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C Jacobsen

Publications and source records attributed to C Jacobsen.

At least 73 records · Page 4Linked to original sources
PubMed1996

A characterisation of dark-field imaging of colloidal gold labels in a scanning transmission X-ray microscope.

While X-ray microscopes provide images of biological specimens for which the contrast is mainly due to the difference in the absorption of carbon and oxygen when X-rays transmitted through the specimen are detected, signals other than absorption can also be used to form images. Using the Stony Brook scanning transmission X-ray microscope at the National Synchrotron Light Source, high-angle dark-field images have been formed of cells labelled with colloidal gold, with and without silver enhancement. The high density of the colloidal gold particles, or the silver particles seeded by the gold, leads to a large scattering signal, and the fact that the particle diameters are comparable to the width of the microscope point spread function results in good localisation of the label with high contrast. The dark-field images can have a greater signal to noise ratio than bright-field images acquired with the same incident X-ray dose. The theory of dark-and bright-field imaging is reviewed. Theoretical calculations of scattering from gold and silver particles are presented and good agreement is found between these and experimental dark-field images of 30 nm diameter gold particles. The signal to noise ratios of experimental bright-and dark-field images are measured and found to be in agreement with theory. Images are presented of cells labelled by immunolabelling and in situ hybridisation.

Fibroblasts↗
PubMed1996

X-ray microscopy: preparations for studies of frozen hydrated specimens.

X-ray microscopes provide higher resolution than visible light microscopes. Wet, biological materials with a water thickness of up to about 10 microns can be imaged with good contrast using soft X-rays with wavelengths between the oxygen and carbon absorption edges (at 24 and 43 A). The Stony Brook group has developed and operates a scanning transmission X-ray microscope (STXM) at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The microscope is used for imaging with a current resolution of 50 nm, and for elemental and chemical state mapping. Radiation damage imposes a significant limitation upon high resolution X-ray microscopy of room temperature wet specimens. Experience from electron microscopy suggests that cryo techniques allow vitrified specimens to be imaged repeatedly. This is due to the increased radiation stability of biological specimens in the frozen hydrated state. Better radiation stability has been shown recently with a cryo transmission X-ray microscope developed by the University of Göttingen, operating at the BESSY storage ring in Berlin, Germany. At Stony Brook, we are developing a cryo scanning transmission X-ray microscope (CryoSTXM) to carry out imaging and spectro-microscopy experiments on frozen hydrated specimens. This article will give an outlook onto the research projects that we plan to perform using the CryoSTXM.

Animals↗
PubMed1995

IGF-II receptors in luminal and basolateral membranes isolated from pars convoluta and pars recta of rabbit proximal tubule.

The binding of 125I-labeled insulin-like growth factor-II (125I-IGF-II) to luminal and basolateral membrane vesicles isolated from pars convoluta and the straight part (pars recta) of rabbit proximal tubule was investigated. Analyses of the binding data by use of the general stoichiometric binding equation revealed, that in all preparations IGF-II was bound to one high-affinity binding site and other sites with lower affinities. The specificity of the high-affinity 125I-IGF-II binding to the membrane vesicles assessed by displacement by unlabeled IGF-II, IGF-I and insulin showed that IGF-I displaced 125I-IGF-II in the range 22.5-47.9 nM (IC50) whereas insulin did not effect 125I-IGF-II binding at all. beta-Galactosidase inhibited the 125I-IGF-II binding with half-maximal inhibition of 20-30 nM beta-galactosidase. D-Mannose 6-phosphate increased the binding of 125I-IGF-II and reversed the inhibitory effect of beta-galactosidase. Analyses of 125I-IGF-II binding curves in the presence of beta-galactosidase or D-mannose 6-phosphate demonstrated that none of these compounds changed the binding affinity of 125I-IGF-II for the membrane vesicles. The IGF-II/M6P receptor content in the luminal membranes was in the range 0.21-0.34 pmol IGF-II/M6P receptor per mg protein and very low compared to 2.27-2.86 pmol IGF-II/M6P receptor per mg protein in basolateral membranes.

Animals↗
PubMed1994

A comparative study on the uptake of alpha-aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule.

We investigated whether an immortalized human kidney epithelial cell line (IHKE), compared with normal embryonic cells (NHKE), can be used as a representative system with which to characterize the transport of neutral amino acids in the proximal tubule of the human kidney. The IHKE cell line, immortalized by treatment with NiSO4, exhibited microvilli and enzyme markers specific for highly specialized tubule cells. The Na(+)-dependent uptake of alpha-aminoisobutyric acid (AIB) by IHKE and NHKE cells occurred by means of a single transport system with identical half-saturation constants, but the capacity for uptake was higher in the immortalized cells. Proton-dependent influx of AIB was also mediated by a single transport component with similar uptake characteristics in both types of cells. Imposition of an H(+)-gradient to a Na(+)-gradient reduced the sodium dependent uptake of AIB with the exception of short incubation time (1 min), where addition of a proton gradient produced a marked increase in the Na(+)-dependent influx of AIB in NHKE but not in IHKE cells. Competition experiments revealed that the Na(+)-dependent uptake at 50 microM AIB was reduced by neutral alpha-amino acids in the two cell lines. L-Glutamate, L-aspartate, L-arginine and the beta-amino acid taurine had no effect. Only in the IHKE cell line did addition of 5 mM L-lysine produce a slight inhibition. Except for L-proline all of the neutral and acidic amino acids tested reduced the H(+)-dependent uptake of AIB in the IHKE cell line. By contrast, addition of L-aspartate did not influence the transport of AIB in NHKE cells. L-Arginine, but not L-lysine decreased the influx in both cell lines. We conclude that the IHKE cell line has retained the capability to accumulate AIB by transport protein(s) similar to those present for neutral alpha-amino acids in NHKE cells.

Aminoisobutyric Acids↗
PubMed1994

Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: membrane currents.

Microglia, the resident macrophages of the central nervous system (CNS), can be distinguished from most other cells of the myelomonocytic lineage by a distinct pattern of membrane currents. In the accompanying paper we have shown that the characteristic morphological feature of microglia, ramification, develops both in microglia and other myelomonocytic cells when they are cocultured with astrocytes. We therefore propose that the electrophysiological properties of microglia also develop under the influence of astrocytes, and, moreover, that these properties can also be induced in other cells of the myelomonocytic lineage. Microglia cultured on poly-d-lysine or on a monolayer of fibroblasts possess an inwardly rectifying K(+)-current only, which is of composite nature. In single-channel recordings two types of K(+)-channels are found: i) a noninactivating channel with a conductance of 43pS, and ii) an inactivating channel with 32pS. Microglia cultured on a monolayer of astrocytes additionally develop an outward K(+)-current and a Na(+)-current. The electric parameters of activation and inactivation of the microglial Na(+)-current are identical to those of the neuronal Na(+)-current. Monocytes from peripheral blood and macrophages from spleen exhibit no inward currents. However, when these cells are cocultured with astrocytes they develop microglia-like membrane currents, including the inward and outward K(+)-rectifyer and the Na(+)-current. By contrast, on fibroblasts they retain their macrophage current profile. The expression of the microglia-like membrane currents in the mononuclear phagocytes is induced by a diffusible factor released from the astrocytes into the culture medium, since monocytes and microglia develop the mature microglial current profile, when cultured in astrocyte conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Luminal and basolateral uptake and receptor binding of IGF-I in rabbit renal proximal tubules.

The aim of the present study was to quantify and compare the luminal and basolateral binding and uptake of 125I-labeled insulin-like growth factor I (IGF-I) by means of 1) isolated, perfused, proximal tubules combined with electron microscope autoradiography and 2) luminal and basolateral membrane vesicles from rabbit proximal tubules. 125I-IGF-I was added to isolated perfused proximal tubules for 30 min in concentrations of 1.6-3.9 micrograms/l to either the perfusate or the bath. The luminal and basolateral uptake in 30 min averaged 447 and 410 fg/mm, respectively. About 20% of the luminally absorbed IGF-I was digested. Addition of excess unlabeled IGF-I (10(-7) M) to the bath produced complete inhibition of the basolateral binding/uptake, whereas no inhibition of the luminal uptake was seen. Electron microscope autoradiography showed that IGF-I after luminal endocytic uptake to a large extent was transported into lysosomes. After basolateral exposure the major portion of the grains was found over the basolateral cell membrane; however, a significant amount was located over endocytic vacuoles and lysosomes in both apical and basal parts of the cells. In both luminal and basolateral membrane vesicles, single-class, high-affinity binding sites for IGF-I were found with dissociation constants of 6.3 and 5.7 nM, respectively. Specific binding capacities averaged 2.7 and 25.7 pmol IGF-I/mg protein in luminal and basolateral vesicles. The biochemical data suggest an asymmetric distribution of specific IGF-I receptors in the luminal and basolateral membranes, with a greater abundance of receptors in the latter. The extensive basolateral endocytic binding/uptake of IGF-I compared with that of the luminal in isolated perfused tubules differs considerably from the processing of other peptide hormones.

Animals↗
PubMed1992

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.

Animals↗
PubMed1991

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.

Amiloride↗
PubMed1991

Peripheral nerve function in persons with asymptomatic or minimally symptomatic HIV disease: absence of zidovudine neurotoxicity.

Nucleoside-induced neuropathy characteristically appears as a painful, symmetric, distal polyneuropathy. To evaluate the neurotoxic potential of zidovudine (ZDV, or azidothymidine), in persons with little confounding human immunodeficiency virus (HIV) neuropathy, we evaluated peripheral nerve function in persons completing placebo-controlled studies of ZDV in early HIV disease. Participants had been receiving placebo or ZDV at doses of 800-1,200 mg daily for a median 52 weeks at the time of evaluation. Neuropathic symptoms and abnormalities of motor and sensory function were present in fewer than 10% of both treatment groups. Depressed reflexes were found in 19% of the ZDV group and 18% of the placebo group. Quantitative sensory testing for vibration was abnormal in fewer than 10% of participants and the absolute scores favored the ZDV group. We thus found a low prevalence of peripheral nerve abnormalities and no evidence of ZDV neurotoxicity in this population.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Non-selective cation channels in basolateral-membrane vesicles from pars recta of rabbit kidney proximal tubule.

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars recta of rabbit kidney proximal tubule were investigated. In RbCl-, KCl- and NaCl-loaded vesicles a transient and almost equal accumulation of 86Rb+ was observed. The uptakes of 86Rb+ were inhibited to the same extent by 10 mM-BaCl2 in all loadings. The accumulation was driven by an electrical diffusion potential. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake. At 10 microM-Ca2+ the radioisotope flux was below 20% of control. The vesicles containing the channel showed very low selectivity among the univalent cations K+, Rb+, Li+, Na+ and choline+.

Animals↗
PubMed1990

Potassium channels in basolateral membrane vesicles from pars convoluta of rabbit proximal tubule.

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars convoluta of rabbit proximal tubule were investigated. In KCl loaded vesicles a transient accumulation of 86Rb+ was observed which was inhibited by BaCl2. The accumulation was driven by an electrical diffusion potential, as shown in experiments using membrane vesicles loaded with Li2SO4 and an outwardly directed Li+ gradient established with a Li(+)-ionophore. The vesicles containing the channel showed a cation selectivity with the order K+ = Rb+ much greater than Li+ greater than or equal to Na+ greater than choline+. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake.

Animals↗
PubMed1990

Proton gradient-dependent renal transport of glycine: evidence for vesicle studies.

The characteristics of renal transport of glycine by luminal membrane vesicles isolated from either proximal convoluted part (pars convoluta) or proximal straight part (pars recta) of rabbit proximal tubule were investigated. In vesicles from pars convoluta two transport systems have been characterized: a Na(+)-dependent system with intermediate affinity (half-saturation 3.64 mM) and a Na(+)-independent system that, in the presence of H+ gradient (extravesicular greater than intravesicular), can accelerate the transport of glycine into these vesicles. This is the first demonstration of H(+)-glycine cotransport across the luminal membrane of rabbit kidney proximal convoluted tubule. By contrast, in membrane vesicles from pars recta, transport of glycine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.34 mM) and a low-affinity system (half-saturation 8.56 mM). The demonstration of competition between the H(+)-gradient dependent uptake of glycine, L-alanine, and L-proline, but insignificant inhibition with L-phenylalanine in vesicles from pars convoluta suggests that glycine, L-proline, and L-alanine probably share a common proton gradient-dependent transport system. In vesicles from pars recta, the Na(+)-dependent uptake of glycine was inhibited by low concentrations of L-alanine and L-phenylalanine, whereas addition of L-proline to the incubation medium did not significantly alter the uptake of glycine, suggesting that the Na(+)-dependent high-affinity system for glycine located in pars recta is shared with the high-affinity L-alanine and L-phenylalanine but not L-proline transport system.

Amino Acids↗
PubMed1990

Ca2+ and pH regulation of K+ channels in membrane vesicles of rabbit proximal tubule.

The effect of Ca2+ and pH on the renal epithelial K+ channel was investigated by measuring the Ba2(+)-sensitive 86Rb+ fluxes in membrane vesicles from pars convoluta of rabbit proximal tubule. It was found that the presence of nanomolar concentrations of Ca2+ in the internal compartment (cytoplasmic) of the vesicles ([Ca2+]i) substantially lowered the channel-mediated flux. Ba2(+)-sensitive 86Rb+ uptake was completely blocked by 10 microM [Ca2+]i. This inhibitory effect of Ca2+ was strongly dependent on pH. Thus 0.1 microM [Ca2+]i produced a maximal inhibition of 86Rb+ uptake at pH greater than 7.4 but had no effect at pH less than 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data are compatible with the model that Ca2+ blocks K+ channels by binding to a site composed of one or several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself block transport.

Animals↗
PubMed1990

Internalization of growth hormone-releasing factor by rat anterior pituitary cells: inhibition by cerulenin, an inhibitor of fatty acid acylation.

The GH-releasing factor (GRF) analogue [His1,Nle27]-GRF(1-29) amide was used to study GRF receptor internalization in cultured rat anterior pituitary cells. The half-life of occupied receptors on the surface was approximately 10 min. Uptake of the analogue was followed by lysosomal breakdown, and receptors taken up were replaced to some extent by newly synthesized receptors, as indicated by reduced surface binding in the presence of cycloheximide. 2,3-Epoxy-4-oxo-7,10-dodecadienamide (cerulenin) inhibited internalization without affecting breakdown of the reduced amount of GRF analogue that entered the cells. The effect was half-maximal at 3 micrograms/ml for 1 h. Cerulenin inhibits fatty acid acylation of proteins. One explanation for its effect on GRF receptor internalization is that fatty acid acylation of a protein (possibly the receptor) is necessary for internalization, because cerulenin also inhibited internalization of the transferrin receptor which is known to be acylated. Cerulenin did not affect internalization of the somatostatin receptor present on the same cells, indicating the specificity of the inhibition.

Acylation↗
PubMed1990

Analysis of the secondary structure of the human immunodeficiency virus (HIV) proteins p17, gp120, and gp41 by computer modeling based on neural network methods.

A neural network computer program, trained to predict secondary structure of proteins by exposing it to matching sets of primary and secondary structures from a database, was used to analyze the human immunodeficiency virus (HIV) proteins p17, gp120, and gp41 from their amino acid sequences. The results are compared to those obtained by the Chou-Fasman analysis. Two alpha-helical sequences corresponding to the putative fusigenic domain and to the transmembrane domain of gp41 could be predicted, as well as a possible binding site between p17 and gp41. On the basis of the secondary structure predictions, a three-dimensional model of p17 was constructed. This model was found to represent a stable conformation by an analysis using an energy-minimization program. The model predicts that p17 is attached to the membrane only by the acylated N-terminus, in analogy with the N-terminus of the gag protein of other retroviruses and also with the src oncogene protein p60src. The intracellular C-terminal part of gp41 may act as a receptor by electrostatic interaction with p17.

Algorithms↗
PubMed1990

A regional approach to entry-level critical care education.

The education and training of the novice critical care nurse place a constant financial demand on many Puget Sound area hospitals. Critical care educators from this area, committed to cost-effective, high-quality education, enhanced through precepted clinical experience, assessed the need for a collaborative approach among institutions. Jointly we have successfully planned, implemented, and evaluated a cooperative educational program. To date, the program has served to reduce the costs of preparing the RN entering critical care in the Puget Sound area. Application of principles of adult education, timely application of theory to practice, and expansion of the professional role of critical care clinical nurse specialists and educators have also been recognized as outcomes of the CCEC.

Critical Care↗