Biomedical subjects

R B Ciccarelli

Publications and source records attributed to R B Ciccarelli.

34 records · Page 2Linked to original sources

PubMed1995

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Base Sequence↗

PubMed1995

Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

Amino Acid Sequence↗

PubMed1995

Stable expression and functional characterization of a human cardiac Na+ channel gene in mammalian cells.

In order to develop mammalian cell lines expressing a functional human heart Na+ channel gene (hH1), Chinese hamster ovary (CHO-K1) cells and HeLa cells were transfected with the hH1 gene and the bacterial neomycin (neo) resistance gene. In CHO-K1 cells, direct screening for hH1-positive, G418-resistant colonies by functional patch clamp analysis was complicated due to low-level endogenous expression of a brain-type Na+ channel. Therefore, we developed a stepwise strategy for isolation of cell lines expressing functional hH1 Na+ channels: G418-resistant colonies were sequentially analysed for (1) chromosomal integration of hH1 DNA by PCR, (2) specific hH1 mRNA expression by RT-PCR, (3) hH1 protein production by immunoprecipitation with hH1-specific antisera, and (4) hH1 Na+ channel function by patch-clamp analysis. Using this strategy we obtained two CHO-K1 cell lines which express functional human heart Na+ channels. However, using the same strategy, we were unsuccessful in obtaining functional, hH1-positive HeLa cell lines, even though hH1 mRNA and protein was produced in these cells. The two CHO-K1 cell lines stably express human cardiac Na+ channels which retain normal electrophysiological characteristics with respect to activation and inactivation. In addition, the Na+ channels expressed in these cells are blocked by tetrodotoxin with an IC50 value of 2.5 microM; consistent with known cardiac Na+ channel pharmacology. The density of channels is high enough to permit recording of pseudomacroscopic currents in excised outside-out patches of membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Gene expression, purification and characterization of recombinant human neutrophil collagenase.

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three alpha-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met100 through Gly262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood.

Amino Acid Sequence↗

PubMed1994

Production of active human interleukin-1 beta-converting enzyme in a baculovirus expression system.

The cDNA coding for the precursor form of human interleukin-1 beta-converting enzyme (proICE) was expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The 45-kDa recombinant protein was further processed to several smaller forms of 32, 24, 20, 13 and 10 kDa. Active recombinant ICE derived from the baculovirus expression system (bvICE) was found to be present in soluble lysates of insect cells as an associated heterodimer consisting of 10- and 20-kDa subunits. The activity of bvICE was determined by conversion of precursor interleukin-1 beta (preIL-1 beta) to the mature form (mIL-1 beta) and via site-specific cleavage of a decapeptide which spans the ICE cleavage site in preIL-1 beta. The bvICE system was inhibited by an ICE inhibitor to the same extent as native ICE from the monocytic cell line THP-1. Expression of an active-site mutant (Cys285 to Ser) of proICE in insect cells resulted in the accumulation of partially processed (32-kDa) ICE. The availability of a facile expression system will permit further characterization of the biochemical properties and processing pathway of this unique protease.

Animals↗

PubMed1993

Characterization of endogenous sodium channel gene expressed in Chinese hamster ovary cells.

Chinese hamster ovary (CHO-K1) cells were observed to display transient inward Na+ currents of average amplitude (-92 +/- 20 pA), which activated at voltages more than -40 mV, and peak inward currents were observed at potentials equal to or more than +10 mV. Inward Na+ currents in these cells were eliminated after treatment with 500 or 50 nM tetrodotoxin (TTX), whereas 5 nM TTX resulted in 64 +/- 10% inhibition of Na+ current. Using DNA primers designed to bind to the rat brain IIA Na+ channel subtype, we amplified specific polymerase chain reaction (PCR) fragments from CHO-K1 poly-(A)+RNA. The cloning and sequencing of two of these fragments confirmed the presence of an endogenously expressed Na+ channel gene in these cells, which we have termed cho 1. Comparison of the DNA sequence of cho 1 PCR fragments with other known Na+ channel genes indicated a high degree of homology with rat brain Na+ channel subtypes. Northern blots using riboprobes generated from the cho 1 PCR fragments revealed the presence of a specific 9-kb mRNA in these cells. The molecular and electrophysiological data suggest that the cho 1 Na+ channel gene from CHO-K1 cells is closely related to brain-type Na+ channels.

Animals↗

PubMed1991

Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c.

Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.

Amino Acids↗

PubMed1991

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.

Base Sequence↗

PubMed1990

Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene.

The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.

Amino Acid Sequence↗

PubMed1985

In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication.

The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei.

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.

Animals↗

PubMed1984

Molecular basis for the activity of nickel.

The molecular mechanism for the carcinogenic activity of nickel has been investigated in vitro and in vivo. Formation of stable ternary nickel (II)-DNA-protein complexes occurred in vitro upon incubation of nickel subsulfide (Ni3S2) with DNA and microsomes in 0.05 M tris hydrochloride, pH 7.4. DNA damage in the form of strand breaks and DNA-protein cross-links resulted in vivo following injection of nickel carbonate in rats. Kidney was the preferred tissue for nickel accumulation and DNA damage. A gentle isolation procedure was developed for the isolation of nucleic acids containing bound nickel from tissues of rats injected with nickel carbonate. Higher levels of nickel were bound to kidney nucleic acids as compared with liver nucleic acids. The amount of protein associated with kidney and liver DNA correlated with the amount of nickel bound. Removal of protein associated with kidney DNA and RNA resulted in reduction of the amount of nickel bound. Nickel levels remained constant upon removal of associated protein from liver DNA, but were reduced upon removal of protein from liver RNA. These results are discussed relative to the known carcinogenicity, solubilization and aqueous chemistry of nickel compounds.

Animals↗

PubMed1984

Nickel-bound chromatin, nucleic acids, and nuclear proteins from kidney and liver of rats treated with nickel carbonate in vivo.

The in vivo binding of nickel to chromatin, nucleic acids, and nuclear proteins from rat kidney and liver was investigated. Evidence is presented for the direct interaction of nickel with chromatin from rat tissues following i.p. injection of nickel carbonate. A gentle DNA isolation procedure was developed and used to isolate nickel-bound whole chromatin, DNA + histone octamer complex (polynucleosomes), and deproteinized DNA from kidney and liver nuclei. A similar procedure was developed for isolating nickel-bound RNA from the cytoplasm. The level of nickel bound to whole chromatin from kidney was higher than that from liver, and these levels could be related to the nuclear concentration of nickel. Much higher levels of nickel were bound to the DNA + histone octamer complex and purified, deproteinized DNA from kidney as compared to liver. Substantial levels of nickel were bound to nonhistone proteins and/or chromatin-associated RNA from kidney and liver nuclei. Nickel was also associated with histone octamer proteins from kidney; however, little or no nickel was associated with histone octamer proteins from liver. 63Ni binding to nuclear proteins in rats given injections of 63Ni(II) was investigated by using two different gel electrophoretic systems. Electrophoretic conditions in both systems were found to remove protein-bound 63Ni. These results are discussed relative to the molecular mechanism of nickel carcinogenesis.

Animals↗

PubMed1982

Solubilization of the carcinogen nickel subsulfide and its interaction with deoxyribonucleic acid and protein.

Significant concentrations (1-10 mM) of nickel(II) were found in solution after incubation of the potent carcinogen nickel subsulfide in 0.05 M Tris-HCl, pH 7.4, solutions containing DNA, rat liver microsomes, and NADPH. The presence of NADPH decreased the rate of solubilization of nickel subsulfide. The solubilized nickel exhibited electronic absorption spectra and magnetic moments characteristic of octahedral nickel(II). The solubilized nickel(II) bound to DNA with an apparent equilibrium constant of 730 M-1 and with a saturation binding value of one nickel per 2.4 nucleotides. Microsomes lowered the saturation binding of nickel to DNA but dramatically increased the amount of nickel-DNA complex stable to precipitation with salt and poly-(ethylene glycol). The amount of protein associated with DNA precipitated from protein-extracted solutions correlated with the amount of nickel bound to DNA. These results suggest that microsomes mediate the binding of nickel to DNA by forming a stable ternary protein-nickel(II)-DNA complex.

Animals↗

PubMed1982

Nickel distribution and DNA lesions induced in rat tissues by the carcinogen nickel carbonate.

DNA lesions in nuclei isolated from rat tissues following i.p. injection of the carcinogen nickel carbonate were measured by the alkaline elution technique. Kidney, liver, lung, and thymus gland nuclei were examined at 3 and 20 hr after treatment for the presence of DNA single-strand breaks and cross-links. Single-strand breaks were detectable in lung and kidney nuclei, and both DNA-protein and DNA interstrand cross-links were detectable in kidney nuclei. No DNA damage was observed in liver or thymus gland nuclei. A dose response to both single-strand breaks and cross-links was observed in kidney nuclei. Time course studies revealed that maximum DNA damage in kidney nuclei occurred at 2 to 4 hr following injection and also revealed the presence of an active repair process in these nuclei. Repair-resistant DNA-protein cross-links were observed to persist through 48 hr. Tissue and intracellular nickel concentrations as measured by electrothermal atomic absorption spectroscopy were observed to correlate with the levels of DNA damage and repair. A dose response to the concentration of nickel in tissues and nuclei was observed. These results are discussed relative to the solubilization, toxicity, and carcinogenicity of nickel compounds.

Animals↗

PubMed1981

Nickel carbonate induces DNA-protein crosslinks and DNA strand breaks in rat kidney.

DNA lesions were observed in kidney nuclei isolated from rats 20 h after i.p. injection with nickel carbonate. Dose-dependent, nickel-induced DNA crosslinks and DNA single strand breaks were detected using the alkaline elution technique. The DNA crosslinks were determined to be completely DNA-protein in nature. The DNA damage induced by nickel carbonate is discussed relative to the carcinogenicity of nickel compounds.

Animals↗