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Biomedical subjects

A Cambi

Publications and source records attributed to A Cambi.

At least 19 recordsLinked to original sources
PubMed2006

C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous glycoconjugates.

Human C-type lectin receptors (CLRs) characteristically bind glycosylated ligands in a Ca(2+)-dependent way via their carbohydrate recognition domain (CRD). Their carbohydrate preference is dependent on the amino acid sequence in the CRD domain and on the ability and flexibility of the CRD domain to accommodate sugar moieties that are located at different distances from each other in the glycoconjugate. Although microbial and vertebrate cells are able to produce similar polysaccharide chains, the density of carbohydrates on microbes is much higher compared to vertebrate cells. Despite this difference, carbohydrates present on both cell types can be recognized by the CLRs. These receptors are predominantly expressed by antigen presenting cells such as dendritic cells. In addition to the Toll-like receptor family, CLRs function as pattern recognition receptors by recognizing glycosylated patterns on pathogens. This usually results in internalization of the pathogen, lysosomal degradation and subsequent loading of pathogen-derived peptides into major histocompatibility complex molecules for antigen presentation. However, several pathogens have developed ways to exploit the CLRs to evade immune eradication by for example escaping from the lysosomal degradation pathway or by inducing anti-inflammatory cytokines. When CLRs bind endogenous glycosylated ligands they mediate several processes like cell-cell adhesion and clearance of aberrant cells like tumor cells or apoptotic cells.

Amino Acid Sequence↗
PubMed2004

Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells.

Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

Cell Adhesion Molecules↗
PubMed2001

Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.

We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.

Amino Acid Sequence↗
PubMed2001

Cell biology beyond the diffraction limit: near-field scanning optical microscopy.

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution--approximately 250 nm--dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

Cytological Techniques↗
PubMed2000

Biomolecular interactions measured by atomic force microscopy.

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.

Biophysics↗
PubMed2000

Possible role of two phenylalanine residues in the active site of human cytidine deaminase.

Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137. The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.

Binding Sites↗
PubMed1999

Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana.

The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio-beta-d-galactopyranoside, showed high cytidine deaminase activity. The nucleotide sequence showed a 903-bp open reading frame encoding a polypeptide of 301 amino acids with a calculated molecular mass of 32,582. The deduced amino acid sequence of AT-CDA1 showed no transit peptide for targeting to the chloroplast or mitochondria indicating that this form of cytidine deaminase is probably expressed in the cytosol. The recombinant AT-CDA1 was purified to homogeneity by a heat treatment followed by an ion-exchange chromatography. The final enzyme preparation was >98% pure as judged by SDS-PAGE and showed a specific activity of 74 U/mg. The molecular mass of AT-CDA1 estimated by gel filtration was 63 kDa, indicating, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits. Inductively coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the E. coli enzymes.

Amino Acid Sequence↗
PubMed1999

Yolk formation and degradation during oocyte maturation in seabream Sparus aurata: involvement of two lysosomal proteinases.

Oocyte growth within the follicle is preponderantly due to the accumulation of hepatically derived yolk protein (vitellogenin, VTG) by receptor-mediated endocytosis; once in the oocyte, VTG is partially processed and stored in yolk globules. In some pelagic egg-laying marine teleosts, additional cleavages of yolk proteins followed by a pronounced water uptake occur concomitantly with final oocyte maturation. The aim of this study was to establish the lysosomal enzymes involved in these two proteolytic processes that characterize oocyte maturation of seabream Sparus aurata. The enzymatic activities of several cathepsins were assessed in the various classes of oocytes. Changes in cathepsin B, D, and L activity were found depending on the oocyte maturation stage; cathepsin B and D were found to be at maximum level in early-vitellogenesis oocytes, and cathepsin L in mid-vitellogenesis ones. Cathepsin D and L were purified from seabream ovary, and their roles in VTG and lipovitellin (LV) proteolysis, respectively, were analyzed. Here we demonstrate directly that one of the catalysts for the intraoocytic processing of VTG in yolk proteins is cathepsin D; however, we cannot exclude also a role of cathepsin B in the same process. On the other hand, cathepsin L is responsible for the second proteolytic cleavage of the LV components. We postulate that the acquisition of buoyancy by eggs through the hydration process may be regulated by enzymatic activation at the appropriate time of oocyte maturation, this process probably being the key event in the reproduction of this marine pelagic egg spawner.

Amino Acid Sequence↗
PubMed1998

A comparison of the enantioselectivities of human deoxycytidine kinase and human cytidine deaminase.

The stereoselectivities of recombinant human deoxycytidine kinase (EC 2.7.1.74) (dCK) and of recombinant human cytidine deaminase (EC 3.5.4.5) (CDA) were investigated with respect to a series of cytidine analogs, most of them having the unnatural L-stereochemistry. The enantioselectivity of dCK was always low and generally favored the L-enantiomers in the case of beta-2',3'-dideoxycytidine (beta-ddC), 5-fluoro-beta-2',3'-dideoxycytidine (beta-FddC) and beta-cytidine (beta-riboC). Concerning beta-2'-deoxycytidine, dCK showed a preference for the D-enantiomer. All other examined beta-L-cytidine analogs, [1-beta-L-lyxofuranosyl cytosine (beta-L-lyxoC), l-beta-L-xylofuranosyl cytosine (beta-L-xyloC), and 5-fluoro-1-beta-L-xylofuranosyl cytosine (beta-L-Fxylo C)], were substrates of dCK regardless of the nature of the pentose. None of the studied alpha-L-anomers (alpha-L-riboC, alpha-L-araC, alpha-L-lyxoC, or alpha-L-xyloC) was a substrate of dCK. Contrasting with the relaxed enantioselectivity of dCK, CDA had a strict requirement for D-cytidine analogs since none of the already listed beta-L- or alpha-L analogs was a substrate or an inhibitor of the enzyme. The conjunction of the preceding stereochemical properties of dCK and CDA confers to L-cytidine analogs important potentialities in antiviral and anticancer therapies.

Antiviral Agents↗
PubMed1998

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications.

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.

Amino Acid Substitution↗
PubMed1997

Human placenta cytidine deaminase: a zinc metalloprotein.

Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10-phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolinic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.

Amino Acid Sequence↗
PubMed1997

Human placenta cytidine deaminase: proton-linked enzyme activity and substrate binding.

In this work we focused our attention on some catalytic site requirements for human placenta cytidine deaminase (CDA). The effect of pH on substrate binding and catalysis was studied between pH 3.0 and pH 11.0. The results could be discussed postulating the presence of two classes of ionizable groups in the active site of CDA. The kinetic parameters pH-dependence has been discussed considering the presence of four zinc atoms per each enzyme tetramer. Furthermore fluorescence studies on the enzyme and on enzyme-inhibitor complexes, examined by using polar and non polar quenchers, allowed to define the substrate(inhibitor)-dependent accessibility of the tryptophan-containing pocket on each CDA monomer.

Binding Sites↗
PubMed1996

Recombinant human cytidine deaminase: expression, purification, and characterization.

The complementary DNA (cDNA) coding for human cytidine deaminase (CDA) was obtained using two specific primers to screen RNA from peripheral blood polymorphonuclear leukocytes by reverse transcriptase PCR. The cDNA fragment was ligated into the expression vector pTrc99-A and expressed in Escherichia coli following induction with isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The nucleotide sequence of the cDNA corresponded to that published by Laliberté and Momparler (Cancer Res. 54, 5401-5407, 1994). It contained a 438-bp open reading frame encoding a polypeptide of 146 amino acids with a predicted molecular mass of 16.2 kDa. The protein expressed in E. coli showed high cytidine deaminase activity and its molecular mass was estimated to be 57 kDa by gel filtration and 16 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both values are in agreement with those already published and suggest that human CDA contains three or four identical subunits. Cross-linking experiment indicated that the enzyme is a tetramer. The recombinant CDA has been purified to homogeneity by a rapid procedure consisting of heat inactivation followed by affinity chromatography. The final enzyme preparation showed a specific activity of 105 U/mg, corresponding to about 88-fold purification with respect to the crude extract and was judged to be >98% pure by SDS-PAGE. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis revealed the presence of 1 atom of Zn per subunit. Since CDA causes the deamination of several antitumoral cytidine-analog drugs, the recombinant enzyme was characterized kinetically and several pyrimidine nucleoside analogs were tested as potential substrates and inhibitors. The results obtained agreed closely with those previously reported for the purified human placenta CDA.

Amino Acid Sequence↗