Search PubMed⌕ Search

Biomedical subjects

C Jacobsen

Publications and source records attributed to C Jacobsen.

At least 55 records · Page 3Linked to original sources
PubMed1998

The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine.

Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site. Furthermore, the binding analysis revealed that the interaction is sensitive to calcium in addition to lysine.

Binding Sites↗
PubMed1998

beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Animals↗
PubMed1998

The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin.

Binding of the receptor-associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor-related protein (LRP). The data show that the RAP-binding site in sortilin is localized in the cysteine-rich lumenal part homologous to yeast vacuolar protein-sorting 10 protein (Vps10p), and the sortilin-binding site in RAP is localized in the carboxy-terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III >domain I > domain II.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region.

Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

Amino Acid Sequence↗
PubMed1998

Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths.

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.

Crystallization↗
PubMed1998

Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers.

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

A numerical study of resolution and contrast in soft X-ray contact microscopy.

We consider the case of soft X-ray contact microscopy using a laser-produced plasma. We model the effects of sample and resist absorption and diffraction as well as the process of isotropic development of the photoresist. Our results indicate that the micrograph resolution depends heavily on the exposure and the sample-to-resist distance. In addition, the contrast of small features depends crucially on the development procedure to the point where information on such features may be destroyed by excessive development. These issues must be kept in mind when interpreting contact microradiographs of high resolution, low contrast objects such as biological structures.

Journal Article↗
PubMed1997

Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein.

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.

Animals↗
PubMed1997

Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule.

1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine homeostasis occurs predominantly via changes in the activity of the high-affinity taurine transport system by alterations in the uptake capacity and with an unaffected half-saturation constant. An adaptive response was not observed for the structurally related beta-alanine. 3. Only colchicine, which interferes with microtubule organization, was capable of blocking the response to alterations of taurine in cell medium, whereas inhibition of protein and nucleic acid synthesis by cycloheximide and actinomycin D, respectively, did not change the adaptive pattern. 4. Phorbol 12-myristate 13-acetate (PMA), mimicking the effects of diacylglycerol, induced inhibition of both beta-alanine and taurine uptake. By contrast, the Ca2(+)-ionophore A23187, mimicking the effects of IP3, only stimulated the uptake of taurine but not the influx of beta-alanine. However, the effect of PMA down-regulation and A23187 up-regulation was rapid and short-lived in contrast to the adaptive response, suggesting that the inositol phospholipid pathway involving diacetylglycerol and IP3 is less likely to be linked directly to the adaptive regulation, but rather plays a role in short-term regulation.

Adaptation, Physiological↗
PubMed1996

The receptor-associated protein (RAP) binds calmodulin and is phosphorylated by calmodulin-dependent kinase II.

The receptor-associated protein, RAP, is an intracellular protein that may function as a chaperone for the LDL-receptor family receptors. Here we report calmodulin as the first identified RAP binding protein outside of the LDL-receptor family members. We demonstrate that RAP binds calmodulin in a Ca2+- and pH-dependent manner characteristic of calmodulin-dependent enzymes, and present evidence that RAP is a substrate for calmodulin-dependent enzymes. Thus, CaM-kinase II and calcineurin readily phosphorylate and dephosphorylate, respectively, serine residues in RAP, and in the individual RAP domains D2 (amino acids 113-218) and D3 (amino acids 219-323) which both contain sites for CaM-kinase II-mediated phosphorylation and for calmodulin binding. In addition, we provide evidence that RAP is phosphorylated by other kinases such as casein kinase II. Studies of 32[ortho]P-labelled cell cultures demonstrate that RAP is phosphorylated in vivo. Our results suggest that RAP may have hitherto unknown functions implicating phosphorylation and calmodulin-mediated modulation.

Amino Acid Sequence↗
PubMed1996

Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells.

The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using alpha-aminoisobutyric acid (AIB) and beta-alanine as representatives of alpha- and beta-amino acids, respectively. The Na(+)-gradient dependent influx of AIB occurred by a single, saturable transport system, whereas the Na(+)-gradient dependent uptake data for beta-alanine could be described in terms of two-independent transport components as well as one-transport one-leak model with identical kinetic constants for the high-affinity system. Competition experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids was relatively low. Nor did L-arginine and L-aspartic acid affect the uptake of beta-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral alpha- and beta-amino acids in the embryonic and AHKE cells. However, the uptake capacity of the above-mentioned transport proteins was relatively smaller in the embryonic kidney compared with the adult human kidney, which may explain, at least partly, the phenomenon of physiologic amino aciduria in neonates.

Adult↗
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↗