral observations of [3H] thymidine and [14C] orotic acid incorporation into liver DNA and RNA, respectively, fail to demonstrate comparable changes confirming the probable specificity of the trypsin inhibitor induced effect on the exocrine pancreas. It is suggested that the pronounced trophic effect of orally administered soybean trypsin inhibitor in the neonatal rat pancreas may serve as a useful model for the study of regulatory mechanisms of nucleic acid and protein synthesis in the mammalian pancreas.

Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail.

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

ESTA: a bioassay system for the determination of the potencies of hormones and antibodies which mimic their action.

A bioassay system named ESTA (eluted stain assay) has been developed to measure hormones and antibodies which mimic their action. It is derived from approaches used for cytochemical bioassays. Unlike the latter which use tissue segments or sections, ESTA is based upon uniform microcultures of target cells maintained in microtitre plates. Direct elution of the cytochemical stain from these microcultures into the wells of the microtitre plates permits rapid quantification with a microtitre plate reader. We describe ESTA systems for GH, prolactin, thyroid stimulators and human chorionic gonadotrophin which utilize the reduction of a tetrazolium salt to a formazan by intracellular dehydrogenase as the cytochemical system. These provide examples of ESTA systems in which the assay signal depends solely upon an increase in cell number in response to the hormone, or in which there is additional enzymic amplification.

Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

Epidermal growth factor induced tyrosine phosphorylation of nuclear proteins associated with translocation of epidermal growth factor receptor into the nucleus.

Treatment of human squamous carcinoma cells (HN5 cells) with epidermal growth factor (EGF) caused a time-dependent increase in tyrosine phosphorylation of six nuclear proteins of molecular mass 166, 140, 117, 95, 86 and 79 kDa. The major tyrosine phosphorylated protein was indistinguishable from the plasma membrane form of the epidermal growth factor receptor and was shown by enzyme linked immunosorbent assay (ELISA) to be translocated into the nucleus from extra-nuclear sites upon ligand stimulation. Using immunoelectron microscopy of both isolated nuclei and whole cells, epidermal growth factor receptor (EGF-R) was found to be associated with the chromatin and, to a lesser extent, with the inner surface of the nuclear membrane. Tyrosine phosphorylation of proteins other than EGF-R was particularly notable in the nucleoli. These observations suggest that EGF-R may exert some of its physiological functions by directly inducing tyrosine phosphorylation of specific nuclear proteins. Translocation of EGF-R to the nucleus may provide a vital link between plasma membrane signalling and gene activation.

Studies on the sympathetic efferent nerves of brown adipose tissue of lean and obese Zucker rats.

Previous studies have suggested that the sympathetic tone to brown adipose tissue (BAT) is reduced in the genetically obese (fa/fa) rat. The following experiments were designed to examine with electrophysiological techniques the activity of the sympathetic nerve innervating the interscapular BAT. The spontaneous activity of the efferent nerves was reduced in the obese (fa/fa) rat compared with the lean control. The activity of the nerve showed a linear relationship with changes in core temperature in both genotypes. Electrical stimulation of the ventromedial hypothalamus resulted in similar heat increments in BAT temperature for lean and obese, but this was associated with a smaller increase in nerve firing in the obese rat. Intracerebroventricular administration of glucose enhanced the nerve activity, whereas 2-deoxy-D-glucose reduced the nerve activity in both lean and obese rats. These data suggest that the sympathetic tone is suppressed in the genetically obese rat, but the response to temperature and central glucose metabolism is intact.

Interaction of intracerebroventricular insulin and glucose in the regulation of the activity of sympathetic efferent nerves to brown adipose tissue in lean and obese Zucker rats.

The effects of injection of insulin and glucose into the third cerebral ventricle on the firing rate of the sympathetic efferent nerves to intercapsular brown adipose tissue was investigated in anaesthetized lean and obese Zucker rats. Injection of insulin resulted in a dose-dependent (70-480 pmol) inhibition of nerve firing rate, whereas in combination with glucose (140 pmol of insulin and 139 nmol of glucose), insulin strongly potentiated the increase in firing rate seen with glucose alone. Although basal levels of nerve firing rates were lower in the obese rat, responses to insulin, glucose, and insulin plus glucose were qualitatively similar to those seen in the lean rat. These results are consistent with the hypothesis that insulin acts in the central nervous system as a physiological signal in the control of thermogenesis after feeding, and that this effector system is intact in the obese rat.

Hypothalamic control of brown adipose tissue in Zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres.

Electrophysiological stimulation of the hypothalamic ventromedial nucleus (VMN) resulted in an increase in interscapular brown adipose tissue (BAT) temperature in both lean and obese (fa/fa) rats. Graded stimulations resulted in progressively larger temperature increases in both lean and obese (fa/fa) groups. Both intraperitoneal injection of propranolol and surgical denervation (but not sham denervation) abolished the increase in BAT temperature following VMN stimulation, in both lean and obese (fa/fa) groups. Electrical stimulation of the supraoptic region, and certain anterior hypothalamic regions also resulted in increases in BAT temperature of lean and obese (fa/fa) rats, but stimulation of the dorsomedial nucleus and regions of the lateral hypothalamus did not affect BAT temperature. All hypothalamic regions capable of activating BAT gave a similar maximum rise in temperature for a given stimulus in lean and obese (fa/fa) rats. These results suggest that the efferent sympathetic pathway from the VMN and other hypothalamic regions of BAT is normal in the obese (fa/fa) rat.

Tumour necrosis factor-alpha and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system.

Intracerebral injection of recombinant human tumour necrosis factor-alpha (rTNF alpha) decreased, whereas lymphotoxin increased the activity of the sympathetic efferent nerves to interscapular brown adipose tissue (IBAT). The changes in firing rates were accompanied by a significant dose-dependent change in IBAT and rectal temperatures (which were decreased with rTNF alpha and increased with lymphotoxin treatment). These results indicate that rTNF alpha and lymphotoxin act directly in the CNS to modulate the sympathetic outflow to IBAT. The opposite effects of the two peptides also suggest that in the CNS, separate receptor systems exist.

Response of brown adipose tissue to electrical stimulation of hypothalamic centres in intact and adrenalectomized Zucker rats.

Electrical stimulation of the ventromedial nucleus of the hypothalamus (VMN) increased brown adipose tissue (BAT) temperature in lean and obese Zucker rats, whereas stimulation of the paraventricular nucleus (PVN) was without effect in either genotype, before or after adrenalectomy. The prolonged duration of the increase in BAT temperature in the obese (fa/fa) rat observed after VMN stimulation was normalized after adrenalectomy.

The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

Work of breathing associated with pressure support ventilation in two different ventilators.

The purpose of this study was to compare the work of breathing during pressure support ventilation (PSV) with positive end expiratory pressure (PEEP) utilizing the Siemens SV300 (SV300) and Dräger Evita 4 (EV4) ventilators. Our hypothesis was that patients' work of breathing (WOB(P)) would be unchanged in PSV utilizing flow triggering (FT) in both the SV300 and EV4. We compared two ventilators using six healthy, intubated, sedated, spontaneously breathing pigs weighing approximately 10 kg each. WOB(P) (j/L) and ventilator work of breathing (WOB(V)) (j/L) were measured using a portable monitor which utilizes an esophageal balloon and flow transducer. Each breath was further analyzed for duration of inspiratory effort and negative deflection of pressure needed to trigger PSV. Animals were studied with the SV300 and EV4 on a pressure support of 5 cmH(2)O and PEEP settings of 0 and 5 cmH(2)O. Data were analyzed using the Wilcoxon signed rank test with significance set at P

Effect of lateral hypothalamic lesion on brown adipose tissue of Zucker lean and obese rats.

Acute (10-day) lateral hypothalamic (LH) lesion induced a reduction of food intake in both lean and obese Zucker rats which averaged about 50% over the course of the first 10 days. The aphagia associated with a fall in body weight in both genotypes which was greater than their respective pair-fed controls, indicating a change in energetic efficiency. The reduced level of BAT protein, mitochondria and GDP binding observed in the obese rat was restored after LH lesion, suggesting the reestablishment of a normal sympathetic drive to the tissue. The markedly lower plasma insulin concentration in the LH lesioned obese rat is consistent with a reduction in parasympathetic activity in these animals. Food restriction in the sham lean rat reduced BAT protein content and mitochondrial GDP binding, whereas no such changes were observed in the food restricted obese rat. This demonstrates the insensitivity of the obese rat to dietary signals and may imply that LH lesion restores diet-induced BAT thermogenesis in the obese rat.

The effects of adrenalectomy, corticotropin releasing factor and vasopressin on the sympathetic firing rate of nerves to interscapular brown adipose tissue in the Zucker rat.

The firing rate of the sympathetic efferent nerves to interscapular brown adipose tissue (IBAT) is lower in the obese rat compared with the lean rat. The present experiments show that adrenalectomy has no effect on nerve firing rate in the lean rat and a small but statistically nonsignificant effect in the obese rat. Injection of corticotropin releasing factor (CRF) into the IIIrd ventricle produced a dose dependent increase in the firing rate of the sympathetic nerves to interscapular brown adipose tissue (IBAT) in both lean and obese rats. The basal (unstimulated) level of firing was lower in the obese rat compared with the lean rat and remained significantly below lean values at each dose. The minimum dose of CRF to see an effect (125 ng) and the dose at which maximum effect on nerve firing rate was observed (500 ng) was similar in both genotypes. Injection of adrenocorticotropic hormone (ACTH) had no effect on nerve firing rate to IBAT. Central administration of vasopressin produced a significant increase in sympathetic firing rate to IBAT in both lean and obese rats. The temperature of IBAT was also significantly increased with vasopressin and the duration of the response was longer compared with CRF, but the minimum dose to see an effect was higher (2.5 micrograms). The response to vasopressin was greater in the obese rat compared with the lean rat but the maximum firing rate did not achieve levels observed in lean rats. Chronic infusion of CRF into the IIIrd ventricle of obese rats resulted in a reduction of food intake and body weight gain but IBAT mitochondrial GDP binding was unaltered by the treatment. These data are consistent with the hypothesis that the defect in the obese Zucker rat may be due to a glucocorticoid inhibition of CRF and/or vasopressin action in the CNS.

High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese 'fa/fa' rats.

The number of high affinity [3H]GDP binding sites in brown adipose tissue mitochondria is normal in obese (fa/fa) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.