Biomedical subjects

W Chang

Publications and source records attributed to W Chang.

At least 55 records · Page 3Linked to original sources

PubMed1999

Passive Ca(2+) transport and Ca(2+)-dependent K(+) transport in Plasmodium falciparum-infected red cells.

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca(2+) permeability. The magnitude of the increase is greater than that normally required to activate the Ca(2+)-dependent K(+) channel (K(Ca) channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K(Ca) channel and the Ca(2+) permeability properties of pRBC. For pRBC suspended in media containing Ca(2+), K(Ca) channel activation was elicited by treatment with the Ca(2+) ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca(2+) into pRBC in which the Ca(2+) pump was inhibited by vanadate was found to be within the normal range (30-55 micromol (10(13) cells. hr)(-1)), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca(2+) influx increased to over 1 mmol (10(13) cells. hr)(-1), almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca(2+) into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni(2+). Possible roles for this pathway in pRBC are considered.

Animals↗

PubMed1999

A negative regulatory element in a prespore-specific promoter of dictyostelium discoideum(1).

We previously isolated several 'promoter-trap' transformants in which insertion of a promoterless beta-galactosidase gene into the genome caused expression of beta-galactosidase in specific cell types. The upstream flanking region was rescued from one transformant specifically expressing beta-galactosidase in prespore cells. We sequenced the promoter of the gene that is fused in-frame with lacZ and characterised a negative element that inhibits expression in pstO cells (a subtype of prestalk cells). Gel-retardation assays show that a developmentally regulated factor(s) recognises and binds to this element.

Amino Acid Sequence↗

PubMed1999

Measurement of firefly luciferase reporter gene activity from cells and lysates using Escherichia coli arsenite and mercury sensors.

The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors. Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro). We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a mercury-sensing strain E. coli MC1061(pTOO11), and two new arsenic sensor strains MC1061(pTOO31) and AW3110(pTOO31) which were constructed for this study. Plasmid pTOO31 was constructed by inserting the ars promoter and the arsR gene from plasmid R773 to control firefly luciferase gene expression. Both in vivo and in vitro methods correlated well with the strains tested [correlation coefficients R = 0.99484 and 0.99834] and gave highly comparable results with standard solutions of arsenite or mercury ions and from six environmental water samples spiked with the ions. Use of the in vivo method resulted in lower variation between replicates of the same sample (CVs ranging from 3.9 to 7.2%) and also between different samples (from 8.6 to 25.9%) compared to the in vitro method (CVs ranging from 8.6 to 17.8% for replicates and from 13.1 to 36.3% for different samples).

Animals↗

PubMed1999

An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens.

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its coregulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-activated transcription factor that mediates the stimulatory effects of estrogens and the inhibitory effects of antiestrogens such as tamoxifen in breast cancer and other estrogen target cells. To understand how antiestrogens and dominant negative ERs suppress ER activity, we used a dominant negative ER as bait in two-hybrid screening assays from which we isolated a clone from breast cancer cells that potentiates the inhibitory activities of dominant negative ERs and antiestrogen-liganded ER. At higher concentrations, it also represses the transcriptional activity of the estradiol-liganded ER, while having no effect on other nuclear hormone receptors. This clone, denoted REA for "repressor of estrogen receptor activity," encodes a 37-kDa protein that is an ER-selective coregulator. Its competitive reversal of steroid receptor coactivator 1 enhancement of ER activity and its direct interaction with liganded ER suggest that it may play an important role in determining the sensitivity of estrogen target cells, including breast cancer cells, to antiestrogens and estrogens.

Amino Acid Sequence↗

PubMed1999

PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells.

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.

Antineoplastic Agents, Alkylating↗

PubMed1999

Sino-orbital aspergillosis in acquired immunodeficiency syndrome.

OBJECTIVE: To describe the clinical features, causes, imaging characteristics, treatment, and outcome of patients with the acquired immunodeficiency syndrome (AIDS) and sino-orbital aspergillosis. DESIGN: Records of 5 patients were reviewed. Results of imaging and histopathologic examinations and clinical courses of the patients were studied. RESULTS: There were 3 women and 2 men (mean age, 34.0 years). All had received a diagnosis of AIDS, and mean CD4+ cell count was 0.014 x 10(9)/L (14 cells/mm3). Computed tomographic scanning exhibited heterogeneous, enhancing sino-orbital soft tissue lesions with bony erosion, and magnetic resonance imaging disclosed soft tissue masses hypointense on T1- and T2-weighted images. The infection involved 1 or more paranasal sinuses, with extension into the right orbit in 3 patients and into the left orbit in 2. Patients were treated with aggressive surgical debridement and intravenous antifungal agents. In addition, local irrigation of amphotericin B was performed in 3 patients. Aspergillus fumigatus was found to be the cause in all 5 patients. Intracranial extension developed in 4 patients, and all subsequently died. The 2 longest surviving patients were the only ones being treated with protease inhibitors. Three patients had a history of frequent marijuana smoking. CONCLUSIONS: Sino-orbital aspergillosis is a progressive, relentless, and usually fatal opportunistic infection of advanced AIDS. Patients are first seen with long-standing headache and proptosis with minimal external inflammatory signs. Marijuana smoking may increase the risk for development of sino-orbital aspergillosis in these patients. Aggressive surgical and medical treatment, combined with newer combination therapies using protease inhibitors, may improve the longevity of these patients.

AIDS-Related Opportunistic Infections↗

PubMed1999

Metal binding to the HIV nucleocapsid peptide.

Co(II) and Zn(II) binding constants have been measured for binding to the HIV-1 nucleocapsid N-terminal metal binding domain (residues 1-18), using competition titration methods and monitoring Co(II) binding by visible absorbance spectroscopy. Enthalpies for binding were directly measured by isothermal titration colorimetry. The results are compared with recent studies of related systems, including a study of Zn(II) binding by the full length protein.

Amino Acid Sequence↗

PubMed1999

The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor.

CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi.

Bacterial Proteins↗

PubMed1999

Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells.

We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L(+) D8L(+)) to be defective in expression of either the A27L or the D8L gene (A27L(+) D8L(-) or A27L(-) D8L(+)) or both (A27L(-) D8L(-)). The A27L(+) D8L(+) and A27L(-) D8L(+) mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L(+) D8L(-) and A27L(-) D8L(-) viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L(+) D8L(-) and A27L(-) D8L(-) mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L(+) D8L(-) and A27L(-) D8L(-) mutant virions were 6 to 10% of that of the A27L(+) D8L(+) control virus. Virion binding assays revealed that A27L(+) D8L(-) and A27L(-) D8L(-) mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.

Animals↗

PubMed1999

Induction of Th-1 and Th-2 responses by respiratory syncytial virus attachment glycoprotein is epitope and major histocompatibility complex independent.

In BALB/c mice, sensitization to respiratory syncytial virus (RSV) attachment (G) glycoprotein leads to the development of lung eosinophilia upon challenge infection with RSV, a pathology indicative of a strong in vivo induction of a Th-2-type response. In this study, we found that a strong, RSV G-specific, Th-1-type cytokine response occurred simultaneously with a Th-2-type response in G-primed mice after RSV challenge. Both Th-1 and Th-2 effector CD4(+) T cells recognized a single immunodominant site on this protein, implying that the differentiation of memory CD4(+) T cells along the Th-1 or Th-2 effector pathway was independent of the epitope specificity of the T cells. A similar observation was made in G-primed H-2(b) haplotype mice after RSV challenge, further suggesting that this process is not dependent on the peptide epitope presented. On the other hand, genes mapping to loci outside of the major histocompatibility complex region are crucial regulators of the development of a Th-2-type response and lung eosinophilia. The implication of these findings for the immune mechanisms underlying the pathogenesis of RSV is discussed.

Animals↗

PubMed1999

Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

Animals↗

PubMed1999

Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells.

The availability of Ca2+ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular [Ca2+] ([Ca2+]o) and modify their function. The effects of changing [Ca2+]o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca2+]o in a dose-dependent and reversible manner (ID50, approximately 2 mM Ca2+). RNA levels for aggrecan and type II collagen decreased with increasing [Ca2+]o (ID50, approximately 2.0 and 4.1 mM Ca2+, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca2+]o with ID50 values of approximately 2.9 and 1.6 mM Ca2+, respectively. These responses were rapid, in that increasing [Ca2+]o from 1.0 to more than 6 mM suppressed aggrecan RNA levels by about 50%, and lowering [Ca2+]o from 2.9 to 1.0 mM increased aggrecan RNA levels by about 300% within 4 h. As Ca2+ receptors (CaRs) mediate extracellular Ca2+ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.

Aggrecans↗

PubMed1999

GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo.

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.

Animals↗

PubMed1999

Prospective evaluation of perinatal risk factors for cerebral palsy and delayed development in high risk infants.

Prematurity, intrauterine infection and perinatal brain injury have been reported to be significant risk factors of cerebral palsy (CP). We examined the perinatal predictors of cerebral palsy and delayed development (DD) in 184 high risk infants. Thirty-five infants were diagnosed as cerebral palsy and delayed development at 12 months corrected age. Antenatal, intrapartum, and neonatal factors were prospectively evaluated in 2 groups of high risk infants compared with controls; Group A (n = 79), infants weighing less than 2,000 g; Group B (n = 43), infants weighing 2,000 g or more. In univariate analysis, there were no significant antenatal and intrapartum factors associated with cerebral palsy and delayed development in either group. We found that significant postnatal risk factors of CP in group A included sepsis (p = 0.008), BPD (bronchopulmonary dysplasia) (p = 0.028), IVH (intraventricular hemorrhage) (p = 0.042), ventriculomegaly (VM) (p = 0.001) and a longer duration of mechanical ventilation (p = 0.001); while in group B, sepsis (p = 0.047) and neonatal seizure (p = 0.027) were significant risk factors. In multivariate analysis, sepsis in group B was a moderate risk factor of CP (OR (odds ratio) 1.47; 95% CI (confidence interval) 1.02-2.13). In conclusion, neonatal sepsis may contribute to the development of cerebral palsy and delayed development. We suggest that high risk infants who have sepsis should be carefully followed for cerebral palsy and delayed development. The prevention of cerebral palsy may be feasible by decreasing neonatal risk factors such as sepsis during the neonatal period.

Cerebral Palsy↗

PubMed1999

Excimer laser effects on outflow facility and outflow pathway morphology.

PURPOSE: To determine the relative contributions to aqueous outflow resistance of the tissues distal to the inner wall of Schlemm's canal. METHODS: While performing constant pressure perfusion at 10 mm Hg, a 193-nm excimer laser (Questek) was used to precisely remove portions of sclera, unroofing Schlemm's canal while leaving the inner wall intact. The laser beam was masked to produce a beam 2 mm by 1 mm. The laser output was constant at a fluency of 75 mJ/cm2 and 20 Hz. The excimer laser at a frequency of 1 Hz was used as the aiming beam. Photoablation was performed on human cadaver eyes at the limbus at an angle of 0 degrees to 45 degrees from the optical axis. As the excimer photoablations progressed, Schlemm's canal was visualized by the fluorescence of the Barany's solution containing fluorescein dye. After perfusion fixation the eyes were immersion-fixed overnight. The facility of outflow before (Co) and after (Ce) the excimer ablation was measured in 7 eyes. RESULTS: The facility of outflow increased in all eyes after the excimer sinusotomy, from a mean of 0.29+/-0.02 before the sinusotomy to 0.37+/-0.03 microl/min per mm Hg after (P < 0.05). The mean ratio of outflow facility after and before ablation (Ce/Co) was 1.27+/-0.08 (range, 1.20-1.39), a reduction of outflow resistance of 21.3%. Using the formula of Ellingsen and Grant (1972), percentage of resistance to outflow eliminated = 100 [1 - alphaCo/Ce - (1 - alpha)Co], where alpha = fraction of the circumference dissected. Assuming that because of circumferential flow approximately 50% of Schlemm's canal is drained by the single opening made in the outer wall ablation studies, this results in resistance to outflow eliminated of 35%, which is consistent with the calculated eliminated resistance derived from the data of Rosenquist et al., 1989. Light and scanning electron microscopy confirmed the integrity of the inner wall Schlemm's canal underlying the area of ablation. CONCLUSIONS: The results provide direct evidence indicating that approximately one third of resistance to outflow in the human eye lies distal to the inner wall Schlemm's canal in an enucleated perfused human eye.

Anterior Chamber↗

PubMed1999

[The effect of cAMP on the transcription and regulation of angiotensin II type 1 receptor gene in cardiomyocytes of neonatal rats].

OBJECTIVE: To clarify the effect of cAMP on the transcription of angiotensin II type 1 (AT1) receptor mRNA in cardiomyocytes. METHODS: Cardiomyocytes from SD neonatal rats were cultured in DMEM containing 10% fetal bovine serum (FBS) with 0.1 mmol/L 5-bromodeoxyuridine (Brdu). At day 5-7, primary cultured cells were used. FBS and Brdu were deprived 24 h before the stimulation. The levels of AT1 mRNA in primary cultured cardiomyocytes were examined by RT-PCR. RESULTS: Stimulation of forskolin (30 mumol/L) plus 3-isobutyl-1-methyl-xanthine (IBMX, 30 mumol/L) which induced the accumulation of cellular cAMP decreased the expression of AT1 receptor mRNA to (64.4 +/- 20.3)% of control at 6 h treatment; while it increased the expression of AT1 receptor mRNA to (209.6 +/- 24.0)% of control at 48 h treatment. CONCLUSIONS: cAMP regulated the transcription of AT1 receptor gene.

Animals↗

PubMed1998

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells.

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.

Amino Acid Sequence↗

PubMed1998

Luminescent bacterial sensor for cadmium and lead.

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 microM, and 100 microM, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients.

Animals↗