Biomedical subjects

D Robertson

Publications and source records attributed to D Robertson.

At least 19 recordsLinked to original sources

PubMed1992

A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat.

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).

Amino Acid Sequence↗

PubMed1992

Time-course and extent of retinal ganglion cell death following ablation of the superior colliculus in neonatal rats.

This study has examined the deleterious effect of superior colliculus (SC) ablation on the viability of identified retinotectally projecting ganglion cells in the neonatal rat retina. The time-course and extent of lesion-induced retinal ganglion cell (rgc) death has been determined and an estimate obtained for the rate of clearance of individual dying neurons. In order to demonstrate the projection of rgcs to the SC and the subsequent death of these same neurons after SC lesions, the fluorescent dye diamidino yellow (DY) was injected into the left SC of anesthetized 2 day old Wistar rats (P2: day of birth = P0). DY retrogradely labels the nuclei of tectally projecting rgcs; if these identified rgcs subsequently die, their DY-labelled nuclei become pyknotic and can be visualized in retinal wholemounts. At P4 the rats were again anesthetized and the injected area, seen as a yellow patch in the SC, was removed by aspiration. Rats were perfused 2 to 336 hours after the lesion and retinal wholemounts of the right eye were prepared. Control rats received only DY injections and were perfused at times corresponding to the lesioned animals. In three sham-operated rats; the injected SC was reexposed at P4 but the tectal tissue was not removed. In each of the 42 rats that were analyzed, about 10% of the retina containing retrogradely labelled rgcs was counted; the number of pyknotic versus normally labelled rgcs was determined and changes in normal cell density were also assessed. Pyknotic rates in control and sham-operated rats were similar (average 0.8%, n = 11). In SC-lesioned rats, the proportion of pyknotic DY-labelled rgcs increased to about 2.5% 4 to 8 hours postlesion (PL); the peak period of death occurred at 23 hours PL (8.0%). The amount of pyknosis decreased thereafter and most dying cells had been eliminated by 50 hours PL. Phagocytosis of dying cells was a common feature of retinae in SC lesioned rats. In the long-term (336 hours) rats, counts of normal DY-labelled rgcs in corresponding regions of control and lesioned rats revealed an average decrease in rgc density of 47.3% after P4 tectal ablation. Calculations suggest a clearance time of about 3 hours for dying neonatal rgcs.

Amidines↗

PubMed1992

Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a 'sub-G1' peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

Animals↗

PubMed1992

Early efferent innervation of the developing rat cochlea studied with a carbocyanine dye.

The olivocochlear pathway in the developing rat was visualized in fixed material. The fluorescent carbocyanine dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to the cut central axons of the olivocochlear neurones at the floor of the fourth ventricle, and the termination pattern within the cochlea was examined after dye diffusion. From the day of birth (P0) to postnatal day 2 (P2), efferent innervation of the cochlea was exclusively in the region of the inner hair cells. Between P2 and P11, progressive outgrowth of neuronal processes to the outer hair cell region occurred; possible connections with the outer hair cells were occasionally seen at P4 and approached the mature pattern by P6. The efferent innervation of the organ of Corti appeared to mature progressively from the cochlear base to the apex, with outgrowth to the outer hair cells occurring earlier in the basal turn of the cochlea than in the second and third cochlear turns. Numerous blind axonal endings were observed in the spiral lamina especially at early postnatal ages. These findings may be consistent with a sequential pattern of arrival of efferent axons at the organ of Corti and ongoing death of efferent neurones in the brainstem during this period of development.

Animals↗

PubMed1992

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Animals↗

PubMed1992

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.

Adenofibroma↗

PubMed1992

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment.

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for beta-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.

Base Sequence↗

PubMed1992

Electron microscopic immunocytochemistry of GAP-43 within proximal and chronically denervated distal stumps of transected peripheral nerve.

Growth-associated protein, GAP-43 was initially described as a neuron-specific molecule thought to play a critical role in axonal growth and regeneration. However, it is also expressed in vitro in certain CNS glia, Schwann cell precursors and non-myelinating Schwann cells. In this paper, we report the subcellular localization of GAP-43 in vivo in chronically-denervated Schwann cells in the distal stumps of previously transected rat sciatic nerve. We have used a progressive lowering of temperature method combined with the non-polar acrylic resin Lowicryl HM20 and a post-embedding labelling regime to visualize the distribution of GAP-43, S-100 (marker for Schwann cells), RT97 and NF68 (markers for different subunits of the neurofilament molecule). We report that (1) the smallest calibre regrowing axons were GAP-43-positive, sometimes NF68-positive but always RT97-negative; (2) regenerating myelinated axons and larger unmyelinated axons (> 0.7 microns diameter) were NF68-positive, RT97-positive but GAP-43-negative; (3) cytoplasmic processes within Schwann cell basal lamina tubes in the distal stumps were S-100-positive, GAP-43-positive but RT97- and NF68-negative. The similar localization of GAP-43 within regrowing axons and denervated Schwann cells suggests that GAP-43 may function similarly in both situations, and may thus be involved in motility and/or elongation of axons and Schwann cells during regeneration.

Animals↗

PubMed1992

Mechanical preprocessing in the mammalian cochlea.

The mammalian cochlea responds with exquisite sensitivity to the small fluctuations in air pressure that make up the stimulus of sound. Moreover, it responds to pressure fluctuations that occur extremely rapidly and that vary over a wide range of intensities--in both cases, to an extent outside the capabilities of unaided nerve fibres. Research performed during the past decade has shown that these properties are dependent on a physiological source of mechanical energy that operates probably within the outer hair cells of the organ of Corti. These cells, which are anatomically and functionally similar to the primary receptor cells, the inner hair cells, are believed to function as a source of mechanical power to assist the mechanical sensitivity of the cochlea, by mechanisms that currently are not understood. Several possible mechanisms have been proposed, but each has limitations that may make it an unsuitable candidate. Recent work has also demonstrated the likely role of mechanoelectrical transduction in outer hair cells in controlling the power source and thereby influencing the sensitivity and amplitude range of the cochlea.

Animals↗

PubMed1992

Role of cyclic AMP in adenosine inhibition of intracellular calcium rise in human platelets. Comparison of adenosine effects on thrombin- and epinephrine-induced platelet stimulation.

Thrombin-induced platelet aggregation is associated with an increase in intracellular calcium. Epinephrine provokes aggregation in the absence of a rise in intracellular calcium. Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. In this study, the authors examine the effect of adenosine on the rise in intracellular calcium and on platelet aggregation, and the role of cyclic AMP (cAMP) in these actions. Human platelets were obtained from citrated plasma containing 5 micrograms/mL of indomethacin. Intracellular calcium was determined by fura-2 fluorescent dye. Adenosine inhibited thrombin-induced platelet aggregation and the rise in intracellular calcium in a dose-dependent manner. At a concentration of 100 mumol/L, adenosine completely inhibited thrombin-induced aggregation, but only partly inhibited the rise in intracellular calcium (55%). Adenosine also partially inhibited the rise in calcium produced by thrombin in both calcium-containing and calcium-free media, suggesting that adenosine inhibits both calcium influx and calcium mobilization. The effects of adenosine on intracellular calcium, as in the case of platelet aggregation, appear to be linked to adenylate cyclase, since they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1-mmol/L) and were potentiated by phospho-diesterase inhibition with papaverine (1 mumol/L). Adenosine and dibutyryl-cAMP also inhibited epinephrine-stimulated platelet aggregation in a dose-dependent manner. Thus, it appears that adenosine may inhibit platelet aggregation independently of its ability to decrease cytosolic free calcium.

Adenosine↗

PubMed1992

Histological and angiographic effects of a pulsed holmium:YAG laser in normal and atherosclerotic human coronary arteries and aorta.

OBJECTIVE: The aims were (1) To determine the histological and angiographic effects of holmium:YAG laser energy delivered through clinical multifibre laser catheters on fresh cadaveric coronary arteries; and (2) to relate the placement of optical fibres in the catheter to patterns of tissue ablation in cadaveric aorta. METHODS: Eight fresh cadaveric hearts and segments of aorta were used. Hearts were mounted on a new pressure perfusion device. The laser catheter was delivered over a guidewire in the lumen until it met an area of resistance. The coronary artery lumen was perfused at approximately 100 mm Hg mean pressure. These arterial areas were identified on angiography, marked, and then exposed to laser energy in the range 600-3000 mJ.mm-2. Normal and atherosclerotic areas of fresh cadaveric aortic strips were exposed to increasing laser energies using either constant or increasing fluence. Coronary arteries were pressure perfused with formalin for 18-24 h at 100 mm Hg mean pressure, and aortic strips were immersed in 5% formalin. Light and scanning electron microscopy studies were carried out. RESULTS: There were no perforations or dissections by angiography in the fresh coronary arteries. One of 15 normal coronary artery segments and 10 of 16 of the pressure perfused, fixed, atherosclerotic coronary artery segments showed thermal changes associated with atherosclerotic plaque ablation. In aortic tissue, thermal effects extended 0 to 0.6 mm lateral to the ablated crater. Acoustic effects were seen only in the aortic strips after ablation at fluences > 1000 mJ.mm-2. The "dead spaces" around the optical fibres in the catheter resulted in significant amounts of coagulated tissue fragments remaining in the crater. CONCLUSIONS: Holmium:YAG laser energy delivered through multifibre catheters ablated atherosclerotic tissue in coronary arteries with minimal damage to the normal walls. The cadaveric coronary artery perfusion apparatus is useful for assessing catheter delivery and mobility and the effects of laser energy on the coaxially orientated normal and atherosclerotic coronary arterial wall.

Aorta↗

PubMed1992

An appraisal of low-temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies.

The progressive lowering of temperature (PLT) method of embedding for electron microscope immunolabelling has been examined with the objective of formulating a standardized protocol which can be applied to a wide variety of samples. The methods described cover fixation, processing of samples by the PLT method, embedding in Lowicryl HM20 and subsequent immunolabelling. Each of the steps in the fixation and embedding protocol has been assessed for its potential to retain both morphology and antigenicity. Comparison of samples embedded in Lowicryl K4M and HM20 at -25 degrees C indicate an increased membrane contrast in HM20 sections, and a further improvement in morphology when samples were embedded in HM20 at -50 degrees C rather than at -25 degrees C. The results of applying the methods described are demonstrated in a range of samples of both mammalian and botanical origin, which includes solid tissues, cells in suspension, and cells grown in vitro as a monolayer culture and embedded in situ. Samples processed by this method have been immunolabelled using a wide range of antibodies recognizing nuclear, cytoplasmic, cell membrane and extracellular matrix antigens.

Acrylic Resins↗

PubMed1992

Ultrastructural localization of bcl-2 protein.

Previous cell subfractionation studies have indicated that bcl-2 is an inner mitochondrial membrane protein. We have sought to determine the ultrastructural localization of bcl-2 protein in lymphoma and breast carcinoma cell lines and biopsy material known to overexpress bcl-2 using immunoelectron microscopy. To avoid the possibility of processing artifacts, samples were prepared by three different methods: progressive lowering of temperature, cryosectioning, and freeze-substitution. In all instances the labeling of bcl-2 protein was relatively weak but the distribution the same. In both lymphoma and breast carcinoma tissues, bcl-2 protein was detected on the periphery of mitochondria: little labeling of either the mitochondrial matrix or cristae could be detected. Labeling was also detected on the perinuclear membrane and throughout the cytoplasm, as also indicated by confocal microscopy. These data therefore indicate that bcl-2 protein can be detected at several intracellular sites and that at the likely functional destination, the mitochondria, there appears to be, contrary to expectations, a preferential association with the outer membrane.

Breast Neoplasms↗

PubMed1992