Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea)

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

ULTRASTRUCTURAL-LOCALIZATION OF PANCREATIC POLYPEPTIDE AND FMRFAMIDE IMMUNOREACTIVITIES WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE NEMATODE, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

A post-embedding immunogold technique has been used to examine the subcellular distribution of immunoreactivities to vertebrate pancreatic polypeptide (PP) and to the invertebrate peptide, FMRFamide within the central nervous system (CNS) of the nematode, Ascaris suum. Gold labelling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the main ganglia and nerve cords in the CNS. Double-labelling of peptides demonstrated an apparent co-localization of PP and FMRFamide immunoreactivities in the same dense-cored vesicles, although populations of dense-cored vesicles that labelled solely for FMRFamide were also evident. Antigen preabsorption studies indicated little or no cross-reactivity between the two antisera.

ULTRASTRUCTURAL-LOCALIZATION OF FMRFAMIDE-IMMUNOREACTIVITY AND PANCREATIC POLYPEPTIDE-IMMUNOREACTIVITY WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE LIVER FLUKE, FASCIOLA-HEPATICA (TREMATODA, DIGENEA)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMRFamide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode, Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and 'giant' nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE CENTRAL-NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

Development of XUV lasers at the RAL central laser facility

Recent progress in the development of XUV lasers by research teams using high-power and ultrashort-pulse Nd:glass and KrF laser facilities at the Rutherford Appleton Laboratory is reviewed. Injector-amplifier operation and prepulse enhanced output of the Ge XXIII collisional laser driven by a kilojoule glass laser, enhanced gain in CVI recombination with picosecond CPA drive pulses from a glass laser, and optical field ionization and XUV harmonic generation with a KrF CPA laser are described.

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

A novel GUCY2D mutation, V933A, causes central areolar choroidal dystrophy

Purpose: To identify the genetic cause of central areolar choroidal dystrophy (CACD) in a large Northern Irish family.
Methods: We previously reported linkage of the locus for CACD in this family to an interval of approximately 5 cM on chromosome 17p13 flanked by polymorphic markers D17S1810 and CHLC GATA7B03. We undertook sequence capture, massively-parallel sequencing and computational alignment, base-calling and annotation to identify a causative mutation. Conventional sequencing was used to confirm the results.
Results: Deep sequencing identified a single-base substitution in guanylate cyclase 2D, membrane (retina-specific) (GUCY2D). The novel mutation segregated with the disease phenotype and resulted in substitution of valine by alanine at position 933, within the catalytic domain of the protein. It altered a motif that is strongly conserved in a large number of distantly related proteins across several species, and was predicted to have a damaging effect on protein activity.
Conclusion: Mutations in GUCY2D have previously been associated with dominant cone rod dystrophies (CORD6) and recessive forms of Leber's congenital amaurosis (LCA). This is the first report of GUCY2D mutation causing CACD and adds to our understanding of genotype-phenotype correlation in this heterogeneous group of choroidoretinal dystrophies.

Off-central

Edited version of keynote address to AHRA 'Peripheries' conference, October 2011

Isolation basin records of late Quaternary sea-level change, central mainland British Columbia, Canada.

Isolation basin records from the Seymour-Belize Inlet Complex, a remote area of central mainland British Columbia, Canada are used to constrain post-glacial sea-level changes and provide a preliminary basis for testing geophysical model predictions of relative sea-level (RSL) change. Sedimentological and diatom data from three low-lying (<4 m elevation) basins record falling RSLs in late-glacial times and isolation from the sea by ~11,800–11,200 14C BP. A subsequent RSL rise during the early Holocene (~8000 14C BP) breached the 2.13 m sill of the lowest basin (Woods Lake), but the two more elevated basins (sill elevations of ~3.6 m) remained isolated. At ~2400 14C BP, RSL stood at 1.49 ± 0.34 m above present MTL. Falling RSLs in the late Holocene led to the final emergence of the Woods Lake basin by 1604 ± 36 14C BP. Model predictions generated using the ICE-5G model partnered with a small number of different Earth viscosity models generally show poor agreement with the observational data, indicating that the ice model and/or Earth models considered can be improved upon. The best data-model fits were achieved with relatively low values of upper mantle viscosity (5 × 1019 Pa s), which is consistent with previous modelling results from the region. The RSL data align more closely with observational records from the southeast of the region (eastern Vancouver Island, central Strait of Georgia), than the immediate north (Bella Bella–Bella Coola and Prince Rupert-Kitimat) and areas to the north-west (Queen Charlotte Sound, Hecate Strait), underlining the complexity of the regional response to glacio-isostatic recovery.

Holocene climate change, vegetation history and human impact in the Central Mediterranean: evidence from the Maltese Islands

Holocene climates and human impact in the Mediterranean basin have received much attention, but the Maltese Islands in the Central Mediterranean, although a pivotal area, have been little researched. Here, sedimentary and palynological data are presented for three cores from the Holocene coastal and shallowmarine
deposits of the Maltese Islands. These show deforestation from Pinus-Cupressaceae woodland in the early Neolithic, and then a long, but relatively stable history of agriculturally degraded environments to the present day. The major climate events which have affected the Italian and Balkan peninsulas to the
north, and Tunisia to the south, are not reflected in the pollen diagrams from the Maltese Islands because of the strong anthropogenic imprint on the Maltese vegetation from early in the Neolithic. Previous suggestions of environmentally-driven agricultural collapse at the end of the Neolithic appear, however,
to be substantiated and may be linked to regional aridification around 4300 cal. BP. Depopulation in early Medieval times is not supported by the current palynological evidence.

Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.