Showing papers by "University of Liverpool published in 1987"

Fluorescence and bioluminescence measurement of cytoplasmic free calcium.

Abstract: The first measurements of cytosolic free Ca2\", [Ca2\"]1, in living cells were made with aequorin in giant muscle cells (Ridgway & Ashley, 1967) in the late 1960s. During the 1970s two further methods were introduced, bis-azo absorbance dyes (mainly arsenazo III) and calciumselective microelectrodes (Ashley & Campbell, 1979). However, even as late as 1981, [Ca'1] had been reliably measured in very few cell types other than giant cells of invertebrates and the techniques were largely confined to the laboratories of those specialized in excitable cell physiology and familiar with microelectrodes or microinjection and advanced optical or electronic technologies. In 1982 a new generation of fluorescent dyes was introduced, with quin2 (Tsien et al., 1982a) and a chemical trick for loading it non-disruptively into populations of cells of any size (Tsien, 1981). This technique in its basic form is simple enough, and needs only basic laboratory instrumentation, so that it is now routinely used in hundreds oflaboratories. The technique has been rapidly developed with the invention of the superior dyes, fura-2 and indo1, and technologies for monitoring [Ca2\"], in single cells, whole fields ofidentified cells, and even localized areas within cells, and also improvements in time resolution down to the millisecond range. An outline of these developments forms part of this review. Clearly, with hundreds of papers already published we can only point out the major features, advantageous and problematic, of the technique and hope to provide a critical guide to the literature that details the important points. Another development has been the refinement of the use of the calcium sensitive photoproteins aequorin and obelin. Important novel data on microinjected small single cells are now coming from specialist laboratories and the techniques are not as fearsomely difficult as commonly thought. Part of this review is therefore devoted to an explanation of this method, its advantages and pitfalls, and its successes. We will not consider bisazo dyes or calcium-selective microelectrodes. There are several detailed accounts of these techniques and their place in relation to other approaches (e.g. Thomas, 1982; Blinks et al., 1982; Rink, 1983; Tsien & Rink, 1983). Nor will we review the fluorine derivatives of the fluorescent dyes that have provided n.m.r. probes (Metcalf et al., 1985). This review covers the technical considerations of using fluorescent and bioluminescent probes for measuring [Ca2+]1; we make no attempt to cover the extensive and proliferating data obtained from such measurements.

Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels.

Abstract: Inositol 1,4,5-trisphosphate (Ins P3) is a second messenger releasing intracellular Ca2+ into the cytosol–5. It has recently been proposed that inositol 1,3,4,5-tetrakisphosphate (Ins P4), which is formed from Ins P3 by Ins P3-3-kinase (ref. 6), acts with Ins P3 as a second messenger by promoting extracellular Ca2+ entry7. It has been suggested that Ins P3 itself can act to stimulate Ca2+ uptake from the extracellular fluid, although a physiological function for Ins P4 was not excluded8,9. Transmembrane currents can now be measured in single cells by voltage clamping under conditions where the intracellular perfusion fluid can be changed several times during individual experiments10,11. We have used this method to test the effects of Ins P3 and Ins P4 on the Ca2+-activated K+ current12,13, and now show that neither Ins P3 alone nor Ins P4 alone can activate a sustained current, whereas Ins P3 and Ins P4 in combination evoke a sustained increase in Ca2+-activated K+ current which is dependent on external Ca2+.

The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo

Abstract: A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600) In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo

Range of mental illness among the elderly in the community. Prevalence in Liverpool using the GMS-AGECAT package.

Abstract: A sample of 1070 elderly persons aged over 65 living in the Liverpool community was interviewed using the community version of the Geriatric Mental State (GMS) and the findings processed to provide a computerised diagnosis by AGECAT. Levels of organic disorder, probably dementia, reached 5.2%, intermediate between those of London and New York derived from previous studies. Levels for depressive illness overall were below those of other studies at 11.3% while levels for neurotic disorder were much the same at 2.4%. The rise in the prevalence of dementia with age was further confirmed. The GMS AGECAT Package provides a method for standardising both the collection of data and the diagnostic process for comparative epidemiological studies and other research.

Lignocellulose-degrading actinomycetes

Abstract: Increasing interest in the exploitation of plant biomass as a renewable resource has provided an impetus for research on microbial degradation of lignocellulose. This has traditionally been concentrated on the fungi, but lignocellulose-degrading prokaryotes are beginning to receive more attention. Strain improvement by genetic manipulation, and large-scale cultivation are more easily achieved in prokaryotes, and the actinomycetes are no exception. In addition, their growth as branching hyphae is well adapted to the penetration and degradation of insoluble substrates such as lignocellulose. Actinomycetes form an important part of the microbial community responsible for nutrient recycling in natural substrates. Their specific contribution to lignocellulose degradation in the environment has received only limited attention but their importance can be inferred from the ubiquity and diversity of species in which activity against lignocellulose has been demonstrated. Characterisation of the cellulolytic activity of actinomycetes has understandably received most attention, since cellulose is the major component of plant biomass and a potentially utilisable source of glucose. Actinomycete cellulases are inducible extracellular enzymes which appear to attack cellulose in much the same way as fungal hydrolytic cellulases. Actinomycete xylanases similarly conform to the basic patterns of production and activity established in other bacteria and fungi, but they have been relatively little-studied. Evidence for activity against lignin has only recently been obtained for actinomycetes, with the development of sensitive radiometric assays which permit detection of limited ligninolytic activity. There is little doubt that actinomycetes compare poorly with the whiterot fungi in relation to the extent of delignification achieved, particularly with wood lignocelluloses. Nevertheless, their ability to solubilise grass lignins may have a role in humification and biotechnological applications of lignocellulose conversion. The biochemistry of lignin degradation by actinomycetes remains poorly understood and the enzymes involved have yet to be identified. There is more information on the enzymology of cellulose and hemicellulose degradation, but how these different groups of enzymes and their component proteins interact to solubilise lignocellulose is largely unknown. Elucidation of the biochemistry of lignocellulose degradation and development of actinomycete-based systems for lignocellulose conversion should be greatly assisted by the application of the recombinant DNA techniques recently developed for this group of organisms.

Tissue-biomaterial interactions

Abstract: A wide variety of materials is being increasingly used in medical practice for the treatment of patients in which the materials come into direct and often sustained contact with the tissues of the body. The commonly-known examples of hip replacements, contact and intraocular lenses and pace-makers serve to emphasize the importance of this subject. Because the body is so well equipped to reject any intruding object, whether that is a bacterium or a splinter of wood, the materials which are to stand any chance of success within this hostile yet sensitive milieu must be chosen very carefully. The subject of biomaterials represents an almost unique blend of physical and biological sciences, and it is becoming increasingly important that these aspects are drawn together to help in the development of quality biomaterials that are able to perform optimally in this environment. The key to this subject lies in the interactions that take place between biomaterials and these tissues and this review is aimed at providing, for the materials scientist, an understanding of the mechanisms of these interactions.

Organic complexation and its control of the dissolved concentrations of copper and zinc in the Scheldt estuary

Abstract: Cathodic stripping voltammetry (CSV) is used to determine total (after UV-irradiation) and labile dissolved metal concentrations as well as complexing ligand concentrations in samples from the river Scheldt estuary. It was found that even at high added concentrations of catechol (1 mm for copper and 0·4 mm for iron) and of APDC (1 mm for zinc) only part of the dissolved metal was labile (5–58% for copper, 34–69% for zinc, 10–38% for iron); this discrepancy could be explained by the low solubility of iron which is largely present as colloidal material, and by competition for dissolved copper and zinc by organic complexing ligands. Ligand concentrations varied between 28 and 206 nm for copper and between 22 and 220 nm for zinc; part of the copper complexing ligands could be sub-divided into strong complexing sites with concentrations between 23 and 121 nm and weaker sites with concentrations between 44 and 131 nm. Values for conditional stability constants varied between (logK′ values) 13·0 and 14·8 for strong and between 11·5 and 12·1 for weaker copper complexing ligands, whereas for zinc the values were between 8·6 and 10·6. The average products of ligand concentrations and conditional stability constants (a-coefficients) were 6 × 102 for zinc and 6 × 106 for copper. The dissolved zinc concentration was found to co-vary with the zinc complexing ligand concentration throughout the estuary. It is argued that the zinc concentration is regulated, in this estuary at least, by interactions with dissolved organic complexing ligands. A similar relationship was apparent between the dissolved copper and the strong copper complexing ligand concentration. The total copper complexing ligand concentrations were much greater than the dissolved copper concentrations, suggesting that only strongly complexed copper is kept in solution. These results provide evidence for the first time that interactions of copper and zinc with dissolved organic complexing ligands determine the geochemical pathway of these metals.

Sedimentologic and geomorphic variations in storm-generated alluvial fans, Howgill Fells, northwest England

Abstract: In June 1982, a storm with a return period greater than 100 yr, but lasting less than 2.5 hr, destabilized hillslopes and produced a suite of geomorphologically and sedimentologically diverse alluvial fans. Thirteen major fans were deposited at the tributary junctions between small ( 2 ) catchments and two north-flowing; headwater streams of the River Lune, northwest England. Storm-generated fans spread over or became inset into older stable fans and produced localized vertical accretion of as much as 3 m and lateral accretion of as much as 100 m. Sedimentary processes operating during deposition involved debris flow, transitional flow, and streamflow. Six facies types are recognized on the basis of depositional topography, sedimentary texture and fabric, and matrix content: viscous debris flow (D1), dilute debris flow (D2), transitional flow (T1), fluvial bars and lobes (S1, S2), and fluvial sheet gravels (S3). Regionally, streamflow deposition prevails over debris-flow deposition, and type S3 facies has the greatest areal extent. Temporal and spatial variations in facies deposition during the storm, however, resulted from water:sediment ratio variations. Fan deposition involved an early phase of debris flow to transitional flow due to large inputs of sediment from hillslope failures. This was followed by a systematic change to more dilute conditions, resulting in streamflow deposition and eventually in channel incision. A significant amount of geomorphic work and complex variations in sedimentary processes during the storm resulted, in part, from extensive overland flow and hillslope destabilization. Discriminant analyses indicate that catchment size, channel gradient, and percentage of area eroded during the storm controlled whether debris-flow or streamflow facies dominated a fan sequence. Smaller, steeper catchments had a greater percentage of the area yielding sediment and are dominanted by debris flows, whereas larger catchments produced more runoff resulting in dilution and streamflow. The results indicate that the facies sequences and fan entrenchment in the Howgill Fells, which are typically considered products of longer term climatic change or tectonics in other localities, are here primarily affected by thresholds related to catchment geomorphology, by the type of sediment available, and by the position within the storm cell.

Trace fossils and correlation of late Precambrian and early Cambrian strata

Abstract: Trace fossils are abundant and diverse in many clastic sequences spanning the Precambrian-Cambrian boundary and may prove to be the most useful palaeontological method for global correlation in this stratigraphic interval. The ichnofaunas of the latest Precambrian (Vendian) rocks include some forms whose range does not extend into the Cambrian (e.g. Bilinichnus, Intrites, Palaeopascichnus, Vendichnus, Vimenites) and others which continue throughout most or all of the Phanerozoic (e.g. Arenicolites, Aulichnites, Cochlichnus, Didymaulichnus, Gordia, Neonereites, Planolites, Skolithos). At least 50 ichnogenera make their first appearance below the lowest trilobites in sections with broad geographic spread. A few of these appear to have a short time range, extending to about the incoming of the trilobites (e.g. Astropolichnus, Didymaulichnus miettensis, Plagiogmus, Taphrhelminthopsis circularis), but the majority continue through most or all of the Phanerozoic.For correlation of Precambrian-Cambrian boundary sequences it is therefore possible to use both the occurrence of those ichnogenera with a short time range and the incoming of those with an extended range. Three stratigraphical zones can be recognized with respect to the incoming of trace fossils. Zone I is of Upper Vendian age and includes Arenicolites, Bilinichnus, Cochlichnus, Didymaulichnus, Gordia, Harlaniella, Intrites, Nenoxites, Neonereites, Palaeopascichnus, Skolithos, Vendichnus and Vimenites. In Zone II, of Lower Tommotian age, the earliest examples of Bergaueria, Phycodes, Teichichnus and Treptichnus are encountered. Many trace fossils appear in Zone III, which extends from Upper Tommotian to Lower Atdabanian, but the most important are: Astropolichnus, Cruziana, Diplichnites, Diplocraterion, Dimorphichnus, Plagiogmus, Rusophycus and Taphrhelminthopsis circularis.This vertical zonation of trace fossils allows an attempt at world-wide correlation, from which the most significant conclusions are that the Vendian/Tommotian boundary can probably be placed: (i) near the middle of the McNaughton Formation in the Rocky Mountains, Canada; (ii) at the base of the Deep Spring Formation or in the underlying Reed Dolomite in the White Inyo Mountains, California, U.S.A.; (iii) low in the Chapel Island Formation in the Burin Peninsula, Newfoundland, Canada; (iv) at or close to the base of the Candana Quartzite in North Spain; (v) at or below the base of the Breivik Member in Finnmark, Norway; and (vi) near or below the base of the Zhongyicun Member at Meischucun, China.The sections in the Burin Peninsula, Newfoundland and Meischucun, China are favoured candidates for the global stratotype for the Precambrian-Cambrian boundary. In the Burin Peninsula, the trace fossils suggest that the Tommotian/Atdabanian boundary may be within or at the base of the Random Formation, thereby implying that the Tommotian may include a thickness of 500 m of sediment comprising at least most of the Chapel Island Formation. At Meishucun, the ichnofaunal evidence implies that the Tommotian/Atdabanian boundary is probably no higher than the top of the Zhongyicun Member. The thickness of the Tommotian is therefore possibly only about 20 m here, implying a very condensed sequence, a conclusion consistent with an abundance of phosphorites. Two stratotype reference points for the Precambrian-Cambrian boundary have been suggested in this section. The lower point (0.8 m above the base of the Xiawaitoushan Member) may be near the Vendian/Tommotian boundary or younger, while the higher point (base of Unit 7 of the Zhongyicun Member) is probably Upper Tommotian or even Lower Atdabanian. The higher point would place the boundary above the world-wide dramatic increase in trace fossil abundance and diversity but probably before the first trilobites. This would almost certainly have advantages for correlation. The inference that the Meishucun section is younger than most Chinese work suggests should not therefore, by itself, prejudice its adoption as global stratotype.In general, where comparative data are available, the trace fossil correlations agree well with pre-existing proposals based on small shelly fossils. The degree of resolution of the two methods would appear at present to be similar but trace fossils, being found mainly in clastic facies, may benefit from more frequent occurrence.

Cellular origins of cyclic GMP responses to excitatory amino acid receptor agonists in rat cerebellum in vitro.

Abstract: Incubated slices and freshly dissociated cells from 8-day-old rat cerebellum were used to try to identify the cells that participate in the large increases in cyclic GMP levels that follow activation of excitatory amino acid receptors in this tissue. In the slices, cyclic GMP responses to L-glutamate and related excitants were unaffected by tetrodotoxin and could be replicated by the guanylate cyclase activator nitroprusside. Nitroprusside and the receptor agonists appeared to activate the same pool of the enzyme. Prior destruction of neuroblasts, deep nuclei, or Golgi neurones did not cause loss of responses to L-glutamate. If granule cells were rendered necrotic, however, the cyclic GMP responses to all excitants tested were reduced by greater than or equal to 90%. Substantial losses of responses to veratridine and high K+ levels also occurred, but the nitroprusside-induced elevations were unaffected. In dissociated cell suspensions, the magnitude of responses to receptor agonists, but not those to nitroprusside, was markedly dependent on cell concentration. Responses to L-glutamate were the same in cell suspensions that were Purkinje cell depleted and Purkinje cell enriched. It is concluded that granule cells are primarily involved in the cyclic GMP responses to excitatory amino acids but that the cyclic GMP accumulations occur elsewhere, probably in glial cells.

1987 Volvo award in clinical sciences. The perception of back pain and the role of psychophysical tests of lifting capacity.

Abstract: In order to study the predictive value of pre-employment screening tests, a volunteer population of 1,741 men and 1,150 women was questioned about their experience of low-back pain (LBP) and their perception of physical exertion at work. They undertook a battery of tests, including psychophysical as

Granites and yet more granites forty years on

Abstract: The theme of this synoptic review is the relationship between cause, process, source and the geological context during the genesis of the granitic rocks. A close environmental relationship occurs because granites can arise as the end-stage of several generative processes, involving different source rocks, each appropriate to a particular tectonic situation. Expressed in geological terms the differences can be used to erect a genetic classification, to set in perspective the intervention of basic magma, to reveal the importance of the volcanoplutonic interface, to evaluate the significance of the texture, to predict the type of mineralisation, to understand the mode of emplacement and to determine the contribution of the various source rocks.

Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation.

Abstract: Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.

The differentiation of the Skaergaard intrusion

Abstract: McBirney and Naslund have missed the two central points that we reiterate for clarity. First the composition of the cotectic cumulate extract for the LZc, best estimated from the average composition of adcumulate rocks, had a substantially lower silica content than any conceivable basaltic liquid, so silica enrichment is unavoidable. This is true of all the dyke compositions reported by Brooks and Nielsen, even the anomalous rock E with SiO2=46%. Second it is the normal behaviour of tholeiite basaltic magma to produce increasingly iron-rich cumulate extracts with little change in silica until FeTi oxide precipitation occurs when silica-enrichment and compositional divergence begins. Such divergence between equivalent cumulate plutonic and fractionated volcanic rocks hads been widely documented. Skaergaard magmas do have some uncommon features. However, similar assemblages exist in volcanic rocks as in the andesitic to rhyolitic glasses of the Loch Ba ring dyke, Mull (Sparks 1988). It is also a basic principle that elements enriched in cumulate rocks often become depleted in the corresponding liquids. Iron enrichment in the Skaergaard cumulates is complementary to iron depletion in liquids.

Use of perceived effort ratings to control exercise intensity in young healthy adults

Abstract: The purpose of this study was to evaluate the use of the rating of perceived exertion (RPE) as a means of regulating the intensity of exercise during running. The subjects were healthy, relatively fit young adults (16 men and 12 women). Estimates of effort were recorded using the Borg 6–20 Scale whilst the maximal oxygen uptake of the subjects was measured as they ran on an electrically driven treadmill. In a further session, the same subjects were requested to run on the treadmill at constant exercise intensity based on their interpretation of levels 9, 13 and 17 of the Borg Scale. They regulated their running speed and the treadmill gradient but had no knowledge of performance from the equipment display panel. A linear regression analysis was carried out to examine the relationship between heart rate, perceived exertion and relative metabolic demand. This revealed that the rating of perceived exertion was at least as good a predictor of exercise intensity as heart rate in both the graded exercise test and effort production test. The results support the view that RPE may be used to predict relative metabolic demand, especially at higher workloads and could be a useful medium for controlling intensity of effort during vigorous exercise in such subjects.

Reorganisation of peripheral actin filaments as a prelude to exocytosis

Abstract: Evidence is presented, from studies on the adrenal chromaffin cell, that reorganisation of the cortical actin network is necessary to allow granules to reach exocytotic sites in stimulated cells. This reorganisation may involve changes in actin filament cross-linking, assembly and interactions with secretory granule and plasma membranes. The possibility is discussed that cytoskeletal elements including the membrane-binding proteins caldesmon, p70 and p36 may be involved in granule-plasmalemmal interactions immediately prior to exocytosis.

The effects of magnesium upon adenosine triphosphate‐sensitive potassium channels in a rat insulin‐secreting cell line.

Abstract: 1. The patch-clamp method of single-channel recording was applied to K+ channels which are inhibited by intracellular adenosine 5'-triphosphate (ATP: K+-ATP channels) in membrane patches obtained from the insulin-secreting cloned cell line RINm5F. 2. The magnitude of K+ currents flowing outwards through these K+-ATP channels was reduced by internal Mg2+ ions in a dose-dependent manner. Currents flowing inwards through the channels were not affected by Mg2+. Internal Na+ ions had similar effects. 3. Divalent cations (Mg2+, Sr2+ and Ca2+) applied to the internal surface of the patch membrane inhibited the opening of K+-ATP channels in a dose-dependent manner. Internal Na+ ions had no effect. 4. K+-ATP channel activity was stimulated by adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), guanosine 5'-o-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-o-(2-thiodiphosphate) (GDP beta S) when millimolar Mg2+ bathed the internal surface of the patch membrane. In the virtual absence of internal Mg2+ ions ADP, GTP, and GTP gamma S inhibited K+-ATP channels and GDP and GDP beta S were without effect. Adenosine 5'-o-(2-thiodiphosphate) (ADP beta S) inhibited K+-ATP channel activity in the presence and absence of Mg2+. 5. K+-ATP channel openings could be evoked by either ADP or GDP in the presence of an inhibitory concentration of ATP. These openings were abolished in the absence of internal Mg2+. 6. Run-down K+-ATP channels could be reactivated by ATP in the presence of internal Mg2+, but not in its absence. Analogues of ATP were unable to reactivate K+-ATP channels even in the presence of Mg2+. 7. It is concluded that internal Mg2+ ions (i) cause the rectification of the K+-ATP channel current-voltage relationship, (ii) are required for K+-ATP channel activity to be maintained by a phosphorylation process and (iii) are required for K+-ATP channel activity evoked by ADP, GTP and GDP.

A quantitative study of histological changes in the human parotid gland occurring with adult age.

Abstract: Parotid samples from 63 'sudden death' necropsies of both sexes, aged 17-90 years, were collected after exclusions for chronic illness or medications Advancing age was accompanied by acinar atrophy and ductal irregularities The adipose content varied widely at all ages but, together with fibrovascular tissue, tended to increase with age Stereological analysis demonstrated a linear reduction of acinar proportional volume amounting to 30% over the age range Females tended to have more adipose tissue and less fibrous tissue than males These structural age changes resemble those of other salivary glands, but unlike the latter are not accompanied by age-dependent functional impairments This suggests a greater acinar secretory efficiency or larger secretory reserve volume in the parotid than in other glands The frequent high levels of parotid adiposity encountered suggest this feature is not pathological nor necessarily a reliable indicator of nutritional or hormonal diseases, excluded from the present study