Unexpected electric-field-induced antiferroelectric liquid crystal phase in the SmC_{α}

The unique nanometer-sized helical structure in SmC_{α}^{*} may sometimes evolve continuously to the micrometer-sized one in SmC^{*}; conceivably ferroelectric SmC_{α}^{*} is to be unwound by an applied electric field. By drawing electric-field-induced birefringence contours in the field-temperature phase diagram and by studying the superlattice structure of the field-induced subphase with resonant x-ray scattering, we established that an applied field unexpectedly stabilizes the well-known antiferroelectric four-layer biaxial subphase as well as the other prototypal ferrielectric three-layer one in the SmC_{α}^{*} temperature range; the effective long-range interlayer interaction due to the discrete flexoelectric effect actually plays an important role in stabilizing not only the biaxial subphases but also the optically uniaxial SmC_{α}^{*} subphase, contrary to the notion that the competition between the direct interactions of the nearest-neighbor layers and those of the next-nearest-neighbor layers should be required for the nanometer-sized helical structure.

Definite existence of subphases with eight- and ten-layer unit cells as studied by complementary methods, electric-field-induced birefringence and microbeam resonant x-ray scattering.

A mixture of two selenium-containing compounds, 80 wt. % AS657 and 20 wt. % AS620, are studied with two complementary methods, electric-field-induced birefringence (EFIB) and microbeam resonant x-ray scattering (µRXS). The mixture shows the typical phase sequence of Sm-C_{A}^{*}-1/3-1/2-Sm-C^{*}-Sm-C_{α}^{*}-Sm-A, where 1/3 and 1/2 are two prototypal ferrielectric and antiferroelectric subphases with three- and four-layer unit cells, respectively. Here we designate the subphase as its q_{T} number defined by the ratio of [F]/([F]+[A]), where [F] and [A] are the numbers of synclinic ferroelectric and anticlinic antiferroelectric orderings in the unit cell, respectively. The electric field vs temperature phase diagram with EFIB contours indicates the emergence of three additional subphases, an antiferroelectric one between Sm-C_{A}^{*} and 1/3 and antiferroelectric and apparently ferrielectric ones between 1/3 and 1/2. The simplest probable q_{T}'s for these additional subphases are 1/4, 2/5, and 3/7, respectively, in the order of increasing temperature. The µRXS profiles indicate that antiferroelectric 1/4 and 2/5 approximately have the eight-layer (FAAAFAAA) and ten-layer (FAFAAFAFAA) Ising unit cells, respectively. The remaining subphase may be ferrielectric 3/7 with a seven-layer unit cell, although the evidence is partial. These experimental results are compared with the phenomenological Landau model [P. V. Dolganov and E. I. Kats, Liq. Cryst. Rev. 1, 127 (2014)2168-039610.1080/21680396.2013.869667] and the quasimolecular model [A. V. Emelyanenko and M. A. Osipov, Phys. Rev. E 68, 051703 (2003)1063-651X10.1103/PhysRevE.68.051703].

Alginate nanoparticles protect ferrous from oxidation: Potential iron delivery system.

A novel, efficient delivery system for iron (Fe2+) was developed using the alginate biopolymer. Iron loaded alginate nanoparticles were synthesized by a controlled ionic gelation method and was characterized with respect to particle size, zeta potential, morphology and encapsulation efficiency. Successful loading was confirmed with Fourier Transform Infrared spectroscopy and Thermogravimetric Analysis. Electron energy loss spectroscopy study corroborated the loading of ferrous into the alginate nanoparticles. Iron encapsulation (70%) was optimized at 0.06% Fe (w/v) leading to the formation of iron loaded alginate nanoparticles with a size range of 15-30nm and with a negative zeta potential (-38mV). The in vitro release studies showed a prolonged release profile for 96h. Release of iron was around 65-70% at pH of 6 and 7.4 whereas it was less than 20% at pH 2.The initial burst release upto 8h followed zero order kinetics at all three pH values. All the release profiles beyond 8h best fitted the Korsmeyer-Peppas model of diffusion. Non Fickian diffusion was observed at pH 6 and 7.4 while at pH 2 Fickian diffusion was observed.

Controlled release of diclofenac sodium in glycolipid incorporated micro emulsions.

The effect of the glycolipid, hexadecyl-ß-d-glucopyranoside, incorporated in microemulsions (ME(1)) towards the enhancement of skin absorption and skin permeation of Diclofenac sodium (DS(2)) was evaluated. A Franz diffusion cell with a piece of pig's ear epidermis indicated that the optimized ME formulation with glycolipid (0.05wt%) exhibited significantly higher permeability than the conventional formulations. The releasing profiles of DS from ME formulations exhibited first order release kinetics resembling a diffusion controlled release model for the first 8h. Incorporating hexadecyl-ß-D glucopyranoside in ME formulations shows significant potential as a delivery vehicle in the cosmetics and pharmaceutical industry.

Requirement for the von Hippel-Lindau tumor suppressor gene for functional epidermal growth factor receptor blockade by monoclonal antibody C225 in renal cell carcinoma.

Renal cell carcinoma (RCC) is a cytologically and histologically diverse disease in which a spectrum of distinct molecular alterations occurs, including the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which is specific for the clear cell variant of RCC. The prognosis for RCC is poor, and, to date, no effective systemic treatment is available for this cancer. In the present study, we assessed the extent to which the transforming growth factor alpha-epidermal growth factor receptor (EGFR) autocrine loop could be used as a potential therapeutic target for RCC. Northern blot analysis of transforming growth factor alpha and EGFR revealed variable but consistent expression of these transcripts in cell lines derived from both clear cell and non-clear cell RCC variants, indicating the potential for this autocrine loop in both tumor types. The therapeutic utility of interruption of this feedback loop was determined by examining growth inhibition after the exposure of these cell lines to a humanized anti-EGFR monoclonal antibody, C225. In vitro treatment of clear cell RCC-derived cell lines lacking VHL resulted in only a modest decrease in growth rate. In contrast, non-clear cell RCC-derived cell lines that retained VHL responded significantly to C225 treatment. Transfection of VHL into VHL-negative RCC cell lines restored responsiveness to C225, indicating that this tumor suppressor gene is required for effective EGFR blockade. Growth inhibition by C225 in VHL-positive cells was linked to a requirement for VHL to up-regulate p27 in response to C225. These data provide compelling evidence that treatment modalities for RCC are likely to be strongly influenced by the molecular etiology of this phenotypically diverse cancer.

Mechanisms of epithelial development and neoplasia in the metanephric kidney.

Recent studies on the mechanisms of normal epithelial development in the kidney, and on the aetiology of renal neoplasms, are converging to reveal remarkably close relationships between the phenotypes and behaviours of normally-developing and neoplastic cells. Normal renal epithelia arise from two sources; those of the collecting duct system develop by arborisation of an initially-unbranched ureteric bud, in a manner similar to the development of other glandular organs, while epithelial nephrons develop via an unusual mesenchyme-to-epithelial transition. Both types of development require controlled proliferation, cell-cell and cell-matrix interactions, protease activity etc., but of the two tissues, the development of the nephrons is arguably the more complex. It includes many defined stages, signals and checkpoints that ensure that events happen at the right time, and that processes such as proliferation, apoptosis and differentiation are properly balanced. Detailed investigation of renal neoplasms has revealed some to be caused by mutations in molecules with known roles in normal nephrogenesis (e.g. Wilms' tumour and the WT-1 gene, renal cell carcinoma and the c-met receptor tyrosine kinase gene), some to be caused by mutations in genes expressed during normal development (e.g. renal cell carcinoma and the TSC-2 gene, renal cell carcinoma of the clear cell variety and the VHL gene). Furthermore, these and other tumours of unknown aetiology re-express genes such as Pax-2 that are expressed during the normal mesenchyme-to-epithelium transition but are shut off during terminal differentiation. Their re-appearance in tumours suggests that the cells have 'regressed' in an ontogenic sense, and their biology may therefore be understood most clearly by reference to the properties of normal developing cells rather than cells of a mature kidney.

Ultrastructural studies of neuronal correlates of the pubertal reaugmentation of hypothalamic gonadotropin-releasing hormone (GnRH) release in the rhesus monkey (Macaca mulatta).

This study tested the hypothesis that puberty in primates is triggered by a remodeling of synaptic inputs and/or glial coverage of hypothalamic gonadotropin releasing hormone (GnRH) neurons. Male rhesus monkeys were prepubertally castrated at 16 months of age and were killed and perfused either 1 month later (n = 4, juvenile group) or at 30 months of age, shortly after initiation of the pubertal increase in pulsatile GnRH release (n = 4, adult group). Hypothalami were sectioned, immunocytochemically stained for GnRH, and processed for electron microscopy. Cross-sectional profiles of 77 GnRH cells from the medial basal hypothalamus (MBH) and the region of the organum vasculosum of the lamina terminalis (OVLT) were compared between the two developmental stages. GnRH cell and nucleolus size in the two groups were the same. The percentage of GnRH perikaryal membrane occupied by synaptic density in the MBH of juveniles was significantly greater (P < 0.05) than that of adults. Differences in the percentage of GnRH perikaryal membrane occupied by synaptic density were not observed in the OVLT nor on GnRH dendrites in either brain region. Qualitative analysis, based on synaptic vesicle shape, failed to reveal developmental differences in putatively excitatory or inhibitory synapses on GnRH cells. The degree of glial ensheathment of GnRH neurons did not change significantly during the two developmental stages. These findings provide ultrastructural evidence for the view that, in primates, neuronal plasticity, and specifically a decrease in synaptic input to GnRH perikarya, may underlie the initiation of the pubertal mode of release of this neuropeptide, and therefore, the onset of puberty in these species.

Replacement with recombinant human inhibin immediately after orchidectomy in the hypophysiotropically clamped male rhesus monkey (Macaca mulatta) maintains follicle-stimulating hormone (FSH) secretion and FSH beta messenger ribonucleic acid levels at precastration values.

This study directly tested the inhibin hypothesis by examining the ability of replacement with recombinant human (rh-) inhibin, either alone or in combination with testosterone (T), to maintain FSH secretion and FSH beta messenger RNA (mRNA) at intact levels after orchidectomy in the hypophysiotropically clamped juvenile rhesus monkey. Thirteen male monkeys (11-21 months of age) received an intermittent i.v. infusion of GnRH (0.1 microgram/min for 3 min every 3 h). After 4-6 weeks of GnRH stimulation, 10 animals were orchidectomized, and 3 monkeys were sham castrated. Hormone replacement was initiated at castration and maintained for 4 days. Three monkeys received a combination of inhibin and T replacement, 4 monkeys received replacement with inhibin alone, and 3 monkeys received T replacement alone. A continuous i.v. infusion of rh-inhibin (832 ng/h.kg) was used to replace the testicular protein, whereas SILASTIC capsules were implanted sc for T replacement. The FSH response to castration and hormone replacement was determined by measuring circulating concentrations of this gonadotropin before a GnRH pulse and for 3 h thereafter on the day before surgery and on days 2 and 4 postcastration. Circulating immunoactive inhibin was measured by a RIA that recognizes the free alpha-subunit of inhibin as well as inhibin dimers. At the end of the study, anterior pituitaries were collected for analysis of steady state levels of FSH beta, LH beta, and alpha-subunit mRNAs. Steroid replacement alone, which produced circulating T concentrations in the upper physiological range, failed to prevent the postcastration increases in circulating FSH concentrations and pituitary FSH beta mRNA levels. In contrast, when circulating immunoactive inhibin in T-replaced monkeys was maintained at precastration levels (approximately 2 ng/ml) by infusion of rh-inhibin, FSH secretion and synthesis were held at control values. When T was omitted from combined replacement, the FSH-suppressing action of the recombinant hormone was not compromised. These results demonstrate that rh-inhibin is biologically active in the monkey, and the action of inhibin to suppress FSH synthesis and secretion does not require a concomitant action of T. Moreover, because the hypophysiotropic drive to the pituitary-testicular axis was clamped, the FSH-suppressing action of rh-inhibin must be at the pituitary.

Glutamate-immunoreactive neurons and their gonadotropin-releasing hormone-neuronal interactions in the monkey hypothalamus.

Glutamate (Glu) is the most prevalent excitatory neurotransmitter in the brain and has been implicated in the regulation of GnRH secretion in several mammalian species, including the monkey. To investigate the neuroanatomical basis for Glu-GnRH interactions, we performed an immunocytochemical study at both the light and electron microscopic levels on the brains of four female and five male macaques. Initially, we determined the location of Glu-immunoreactive (-ir) elements using a monoclonal antibody specific for glutaraldehyde-fixed Glu (Glu-2) and 3,3'-diaminobenzidine-4-HCl (DAB). Glu-ir was observed in the cytoplasm and to a variable degree in the nuclei of neurons in the diencephalon. Cytoplasmic staining was particularly intense in numerous neurons in the arcuate nucleus, supraoptic nucleus, and many paraventricular nucleus neurons. Short Glu-ir processes were evident in these and other hypothalamic regions and were extremely dense in the infundibular stalk and median eminence. Prior absorption of the Glu-2 antibody with a Glu-glutaraldehyde-BSA conjugate completely abolished all immunostaining in both neuronal nuclei and cytoplasm. Double label Glu-GnRH immunostaining for light microscopy was performed using Glu-2 and DAB without enhancement, and a polyclonal antibody (LR1 or LR2) with silver-enhanced DAB for Glu and GnRH, respectively. Glu-ir interactions with GnRH-ir cell bodies were not apparent, but a few Glu-ir axons seemed to contact GnRH-ir dendrites in the organum vasculosum of the lamina terminalis, medial septum, and arcuate nucleus regions. Reciprocal interactions occurred more frequently, however, in which GnRH-ir axons and dendritic fibers engaged Glu-ir cell bodies en passant, particularly toward the medial and posterior hypothalamus. For ultrastructural analyses, Glu-ir elements were stained with the Glu-2 antibody and 15 nm immunogold or DAB. Electron microscopy demonstrated that Glu-ir was associated with clear microvesicles within the neuronal cytoplasm. Glu-ir processes made classical asymmetrical synapses with one another and received asymmetrical synapses from unlabeled afferents. In sections double labeled for Glu with immunogold and for GnRH with DAB, axo-somatic interactions were not observed. However, axo-dendritic Glu-GnRH synapses were seen, which usually exhibited Glu-ir labeling of terminal vesicles and inconsistent postsynaptic densities, with GnRH-ir neurosecretory granules sometimes congregated in the apposing dendrite or spine. Surprisingly, reverse GnRH-Glu interactions were observed more frequently.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal expression of polysialic acid-neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta).

Puberty in primates is triggered by a gonad-independent reinitiation of a pulsatile mode of GnRH release. The purpose of the present study was to begin to examine the hypothesis that this neuroendocrine event is the result of structural or plastic changes within the neural network governing the activity of GnRH neurons. Specifically, we sought to determine whether polysialic acid neural cell adhesion molecule (PSA-NCAM), a plasma membrane-associated glycoprotein that has previously been proposed to be a marker for postnatal neuronal plasticity, was expressed within GnRH neuron containing areas of the rhesus monkey hypothalamus. The study employed male monkeys that were castrated prepubertally. Immunocytochemistry of hypothalamic tissue from four animals of pubertal age employing a monoclonal antibody (12F8) specific for PSA-NCAM revealed the presence of PSA-NCAM immunoreactivity within the region of the arcuate nucleus and median eminence of the medial basal hypothalamus (MBH) and in the region of the organum vasculosum of the lamina terminalis of the rostral hypothalamus, two areas in the monkey brain where GnRH neurons are concentrated. As expected, immunostaining for total NCAM using a polyclonal rabbit antibody to mouse total NCAM was uniformly distributed throughout hypothalamic sections containing the MBH. Double staining showed that some, though not all, GnRH cell bodies of the MBH were located within the PSA-NCAM-immunopositive region of the arcuate nucleus and the median eminence. The pattern of PSA-NCAM immunoreactivity in the MBH of three prepubertal monkeys was similar to that seen for the older animals. Western analysis of a membrane extract from the MBH of a monkey of pubertal age, employing antibody 12F8, identified a broad band of staining at the expected molecular weight for this adhesion molecule. A similar, but less intense, immunoreactive band was observed for the preoptic area. In contrast, an immunoblot of a membrane extract of cerebral cortex was only faintly positive for PSA-NCAM. Taken together, the foregoing findings are consistent with the notion that structural changes within the MBH may underlie the pubertal reinitiation of pulsatile GnRH release. Moreover, the presence of PSA-NCAM in the MBH of prepubertal monkeys suggests that the role, if any, of this molecule in the onset of sexual maturation in primates is permissive in nature.

Cholecystokinin stimulates gonadotropin-releasing hormone release in the monkey (Macaca mulatta).

There is evidence to suggest that the arginine vasopressin release observed in association with emesis after i.v. injection of cholecystokinin (CCK) and N-methyl-D-aspartate (NMDA) is mediated by stimulation of emetic centers in the brainstem. That the GnRH-releasing action of NMDA is also sometimes accompanied by emesis led to the suggestion that stimulation of this parvocellular system by the glutamate agonist may also be mediated in part via activation of brainstem pathways. If this is the case, then other nauseogenic agents, such as CCK, should similarly elicit GnRH release. The foregoing prediction was tested in the castrated juvenile male monkey, an experimental model characterized by the absence of spontaneous GnRH release. GnRH discharges were monitored indirectly by measuring changes in circulating LH concentrations after the responsivity of the gonadotroph had been heightened by a chronic intermittent i.v. infusion of the decapeptide. An i.v. bolus of CCK at 10 and 30 micrograms/kg BW led to a distinct discharge of GnRH accompanied by emesis or other behaviors suggestive of nausea. Lower doses of CCK (1 and 3 micrograms/kg BW) failed to significantly perturb GnRH release or cause emesis, although NMDA (5 mg/kg BW; racemic form) injected i.v. 3 h after the CCK challenge led to a robust rise in GnRH. In a parallel study, three repetitive i.v. CCK injections at 2h intervals maintained intermittent GnRH release. Pretreatment with a long-acting GnRH receptor antagonist ([AcD2Nal1,4ClPhe2,DTrp3,DArg6,DAla10]GnRH -HOAc) abolished the LH response to CCK, confirming that the action of this peptide was mediated by GnRH release. Although a direct hypothalamic site of action for these agents remains the most likely possibility, since both NMDA and CCK receptors are present in the infundibular region, the present data are consistent with the notion that CCK and, by inference, NMDA may activate GnRH release in part via the stimulation of brainstem emetic centers. Plasma GH, PRL, and cortisol concentrations were also monitored during the course of some of these experiments, and the release of these hormones was observed after the administration of either the 10 or 30 micrograms/kg BW dose of CCK.

Photoperiodic induction in vitro: the dynamics of gonadotropin-releasing hormone release from hypothalamic explants of the Japanese quail.

The Japanese quail is a photoperiodic animal that under certain experimental conditions can respond to a single long day with a wave of LH secretion. Such a system offers an opportunity to analyze the photoneuroendocrine changes as they occur in real time, especially as all of the neural machinery (photoreceptor, clock, and GnRH system) is believed to lie within the hypothalamus. The first detectable rise in LH occurs at about hour 23 of the long day, and this single inductive event leads to prolonged LH secretion lasting for up to 2 weeks and peaking 2-4 days after the dawn of the long day. The size of the quail's hypothalamus is such that the entire structure, including both the GnRH cell bodies and the median eminence, can be cultured for some hours, and the rates of GnRH release measured therefrom. The present experiments used hypothalamic explants from quail at different times throughout the photoperiodic response, superfused them for up to 7.5 h in vitro, and measured the dynamics of GnRH release. A significant step increase of 80% in GnRH release occurred between hours 22.5 and 23 in quail that had been exposed to a long day: an equivalent change was not found in hypothalami taken from quail maintained only under short day lengths. In explants taken from quail at the peak of LH secretion (53 h after dawn of the long day), the rates of GnRH release were double those found in control quail not exposed to the long day. Explants taken 14 days after the long day, when LH secretion had subsided fully, showed no difference in GnRH release between photo-stimulated and control quail. These results suggest that photoperiodic induction involves a timed increase in GnRH release, and the rise at hour 23 is believed to represent photoperiodic induction actually taking place within the brain in vitro. They also suggest that the wave of LH secretion triggered by the single long day is, at least in part, a neuroendocrine or neural phenomenon; this confirms earlier indirect evidence to this effect.

The neurobiology of primate puberty.

In higher primates the protracted prepubertal phase of development is occasioned by a mechanism that suppresses pulsatile hypothalamic gonadotropin-releasing hormone (GnRH) secretion from late infancy until the onset of puberty and thereby guarantees, in the juvenile, the quiescence of the pituitary-gonadal axis. Studies from our laboratory have employed the rhesus monkey, a representative higher primate, as an experimental paradigm. GnRH release has been measured using luteinizing hormone secretion by the in situ pituitary as a bioassay for the hypothalamic hormone. The nature of the prepubertal brake on pulsatile GnRH release in the monkey has been probed using physiological, neuroanatomical and neuropharmacological approaches. Such studies have led to the view that the prepubertal hiatus in pulsatile GnRH release results from a withdrawal in late infancy of a synchronized frequency-facilitated afferent neural input to the GnRH network, which in all other respects appears to exhibit properties identical to those in the postpubertal animal. The mechanism timing the onset of puberty, i.e. that responsible for the reactivation of synchronous activity in the GnRH network, is posited to be under the control of a central neural time- or growth-tracking device.

[Whole soy bean flour: use of a methodology of response surface for the study of nutritional aspects]. / Farinha de soja integral: aplicação da metodologia da superfície de resposta para estudo de aspectos nutricionais.

Cotyledons of soybeans (Glycine max) of the Paraná variety were subjected to hydrothermal processing. Response surface methodology was used to evaluate the conditions which provided a product of the highest protein quality. The processing variables used in the experimental design were: soaking time (0 - 8 hr); blanching time (5 - 35 min) and bicarbonate concentration (0 - 0.5 g%) in blanching water. Biological evaluations of protein quality were done using the NPR. The mathematical model developed does not distinguish between treatments (p less than 0.05) in the experimental region studied.

Farinha de soja integral: aplicacao da metodologia da superficie de resposta para estudo de aspectos nutricionais. / Integral soy bean flour: use of a methodology of response surface for the study of nutritional aspects

Cotiledones de soja (Glycine max, L. Merril) da variedade Parana, foram processados por metodo hidrotermico, manipulando as condicoes de processamento, atraves de metodologia da superficie de resposta, para escolha de condicoes, que fornecam produto de melhor qualidade proteica. As variaveis de processamento utilizadas no delineamento experimental foram: tempo de hidratacao de 0 a 8h; tempo de escaldamento de 5 a 35 min. e tratamento quimico com bicarbonato de sodio (NaHCO3) de 0 a 0.5 g%. As farinhas de soja integrais obtidas foram submetidas a avaliacao da qualidade nutricional proteica atraves do teste de NPR (razao de proteina liquida) e aminoacidos sulfurados. O modelo matematico desenvolvido nao conseguiu distinguir entre os tratamento (p < 0.05) na regiao experimental estudada

Bioavailability of vitamin B-6 from wheat bread in humans.

Bioavailability of vitamin B-6 from three types of bread was studied in nine men. Each week one of the three types of bread, whole wheat (WHW), white (W) and W enriched with vitamin B-6 (WB6) was fed (570-600 g/day) to each subject using a Latin square design. The WHW, WB6 and W bread supplied 1.20, 1.18 and 0.35 mg of B-6 daily, respectively. The remaining constant diet supplied 0.38 mg of B-6. When W was fed, the subjects also received an oral dose of pyridoxine to maintain a daily intake of 1.5 mg of vitamin B-6. Dietary, urinary, fecal and blood samples were analyzed for vitamin B-6. The predominant forms of vitamin B-6 in the diet and urine were pyridoxine and pyridoxal, respectively. Fecal vitamin B-6 excretion was significantly higher (P smaller than or equal to 0.01) and urinary 4-pyridoxic acid excretion significantly lower (P smaller than or equal to 0.05) when WHW bread was fed than when either WB6 or W bread was fed. The plasma pyridoxal phosphate level was slightly lower when WHW bread was consumed as compared to when WB6 was fed. There were no significant differences in urinary B-6, plasma B-6 or the activity of the erythrocyte transaminases in relation to the type of bread fed. These data suggest that vitamin B-6 is 5-10% less available from WHW than from WB6 or W bread and an oral dose of B-6. The enrichment of refined wheat flour with pyridoxine is feasible based on the nutritional indices determined in this study.