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PubMed1991

Visualisation and measurement of the calcium message in guard cells.

We have applied several novel technologies to investigate the role of cytosolic free calcium [Ca2+]i in signal transduction in guard cells of Commelina communis L. Fluorescence ratio imaging and photometry together with the fluorescent Ca2+ indicator Indo-1 were used to directly visualise and measure dynamic spatial and temporal changes in [Ca2+]i in response to various exogenous stimuli. More subtle manipulation of the Ca2+ signal transduction pathway was achieved through the use of photoactivateable, caged Ca2+ and caged inositol-1,4,5-triphosphate (InsP3) released directly into the cytoplasm of the guard cell after microinjection. In these experiments, changes in [Ca2+]i were simultaneously monitored with the fluorescent Ca2+ indicator, Fluo-3. Resting levels of [Ca2+]i (100-200 nM) increased in response to elevated [Ca2+]e, lowering [K+]e, application of the ionophore A-23187 or cytosolic release of either Ca2+ or InsP3 from their caged forms. Stomatal closure was triggered if [Ca2+]i increased above a threshold of about 600 nM. Abscisic acid (ABA) had little effect on [Ca2+]i in the majority of cells studied, being elevated in only a minority of cells investigated. However, stomatal closure occurred in all cases after ABA application. This suggests that ABA acts through both Ca(2+)-independent and Ca(2+)-dependent pathways. The imaging data revealed a substantial heterogeneity in [Ca2+]i within the guard cell. Cytoplasmic regions, particularly near the nucleus, often showed marked elevations and sometimes oscillations. The origin and kinetics of the Ca2+ fluxes leading to the dynamic spatial patterns is discussed along with several new approaches directed towards identification of the source of the Ca2+. These methods include optical sectioning and 3-D reconstruction of both the endomembrane system and [Ca2+]i in living guard cells using confocal microscopy. Overall, our data is consistent with multiple sources for [Ca2+]i, including uptake across the plasma membrane and InsP3- or Ca(2+)-induced Ca2+ release from internal stores.

Calcium↗

PubMed1992

Immunocytochemical identification of oestrogen receptors in preoptic neurones containing calcitonin gene-related peptide in the male and female rat.

Using single- and double-labelling immunocytochemistry with antibodies specific for the oestrogen receptor and calcitonin gene-related peptide (CGRP), we have demonstrated oestrogen receptor immunoreactivity in the sexually dimorphic CGRP-immunoreactive (IR) population of the medial preoptic area (MPOA). In the short-term gonadectomised female approximately 80% of preoptic CGRP-IR neurones were immunoreactive for the oestrogen receptor. In short-term gonadectomised males, a small population of CGRP-IR cells was visualised in the MPOA only after colchicine treatment. Approximately 30% of CGRP-IR cells in the male were oestrogen receptor-IR, accounting for 2% of the total population of cells containing oestrogen receptors in this area. In the colchicine-treated female, it is estimated that 10-15% of preoptic oestrogen receptor-IR cells contain CGRP. These results indicate that CGRP is synthesised by preoptic neurones with oestrogen receptors. Furthermore, the identification of oestrogen receptors in the sexually dimorphic CGRP population suggests that these neurones may be directly linked with gonadal steroid-dependent, sex-specific functioning of the MPOA.

Animals↗

PubMed1990

Visualisation of the gastrin receptor within rat mucosa using a biotinylated gastrin antagonist.

We have previously demonstrated that the peptide Boc-L-Trp-L-Leu-beta-Ala is a potent and specific antagonist of pentagastrin-stimulated acid secretion in both the rat and the dog. Using conventional solution phase methodology, the analogue biotinyl-L-Trp-L-Leu-beta-Ala was prepared in reasonable yield and purity and applied to cryostat sections of rat intestinal and other tissues. The sections were exposed to 5-10 micrograms of peptide and the bound analogue was visualised using streptavidin-fluorescein. The binding of the analogue was demonstrated in sections from fundus, duodenum, ileum, colon, and lung. However, the analogue failed to bind to tissue from the pancreas, heart, kidney, or liver. The binding of the probe was greatly reduced or completely inhibited by preincubation with Boc-L-Trp-L-Leu-beta-Ala, pentagastrin, or gastrin 1-17. The distribution of the cells recognised by the probe was consistent with the distribution of histamine-containing enterochromaffin-like cells. The results of this study may have some bearing on current theories of the mechanism of gastrin-stimulated acid release.

Amino Acid Sequence↗

PubMed1990

Structural analysis of plant ribosomal 5S RNAs. Visualisation of novel tertiary interactions by cleavage of lupin and wheat 5SrRNAs with ribonuclease H.

A model for the tertiary structure of plant 5S rRNA, previously proposed by our laboratory (Joachimiak, A. et al. (1990) Int. J. Biol. Macromol., in press) was tested by specific cleavage of the plant 5S rRNA in the presence of synthetic oligodeoxynucleotides. The hexanucleotides used in this study were complementary to different portions of loops C, D and E, the nucleotides of which have recently been proposed to be involved in tertiary hydrogen bonds. The results obtained strongly support the interaction of loops C and D by nucleotides C34, C35, C36, A37 and G85, G86, G87, U88, respectively. Digestion pattern of loop E (domain gamma, nucleotides 66-110) suggests a possible different arrangement of this part of the plant 5S rRNA molecule, when compared with other eukaryotes.

Base Sequence↗

PubMed1991

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.

Animals↗

PubMed1988

[Multiple neurologic manifestations of Borrelia burgdorferi infection].

The neurological spectrum of Borrelia burgdorferi infections is still enlarging. We review epidemiological, pathological and serological data of Lyme disease. The course of the disease is divided in three stages: stage 1 during the first month is characterised by erythema chronicum migrans and associated manifestations; stage 2 includes not only the classical European meningoradiculitis but also less specific neurological symptoms: isolated lymphocytic meningitis with an acute or even relapsing course, apparently idiopathic facial palsy, neuritis of other cranial nerves, polyneuritis cranialis, Argyll-Robertson sign, peripheral nerve involvement, acute transverse myelitis, severe encephalitis, myositis. During stage 3, three to five months or longer after the onset of the disease, chronic arthritis, acrodermatitis chronica atrophicans and various neurological symptoms can be observed: chronic neuropathy with mainly sensory or motor signs, recurrent strokes due to cerebral angiopathy and progressive encephalomyelitis; this third stage the central nervous system involvement is characterised by slowly progressive or fluctuating course during months or years, ataxic or spastic gait disorder, bladder disturbances, cranial nerve dysfunction including optic atrophy and hypoacusia, dysarthria, focal and diffuse encephalopathy. This chronic central nervous system disease can mimic multiple sclerosis, anorexia nervosa, psychic disorders or subacute presenile dementia. It is often associated with pleiocytosis, abnormal EEG and evoked potentials, sometimes multifocal and mainly periventricular white matter lesions visualised by CT or MRI, and as a rule high antibody titers against Borrelia burgdorferi. High doses of penicillin can halt the disease, sometimes induce spectacular regression of symptoms or sometimes be inefficient; ceftriaxone could be a more powerful therapy. Similarities between syphilis and Borreliosis are multiple: both of these spirochetes contain plasmids, can be transmitted through the placenta and progress for many years through successive stages, with multiorgan symptoms, including parenchymatous and vascular lesions of the central nervous system. Borrelia burgdorferi is the new great imitator.

Animals↗

PubMed1988

Passive smoking and lung cancer: a publication bias?

To assess the likelihood of publication bias in a recent review of the effect of passive smoking on lung cancer the evidence from the reviewed papers was visualised on a "funnel" plot. In such a plot if the relative risks from various studies are plotted according to sample size they should scatter round some underlying true value, the scatter being greatest where the studies have the lowest statistical power--thus showing a "funnel" pattern. If there is publication bias and studies with non-significant results are not being published there should be a "gap" in the plot. The logarithm of the relative risks was plotted against the standard error of the logarithm of the relative risk (which was used instead of sample size as a measure of statistical uncertainty). The resulting plot was compatible with a publication bias but only in studies on men. Further studies of passive smoking and lung cancer in men seem to be warranted.

Data Interpretation, Statistical↗

PubMed1991

Detection of Borrelia burgdorferi in cerebrospinal fluid by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences from different clinical isolates of Borrelia burgdorferi and from cerebrospinal fluid (CSF) of two patients with Lyme disease of the central nervous system. The amplification products were separated by polyacrylamide gel electrophoresis and visualised by ethidium bromide staining. The definitive identification of amplified DNA as a part of the B. burgdorferi flagellin gene was achieved by hybridisation to a 40-base oligonucleotide probe complementary to a part of the spirochaetal gene but not to the primers. Attempts to cultivate borreliae from either patient were unsuccessful and one patient had no serological marker in serum or CSF to indicate borreliosis. Clinical symptoms of both patients regressed with antibiotic therapy. The PCR system is a powerful and rapid technique to amplify flagellin gene sequences from CSF of patients with neuroborreliosis. Only one-tenth of the time needed for cultivation was required from CSF sampling to diagnosis. Gene amplification might, for the first time, allow effective monitoring of therapy for patients with Lyme disease of the central nervous system.

Base Sequence↗

PubMed1989

Prenatal diagnosis of beta thalassaemia based on restriction endonuclease analysis of amplified fetal DNA.

In the Mediterranean area, 50% of the beta thalassaemia mutations abolish or create a restriction endonuclease site in the beta globin gene. This study describes a new procedure for prenatal detection of these beta thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus. We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the beta globin gene; these are the most frequent beta thalassaemia mutations in this population, accounting for 95% and 2.2% of the beta thalassaemia chromosomes. The main advantages of this procedure are simplicity (no radioactivity), sensitivity (0.2 microgram of DNA), and rapidity (12 hours). The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling. By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.

Amniocentesis↗

PubMed1992

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

AIMS: To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. METHODS: Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. RESULTS: M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. CONCLUSIONS: The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Base Sequence↗

PubMed1990

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor.

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗