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The rare pediatric embryonal tumors retinoblastoma, medulloblastoma and neuroblastoma derive from neuroectodermal tissue and share similar histopathological features despite different anatomical locations and diverse clinical outcomes. As metabolism can reflect genetic and histological features, we investigated whether the metabolism of embryonal tumors reflects their similar histology, shared developmental and neural origins, or tumor location. We undertook metabolic profiling on 50 retinoblastoma, 39 medulloblastoma and 70 neuroblastoma using high resolution magic angle spinning magnetic resonance spectroscopy (1H-MRS). Mean metabolite concentrations identified several metabolites that were significantly different between the tumor groups including taurine, hypotaurine, glutamate, glutamine, GABA, phosphocholine, N-acetylaspartate, creatine, glycine and myoinositol, p < 0.0017. Unsupervised multivariate analysis found that each tumor group clustered separately, with a unique metabolic profile, influenced by their underlying clinical diversity. Taurine was notably high in all tumors consistent with prior evidence from embryonal tumors. Retinoblastoma and medulloblastoma were more metabolically similar, sharing features associated with the central nervous system (CNS). Neuroblastoma had features consistent with neural tissue, but also contained significantly higher myoinositol and altered glutamate-glutamine ratio, suggestive of differences in the underlying metabolism of embryonal tumors located outside of the CNS. Despite the histological similarities and shared neural metabolic features, we show that individual neuroectodermal derived embryonal tumors can be distinguished by tissue metabolic profile. Pathway analysis suggests the alanine-aspartate-glutamate and taurine-hypotaurine metabolic pathways may be the most pertinent pathways to investigate for novel therapeutic strategies. This work strengthens our understanding of the biology and metabolic pathways underlying neuroectodermal derived embryonal tumors of childhood.

Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies—outlined in a validated pathogenicity scoring system—is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt-tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as “definitely pathogenic” mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as ‘possibly pathogenic’ (m.4289T>C, m.7554G>A, and m.8304G>A).

The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1andProk2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16–19).Prok1mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediatorsIl6,Il1b,Tnf,Cxcl2andCxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression ofProk1or its receptor (Prokr1) in fetal membranes. These results suggest that althoughProk1exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.

Biomarkers of early response to treatment have the potential to improve cancer therapy by allowing treatment to be tailored to the individual. Alterations in lipids detected by in vivo MRS have been suggested as noninvasive biomarkers of cell stress and early indicators of cell death. An improved understanding of the relationship between MRS lipids and cell stress in vitro would aid in the translation of this technique into clinical use. Rat BT4C glioma cells were treated with 50 µ m cis-dichlorodiammineplatinum II (cisplatin), a commonly used chemotherapeutic agent, and harvested at several time points up to 72 h. High-resolution magic angle spinning (1) H MRS of cells was then performed on a 600-MHz NMR spectrometer. The metabolites were quantified using a time domain fitting method, TARQUIN. Increases were detected in saturated and polyunsaturated fatty acid resonances early during the exposure to cisplatin. The fatty acid CH(2) /CH(3) ratio was unaltered by treatment after allowing for contributions of macromolecules. Polyunsaturated fatty acids increased on treatment, with the group -CH=CH-CH(2) -CH=CH- accounting for all the unsaturated fatty acid signals. Transmission electron microscopy, in addition to Nile red and 4',6-diamino-2-phenylindole co-staining, revealed that the lipid increase was associated with cytoplasmic neutral lipid droplets. Small numbers of apoptotic and necrotic cells were detected by trypan blue, annexin V-fluorescein isothiocyanate-labelled flow cytometry and DNA laddering after up to 48 h of cisplatin exposure. Propidium iodide flow cytometry revealed that cells accumulated in the G1 stage of the cell growth cycle. In conclusion, an increase in the size of the lipid droplets is detected in morphologically viable cells during cisplatin exposure. (1) H MRS can detect lipid alterations during cell cycle arrest and progression of cell death, and has the potential to provide a noninvasive biomarker of treatment efficacy in vivo.

Using both NADPH diaphorase and anti-nNOS antibodies, we have identified—from retinal flatmounts—neuronal types in the inner retina of the chicken that are likely to be nitrergic. The two methods gave similar results and yielded a total of 15 types of neurons, comprising 9 amacrine cells, 5 ganglion cells, and 1 centrifugal midbrain neuron. Six of these 15 cell types are ubiquitously distributed, comprising 3 amacrine cells, 2 displaced ganglion cells, and a presumed orthotopic ganglion cell. The remaining nine cell types are regionally restricted within the retina. As previously reported, efferent fibers of midbrain neurons and their postsynaptic partners, the unusual axon-bearing target amacrine cells, are entirely confined to the ventral retina. Also confined to the ventral retina, though with somewhat different distributions, are the “bullwhip” amacrine cells thought to be involved in eye growth, an orthotopic ganglion cell, and two types of large axon-bearing amacrine cells whose dendrites and axons lie in stratum 1 of the inner plexiform layer (IPL). Intracellular fills of these two cell types showed that only a minority of otherwise morphologically indistinguishable neurons are nitrergic. Two amacrine cells that branch throughout the IPL are confined to an equatorial band, and one small-field orthotopic ganglion cell that branches in the proximal IPL is entirely dorsal. These findings suggest that the retina uses different processing on different regions of the visual image, though the benefit of this is presently obscure.

Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium.

Human medulloblastomas exhibit diverse molecular pathology. Aberrant hedgehog signalling is found in 20–30% of human medulloblastomas with largely unknown metabolic consequences. Transgenic mice over-expressing smoothened (SMO) receptor in granule cell precursors with high incidence of exophytic medulloblastomas were sequentially followed up by magnetic resonance imaging (MRI) and characterised for metabolite phenotypes by 1H MR spectroscopy (MRS) in vivo and ex vivo using high-resolution magic angle spinning (HR-MAS) 1H MRS. Medulloblastomas in the SMO mice presented as T2 hyperintense tumours in MRI. These tumours showed low concentrations of N-acetyl aspartate and high concentrations of choline-containing metabolites (CCMs), glycine, and taurine relative to the cerebellar parenchyma in the wild-type (WT) C57BL/6 mice. In contrast, 1H MRS metabolite concentrations in normal appearing cerebellum of the SMO mice were not different from those in the WT mice. Macromolecule and lipid 1H MRS signals in SMO medulloblastomas were not different from those detected in the cerebellum of WT mice. The HR-MAS analysis of SMO medulloblastomas confirmed the in vivo 1H MRS metabolite profiles, and additionally revealed that phosphocholine was strongly elevated in medulloblastomas accounting for the high in vivo CCM. These metabolite profiles closely mirror those reported from human medulloblastomas confirming that SMO mice provide a realistic model for investigating metabolic aspects of this disease. Taurine, glycine, and CCM are potential metabolite biomarkers for the SMO medulloblastomas. The MRS data from the medulloblastomas with defined molecular pathology is discussed in the light of metabolite profiles reported from human tumours.

Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.

The visual system of birds includes an efferent projection from a visual area, the isthmo-optic nucleus in the midbrain, back to the retina. Using a combination of anterograde labeling of efferent fibers, reconstruction of dye-filled neurons, NADPH-diaphorase staining, and transmission electron microscopy, we have examined the distribution of efferent fibers and their synaptic structures in the chicken retina. We show that efferent fibers terminate strictly within the ventral retina. In two completely mapped retinas, only 2 fibers from a total of 15,359 terminated in the dorsal retina. The major synapse made by each efferent fiber is with a single efferent target amacrine cell (TC). This synapse consists of 5–25 boutons of 2 μm diameter, each with multiple active zones, pressed into the TC soma or synapsing with a basketwork of rudimentary TC dendrites in the inner nuclear layer (INL). This basketwork, which is sheathed by Muller cell processes, defines a private neuropil in the INL within which TCs were also seen to receive input from retinal neurons. In addition to the major synapse, efferent fibers typically produce several very thin processes that terminate nearby in single small boutons and for which the soma of a local amacrine cell is one of the likely postsynaptic partners. A minority of efferent fibers also give rise to a thicker process, terminating in a strongly diaphorase-positive ball about 5 μm in diameter.

(1)H MRS has great potential for the clinical investigation of childhood brain tumours, but the low incidence in, and difficulties of performing trials on, children have hampered progress in this area. Most studies have used a long-TE, thus limiting the metabolite information obtained, and multivariate analysis has been largely unexplored. Thirty-five children with untreated cerebellar tumours (18 medulloblastomas, 12 pilocytic astrocytomas and five ependymomas) were investigated using a single-voxel short-TE PRESS sequence on a 1.5 T scanner. Spectra were analysed using LCModel to yield metabolite profiles, and key metabolite assignments were verified by comparison with high-resolution magic-angle-spinning NMR of representative tumour biopsy samples. In addition to univariate metabolite comparisons, the use of multivariate classifiers was investigated. Principal component analysis was used for dimension reduction, and linear discriminant analysis was used for variable selection and classification. A bootstrap cross-validation method suitable for estimating the true performance of classifiers in small datasets was used. The discriminant function coefficients were stable and showed that medulloblastomas were characterised by high taurine, phosphocholine and glutamate and low glutamine, astrocytomas were distinguished by low creatine and high N-acetylaspartate, and ependymomas were differentiated by high myo-inositol and glycerophosphocholine. The same metabolite features were seen in NMR spectra of ex vivo samples. Successful classification was achieved for glial-cell (astrocytoma + ependymoma) versus non-glial-cell (medulloblastoma) tumours, with a bootstrap 0.632 + error, e(B.632+), of 5.3%. For astrocytoma vs medulloblastoma and astrocytoma vs medulloblastoma vs ependymoma classification, the e(B.632+) was 6.9% and 7.1%, respectively. The study showed that (1)H MRS detects key differences in the metabolite profiles for the main types of childhood cerebellar tumours and that discriminant analysis of metabolite profiles is a promising tool for classification. The findings warrant confirmation by larger multi-centre studies.

The depletion of ER Ca2+ stores, following the release of Ca2+ during intracellular signalling, triggers the Ca2+ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca2+]i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca2+ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca2+ entry in the form of motes.

GABAergic amacrine cells, cultured from embryonic chick retina, display spontaneous mini frequencies ranging from 0–4.6 Hz as a result of the release of quanta of transmitter from both synapses and autapses. We show here that at least part of this variation originates from differences in the degree to which endocannabinoids, endogenously generated within the culture, are present at terminals presynaptic to individual cells. Though all cells examined scored positive for cannabinoid receptor type I (CB1R), only those showing a low initial rate of spontaneous minis responded to CB1R agonists with an increase in mini frequency, caused by a Gi/o-mediated reduction in [cAMP]. Cells displaying a high initial rate of spontaneous minis, on the other hand, were unaffected by CB1R agonists, but they did show a rate decrease with CB1R antagonists. Such a regulation of spontaneous transmitter release by endocannabinoids might be important in network maintenance in amacrine cells and other inhibitory interneurons.

Neuroblastoma is the most common extracranial solid malignancy in children. The disease possesses a broad range of clinical phenotypes with widely varying prognoses. Numerous studies have sought to identify the associated genetic abnormalities in the tumour, resulting in the identification of useful prognostic markers. In particular, the presence of multiple copies of the MYCN oncogene (referred to as MYCN amplification) has been found to confer a poor prognosis. However, the molecular pathways involved are as yet poorly defined. Metabolite profiles generated by in vitro (1)H MRS provide a means of investigating the downstream metabolic consequences of genetic alterations and can identify potential targets for new agents. Thirteen neuroblastoma cell lines possessing multiple genetic alterations were investigated; seven were MYCN amplified and six MYCN non-amplified. In vitro magic angle spinning (1)H MRS was performed on cell suspensions, and the spectra analysed to obtain metabolite concentration ratios relative to total choline (tCho). A principal component analysis using these concentration ratios showed that MYCN-amplified and non-amplified cell lines form separate classes according to their metabolite profiles. Phosphocholine/tCho and taurine/tCho were found to be significantly raised (p < 0.05) and glycerophosphocholine/tCho significantly reduced (p < 0.05) in the MYCN-amplified compared with the MYCN non-amplified cell lines (two-tailed t test). (1)H MRS of the SH-EP1 cell line and an isogenic cell line transfected with the MYCN oncogene also showed that MYCN oncogene over-expression causes alterations in phosphocholine, glycerophosphocholine and taurine concentrations. Molecular pathways of choline and taurine metabolism are potential targets for new agents tailored to MYCN-amplified neuroblastoma.

Hill races usually include large downhill running sections, which can induce significant degrees of muscle damage in a field setting. This study examined the link between muscle damage, oxidative stress, and immune perturbations following a 7-km mountainous hill race with 457 m of ascent and 457 m of descent. Venous blood samples were taken from 7 club level runners before, immediately after, and 48 hrs postrace. Samples were analysed for total and differential leukocyte counts, markers of muscle damage (CK), lipid peroxidation (MDA), and acute phase proteins (CRP; fibrinogen; α-1-ACT). The total antioxidant status (TEAC) and plasma levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α were also determined. Subjective pain reports, and plasma activities of CK, MDA, and circulatory monocytes reached peak values at 48 hrs postrace (p 0.05). Despite the reports of muscle damage and soreness, no evidence of an acute phase response was observed (p > 0.05), which may be explained by the failure of the race to induce a plasma TNF-α response. Future studies should examine the link between muscle damage, oxidative stress, and the acute phase response following hill races of longer duration with larger eccentric components. Key words: acute phase response, cytokines, antioxidant capacity, creatine kinase, field study

Background — The therapeutic benefits that accompany the continuous administration of organic nitrates are attenuated by the development of tolerance to the compounds. Altered superoxide production and NO bioavailability have been implicated in contributing to the development of tolerance, an effect that may be ameliorated by the administration of antioxidants. Methods and Results — We studied the effect of 3 days of continuous transdermal administration of nitroglycerin (NTG) (10 mg/24 hours) on platelet free radical (NO and superoxide anion [O 2 ·− ] activity) with and without coadministration of supplemental ascorbate (2.4 g/24 hours). NAD(P)H oxidase activity, nitric oxide synthase (NOS) activity, and cyclic guanosine monophosphate (cGMP) content were also assessed. Radial artery pressure pulse waveforms were used to track the hemodynamic actions of NTG. Three days of NTG/placebo was associated with a significant increase in platelet NO and O 2 ·− production from 1.0±1.17 to 2.52±0.88 pmol/10 8 platelets and 13.2±4.8 to 72.5±34.4 pmol/10 8 platelets, respectively ( P 2 ·− min/mg protein and cGMP content from 0.60±0.10 to 0.89±0.16 pmol/10 9 platelets ( P 2 ·− release in platelets. Conclusions — Three days of continuous transdermal administration of NTG was accompanied by increased platelet NO and O 2 ·− production and NADH oxidase activity that was suppressed by coadministration of oral ascorbate. Although a significant degree of tolerance would be expected during continuous nitrate administration, a residual hemodynamic action could be identified by arterial pulse contour analysis.

It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.

The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like gamma-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an approximately 140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the beta2/beta3-subunits of the GABAA receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.

Increases in 1H nuclear magnetic resonance spectroscopy (NMR) visible lipids are a well-documented sign of treatment response in cancers. Lipids in cytoplasmic lipid droplets (LDs) are the main contributors to the NMR lipid signals. Two human primitive neuroectodermal tumour cell lines with different sensitivities to cisplatin treatment were studied. Increases in NMR visible saturated and unsaturated lipids in cisplatin treated DAOY cells were associated with the accumulation of LDs prior to DNA fragmentation due to apoptosis. An increase in unsaturated fatty acids (UFAs) was detected in isolated LDs from DAOY cells, in contrast to a slight decrease in UFAs in lipid extracts from whole cells. Oleic acid and linoleic acid were identified as the accumulating UFAs in LDs by heteronuclear single quantum coherence spectroscopy (HSQC). 1H NMR lipids in non-responding PFSK-1 cells were unchanged by exposure to 10 μM cisplatin. These findings support the potential of NMR detectable UFAs to serve as a non-invasive marker of tumour cell response to treatment.

Miniature postsynaptic currents (minis) in cultured retinal amacrine cells, as in other central neurons, show large variations in amplitude. To understand the origin of this variability, we have exploited a novel form of synapse in which pre- and postsynaptic receptors sample the same quantum of transmitter. At these synapses, mini amplitudes measured simultaneously in the 2 cells show a strong correlation, accounting for, on average, more than half of the variance in amplitude. Two pieces of evidence support the conclusion that variations in the amount of transmitter in different quanta underlie this correlation. First, diazepam, which enhances GABA binding, increases mini amplitude, implying therefore that transmitter concentration is not saturating. Second, we show that amplitude distributions from all cells, even those with a small number of release sites, have the same shape, implying that most or all variance is intrinsic to each release site.

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.

As Cajal discovered in the late 19th century, the bird retina receives a substantial input from the brain. Approximately 10,000 fibers originating in a small midbrain nucleus, the isthmo-optic nucleus (ION), terminate in each retina. The input to the ION is chiefly from the optic tectum which, in the bird, is the primary recipient of retinal input. These neural elements constitute a closed loop, the centrifugal visual system (CVS), beginning and ending in the retina, that delivers positive feedback to active ganglion cells. Several features of the system are puzzling. All fibers from the ION terminate in the ventral retina and an unusual axon-bearing amacrine cell, the target cell, is the postsynaptic partner of these fibers. While the rest of the CVS is orderly and retinotopic, target cell axons project seemingly at random, mostly to distant parts of the retina. We review here the most significant features of the anatomy and physiology of the CVS with a view to understanding its function. We suggest that many of the facts about this system, including some that are otherwise difficult to explain, can be accommodated within the hypothesis that the images of shadows cast on the ground or on objects in the environment, initiate a rapid and parallel search of the sky for a possible aerial predator. If a predator is located, shadow and predator would be temporarily linked together and tracked by the CVS.

The bird visual system includes a substantial projection, of unknown function, from a midbrain nucleus to the contralateral retina. Every centrifugal, or efferent, neuron originating in the midbrain nucleus makes synaptic contact with the soma of a single unique amacrine cell, the target cell (TC). By labeling efferent neurons in the midbrain, we have been able to identify their terminals in retinal slices and make patch-clamp recordings from TCs. TCs generate Na+-based action potentials (APs) triggered by spontaneous EPSPs originating from multiple classes of presynaptic neurons. Exogenously applied glutamate elicited inward currents having the mixed pharmacology of NMDA, kainate, and inward rectifying AMPA receptors. Exogenously applied GABA elicited currents entirely suppressed by GABAzine and therefore mediated by GABAA receptors. Immunohistochemistry showed the vesicular glutamate transporter, vGluT2, to be present in the characteristic synaptic boutons of efferent terminals, whereas the GABA synthetic enzyme, GAD, was present in much smaller processes of intrinsic retinal neurons. Extracellular recording showed that exogenously applied GABA was directly excitatory to TCs and, consistent with this, NKCC, the Cl− transporter often associated with excitatory GABAergic synapses, was identified in TCs by antibody staining. The presence of excitatory retinal input to TCs implies that TCs are not merely slaves to their midbrain input; instead, their output reflects local retinal activity and descending input from the midbrain.

The prostaglandin F2α (PGF2α) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF2α signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF2α increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF2α in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF2α-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF2α-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis. [Cancer Res 2009;69(14):5726–33]

Contains fulltext : 81322.pdf (Publisher’s version ) (Open Access) The retinas of birds receive a substantial efferent, or centrifugal, input from a midbrain nucleus. The function of this input is presently unclear, but previous work in the pigeon has shown that efferent input is excluded from the area centralis, suggesting that the functions of the area centralis and the efferent system are incompatible. Using an antibody specific to rods, we have identified the area centralis in another species, the chicken, and mapped the distribution of the unique amacrine cells that are the postsynaptic partners of efferent fibers. Efferent target amacrine cells are found within the chicken area centralis and their density is continuous across the border of the area centralis. In contrast to the pigeon retina then, we conclude that the chicken area centralis receives efferent input. We suggest that the difference between the two species is attributable to the presence of a fovea within the area centralis of the pigeon and its absence from that of the chicken.

Cells from the embryonic quail retina were immortalized with the v-mil oncogene and cloned by limiting dilution. Their phenotype was examined using the whole-cell patch clamp method. Three membrane currents, IK(IR), INa and IK, were found at different frequencies within a sample of 170 cells drawn from a large clone. Nearly all combinations of these three markers were found and the frequency of combinations showed that the markers assorted independently. Examination of clones of less than 10 cells showed that heterogeneity originates with a high probability within clones, arguing that chromosomal mutation, for example, is unlikely to account for phenotypic diversity. A possible explanation is that phenotypic differences between cells might reflect the local exchange of instructive signals. If so, then the genes for the three phenotypic markers are controlled independently.

1H MRS provides a powerful method for investigating tumour metabolism by allowing the measurement of metabolites in vivo. Recently, the technique of 1H high-resolution magic angle spinning (HR-MAS) has been shown to produce high-quality data, allowing the accurate measurement of many metabolites present in unprocessed biopsy tissue. The purpose of this study was to evaluate the agreement between the techniques of in vivo MRS and ex vivo HR-MAS for investigating childhood brain tumours. Short-TE (30 ms), single-voxel, in vivo MRS was performed on 16 paediatric patients with brain tumours at 1.5 T. A frozen biopsy sample was available for each patient. HR-MAS was performed on the biopsy samples, and metabolite quantities were determined from the MRS and HR-MAS data using the LCModel™ and TARQUIN algorithms, respectively. Linear regression was performed on the metabolite quantities to asses the agreement between MRS and HR-MAS. Eight of the 12 metabolite quantities were found to correlate significantly (P

When mapped with a small spot of light, the central receptive fields of bipolar cells in the salamander retina are much larger than the extent of bipolar cell dendrites. Furthermore responses of bipolar cells to distant spots of light are considerably delayed relative to proximal spots. Using quantitative modelling, electrical coupling between bipolar cells is examined and rejected as a sufficient explanation of the data. An active process appears to shape signal waveform as signals spread laterally in the bipolar cell layer. Chemical synaptic coupling between bipolar cells is considered and shown to be inconsistent with the data. It is suggested that local, transient negative feedback from amacrine cells is involved in shaping bipolar cell signals.

BACKGROUND: Molecularly targeted approaches informing next-generation glioblastoma (GBM) chemotherapy must account for the intra-heterogeneous genome landscape and aim to eradicate invasive residual sub-populations. We outline an analytical strategy that forms a functional genomic corollary to existing genome-wide mutation and gene expression studies. METHODS: Biopsies from spatially distinct intra-GBM regions were surgically resected via Stealth and 5-ALA fluorescence guidance, including core, enhancing and invasive margin regions. Cellular intra-tumour heterogeneity (ITH) was confirmed by immunohistochemistry and qPCR arrays, whilst molecular analytics were conducted using liquid chromatography-mass spectrometry (metabolomic/lipidomic) magnetic resonance spectroscopy (metabolic) and SDS PAGE phosphoprotein enrichment (proteomic). RESULTS: Cellular ITH was confirmed via distinct neural stem cell expression in medial and posterior rims relative to the central enhancement region. Metabolites present in the invasive margin (5-ALA-determined), central enhancement and peri-necrotic core were distinct within each GBM. Controlling for the high proportion of normal brain cells surrounding the invasive margin, high tumour-specific glutamine levels were consistently present. Relative abundance of cytosolic phosphoproteins distinguished active protein pathways within intra-GBM regions, including invasive margin. Metabolomic network analyses confirmed that GBM residual disease is distinct from core ITH regions with respect to metabolic dependence. CONCLUSIONS: This innovative concept is the first to address ITH with multi-region biopsies using advanced analytical bioscience techniques. Identification of metabolites, lipids and phospho-proteins that are functional determinants of GBM infiltration reveal putative molecular targets on residual disease cells and represent a route to expedite new chemotherapy agents to clinic within a precision medicine framework.

The concept of a safe, immunocontraceptive vaccine using the zona pellucida glycoprotein 3 (ZP3) as an immunogen has been marred by the appearance of ovarian dysfunction in several species. However, careful selection of epitopes on mouse ZP3 have demonstrated that it is possible to segregate contraceptive bone marrow-derived (B)-cell epitopes from the cytotoxic thymus-derived (T)-cell epitopes thought to be responsible for inducing ovarian disease. B-cell epitopes on marmoset ZP3 (mstZP3) were identified by epitope mapping studies. Using a panel of polyclonal antibodies against recombinant mstZP3, an immunodominant epitope mstZP3(301-320) was identified. A chimeric peptide was co-linearly synthesized incorporating this sequence with a promiscuous tetanus toxoid T-helper cell epitope. Using the common marmoset (Callithrix jacchus) as an animal model, we have compared the consequences of active immunization with homologous recombinant mstZP3 and mstZP3(301-320) chimeric peptide vaccine. Long-term infertility was achieved using mstZP3 but at the expense of ovarian function. In contrast, no disruption to ovarian function was observed following mstZP3(301-320) immunization. Antibodies to this peptide immunolocalized to the zona pellucida of both marmoset and human ovarian sections and inhibited human sperm-zona binding by approximately 60% in vitro. However, in-vivo studies indicated that targeting a single ZP3 epitope was insufficient to reliably and consistently achieve a contraceptive effect.

PROBLEM: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. METHOD OF STUDY: Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3 45-64 , hZP3 93-110 , hZP3 172-190 and hZP3 341-360 ) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3 45-64 and hZP3 172-190 recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. RESULTS: No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. CONCLUSION: Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.

A fundamental issue in synaptic physiology is whether the postsynaptic response to a quantum of transmitter is limited by the number of receptors available. Fierce debate over the past few years has yielded no consensus. The majority of evidence suggests that the degree of receptor occupancy is likely to be sensitive to a number of factors, including the detailed anatomy of the synaptic cleft and the time course of transmitter clearance, and is probably different from one synapse to the next.

The peptide, neurotensin, is found in a class of amacrine cells synapsing chiefly withother amacrine cells in the chicken retina (Li & Lam, 1990; Watt et al., 1991). Toinvestigate the possible effects of neurotensin, we have used Ca2+imaging to measure cytosolic Ca2+concentrations in cultured chick amacrine cells. Following a delay of about 2 min, neurotensin (300 nM) induced oscillations in Ca2+concentration that typically had a period of 2 min and peak values of about 300 nM when averaged over the cell body. The phospholipase C inhibitors U-73, 112 and 4′-bromophenacyl bromide terminated oscillations induced by neurotensin but the protein kinase inhibitors H7 and staurosporine did not inhibit oscillations, increasing their frequency instead. In the absence of external Ca2+, neurotensin induced only a single Ca2+transient, much briefer than when external Ca2+was present. Together these results suggest that neurotensin activates phospholipase C, thereby producing IP3that triggers Ca2+release from an internal store. Although this released Ca2+contributes to periodic Ca2+peaks, the majority of cytosolic Ca2+, even in the first peak, comes from Ca2+influx across the plasmalemma.

Do graded potential synapses work the same way as action potential synapses? Recent work emphasizes the differences and suggests that graded potential synapses are not all the same either.

IntroductionPhysiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).MethodsCNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.ResultsMacroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.ConclusionsOur comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Cultured retinal amacrine cells show quantal GABAergic synaptic transmission. Voltage clamping pre- and post-synaptic cells of an isolated pair has allowed us to examine the entry and removal of Ca2+ at synaptic terminals. Brief presynaptic Ca2+ currents elicit an initial postsynaptic current that probably reflects the roughly synchronous exocytosis of docked vesicles. Prolonged Ca2+ currents elicit an additional second phase of release whose time course can greatly exceed that of the presynaptic voltage step. The time course of this second phase reflects a sustained increase in cytosolic Ca2+ and is matched closely by the activity of the presynaptic Na-Ca exchanger, as revealed by an exchange current. Eliminating the activity of the exchanger by removal of external Na+ prolongs this second phase of transmission greatly. Because transmitter release at these synapses outlasts Ca+ channel opening, Na-Ca exchange plays a significant role in shaping transmission.

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 x B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by the bar or pat genes, was more efficient than selection on kanamycin, mediated by the nptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants--transgene silencing, as well as poor transgene transmission to progeny was observed in some plant lines in which the parent plants had expressed the transgene.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Background Propionic acidaemia (PA) results from deficiency of Propionyl CoA carboxylase, the commonest form presenting in the neonatal period. Despite best current management, PA is associated with severe neurological sequelae, in particular movement disorders resulting from basal ganglia infarction, although the pathogenesis remains poorly understood. The role of liver transplantation remains controversial but may confer some neuro-protection. The present study utilises quantitative magnetic resonance spectroscopy (MRS) to investigate brain metabolite alterations in propionic acidaemia during metabolic stability and acute encephalopathic episodes. Methods Quantitative MRS was used to evaluate brain metabolites in eight children with neonatal onset propionic acidaemia, with six elective studies acquired during metabolic stability and five studies during acute encephalopathic episodes. MRS studies were acquired concurrently with clinically indicated MR imaging studies at 1.5 Tesla. LCModel software was used to provide metabolite quantification. Comparison was made with a dataset of MRS metabolite concentrations from a cohort of children with normal appearing MR imaging. Results MRI findings confirm the vulnerability of basal ganglia to infarction during acute encephalopathy. We identified statistically significant decreases in basal ganglia glutamate+glutamine and N-Acetylaspartate, and increase in lactate, during encephalopathic episodes. In white matter lactate was significantly elevated but other metabolites not significantly altered. Metabolite data from two children who had received liver transplantation were not significantly different from the comparator group. Conclusions The metabolite alterations seen in propionic acidaemia in the basal ganglia during acute encephalopathy reflect loss of viable neurons, and a switch to anaerobic respiration. The decrease in glutamine + glutamate supports the hypothesis that they are consumed to replenish a compromised Krebs cycle and that this is a marker of compromised aerobic respiration within brain tissue. Thus there is a need for improved brain protective strategies during acute metabolic decompensations. MRS provides a non-invasive tool for which could be employed to evaluate novel treatments aimed at restoring basal ganglia homeostasis. The results from the liver transplantation sub-group supports the hypothesis that liver transplantation provides systemic metabolic stability by providing a hepatic pool of functional propionyl CoA carboxylase, thus preventing further acute decompensations which are associated with the risk of brain infarction.

We have examined synaptic transmission between isolated pairs of chick GABAergic amacrine cells, maintained in sparse culture and identified by their binding of an amacrine cell-selective antibody. Using the perforated-patch method to whole-cell clamp both cells of a pair, postsynaptic currents were examined for step depolarizations of the “presynaptic” cell. Synaptic transmission, frequently reciprocal, was calcium dependent and reversibly blocked by bicuculline. Post-synaptic currents, excluding those due to ohmic electrical coupling, were elicited only for presynaptic voltage steps positive to about -40 mV and were always very noisy, suggesting that they were summed from relatively small numbers of quanta. Postsynaptic currents continued well after the termination of the 100 msec presynaptic voltage step when the step was to -10 mV, or positive to this value. This result is interpreted to imply that presynaptic calcium concentration remains elevated after the membrane is returned to its holding potential. When presynaptic voltages were kept low or else presynaptic voltage was uncontrolled, spontaneous quantal events mediated by GABAA receptors could often be seen. Quanta rose quickly (less than 4 msec) and decayed with a mean time constant of 19.3 msec. The amplitude distributions of quantal currents were positively skewed, sometimes showing rare quanta of exceptionally large amplitude. Peak conductance per quantum was about 300 pS, corresponding to the simultaneous opening of only 17 GABAA channels and corresponding to a net flux of only 32 x 10(3) Cl- ions per millivolt of driving force. Estimates of the maximum sustained release rate at individual release sites suggest an upper bound of between 19 and 42 quanta per second.

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve paris of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (IK) and inward rectifier (IK(IR)) potassium currents. Despite qualitative and quantitative variability in IK and IK(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between –110 and +50 mV. We estimated IK(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated IK(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed IK(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis. © 1993 John Wiley & Sons, Inc.

Contains fulltext : 88654.pdf (Publisher’s version ) (Open Access) BACKGROUND: High-resolution magic angle spinning (HRMAS) NMR spectroscopy allows detailed metabolic analysis of whole biopsy samples for investigating tumour biology and tumour classification. Accurate biochemical assignment of small molecule metabolites that are "NMR visible" will improve our interpretation of HRMAS data and the translation of NMR tumour biomarkers to in-vivo studies. RESULTS: 1D and 2D 1H HRMAS NMR was used to determine that 29 small molecule metabolites, along with 8 macromolecule signals, account for the majority of the HRMAS spectrum of the main types of brain tumour (astrocytoma grade II, grade III gliomas, glioblastomas, metastases, meningiomas and also lymphomas). Differences in concentration of 20 of these metabolites were statistically significant between these brain tumour types. During the course of an extended 2D data acquisition the HRMAS technique itself affects sample analysis: glycine, glutathione and glycerophosphocholine all showed small concentration changes; analysis of the sample after HRMAS indicated structural damage that may affect subsequent histopathological analysis. CONCLUSIONS: A number of small molecule metabolites have been identified as potential biomarkers of tumour type that may enable development of more selective in-vivo 1H NMR acquisition methods for diagnosis and prognosis of brain tumours.

Background Brain and nervous system tumours are the most common solid cancers in children. Molecular characterisation of these tumours is important for providing novel biomarkers of disease and identifying molecular pathways which may provide putative targets for new therapies. 1H magic angle spinning NMR spectroscopy (1H HR-MAS) is a powerful tool for determining metabolite profiles from small pieces of intact tissue and could potentially provide important molecular information. Methods Forty tissue samples from 29 children with glial and primitive neuro-ectodermal tumours were analysed using HR-MAS (600 MHz Varian gHX nanoprobe). Tumour spectra were fitted to a library of individual metabolite spectra to provide metabolite values. These values were then used in a two tailed t-test and multi-variate analysis employing a principal component analysis and a linear discriminant analysis. Classification accuracy was estimated using a leave-one-out analysis and B632+ bootstrapping. Results Glial tumours had significantly (two tailed t-test p < 0.05) higher creatine and glutamine and lower taurine, phosphoethanolamine, phosphorylcholine and choline compared with primitive neuro-ectodermal tumours. Classification accuracy was 90%. Medulloblastomas (n = 9) had significantly (two tailed t-test p < 0.05) higher creatine, glutamine, phosphorylcholine, glycine and scyllo-inositol than neuroblastomas (n = 7), classification accuracy was 94%. Supratentorial primitive neuro-ectodermal tumours had metabolite profiles in keeping with other primitive neuro-ectodermal tumours whilst ependymomas (n = 2) had metabolite profiles intermediate between pilocytic astrocytomas (n = 10) and primitive neuro-ectodermal tumours. Conclusion HR-MAS identified key differences in the metabolite profiles of childhood brain and nervous system improving the molecular characterisation of these tumours. Further investigation of the underlying molecular pathways is required to assess their potential as targets for new agents.

The products of first microspore mitosis were studied in anthers of Hordeum vulgare. These anthers were obtained either directly from plants at the binucleate stage or from tillers which had been estimated visually as containing pollen at the mid-uninucleate stage and then pretreated by being clipped off at ground level and allowed to stand in water for 2 d. In microspores from the latter plants ten-fold increases in the failure of nuclear differentiation, alteration of the axis of division, or both were observed. High frequencies of further microspore response in individual anthers were subsequently induced through culture of the spikes from the pretreated tillers in agitated liquid medium. A five to ten-fold increase in the percentage of anthers producing macroscopic callus was observed in these spikes when compared with the controls. It is suggested that nucleo-cytoplasmic disturbances in the microspores, brought about through the pretreatment period, stimulated the further divisions which eventually resulted in microspore callus formation. Microspore calluses were produced most often following an equal first division, although when mitosis resulted in differentiated nuclei further divisions of the vegetative nucleus also resulted in callus production. Haploid, diploid, polyploid, and mixaploid microspore calluses were produced but no evidence was obtained to link haploid production with any particular developmental pathway. Macroscopic calluses which emerged from the cultured anthers were probably mixtures of cell populations derived from several or many different gametic genotypes.

Suspension cultures of Hevea brasiliensis cells can metabolize the growth regulator 2 chloroethylphosphonic acid to produce a number of compounds, one of which appears to be an acid-labile conjugate. The general metabolic pattern closely resembles that found previously using Hevea leaves. Evidence that the compounds formed are not chromatographic artefacts of a type described by other authors is presented.