Evaluation of commercial essential oil samples on the growth of postharvest pathogen Monilinia fructicola (G. Winter) Honey.

AIM: To assess the effect of several commercial essential oils samples Australian lemon myrtle (Backhousia citriodora), cinnamon bark (Cinnamomum zeylanicum), oregano (Origanum vulgare), thyme oil (Thymus vulgaris), clove bud (Eugenia caryophyllata), valerian (Valeriana officinalis) and Australian tea tree oil (Melaleuca alternifolia) on mycelium growth and spore germination of Monilinia fructicola. The effectiveness of lemon myrtle essential oil as a fumigant for the control of brown rot in nectarines was evaluated. METHODS AND RESULTS: Monilinia fructicola exhibited a different level of sensitivity to each tested essential oil with results suggesting that the essential oils provide excellent control of the pathogen with respect to mycelium growth and spore germination at very low concentrations, whereas for others higher concentrations are needed to reduce significant fungal growth. In vivo application of lemon myrtle essential oil effectively reduced the incidence of M. fructicola on noninoculated fruit. Fumigation of nectarines following inoculation did not reduce the incidence of brown rot in comparison with the inoculated control treatment. No evidence of phytotoxicity on the fruit was recorded. CONCLUSIONS: Lemon myrtle essential oil exhibited the strongest antifungal activity against M. fructicola, in vitro and to a lesser extent, under in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that lemon myrtle essential oil, in particular, has potential as an antifungal agent to control M. fructicola.

Rapid curing of bonding composite with a xenon plasma arc light.

The use of light-cured orthodontic adhesives is an increasingly popular method for the bonding of orthodontic brackets. However, one of the disadvantages of light-cured adhesives is their long curing times. The xenon plasma arc curing light is purported to dramatically reduce the required curing time. The purpose of this study was to test the efficiency of a xenon plasma arc light versus a conventional tungsten-quartz halogen light in producing effective bond strengths for orthodontic brackets. Standardized brackets were bonded to bovine enamel with 3 different orthodontic bonding materials. The bonding materials were exposed to the tungsten-quartz halogen light for 40 seconds and to the xenon light for 3, 6, and 9 seconds. Bond strength was tested 30 minutes and 24 hours after light-curing. The results showed that bond strength with the application of the xenon light was greater with longer exposures. There were no statistically significant differences between the bond strengths of the brackets exposed to the tungsten-quartz halogen light for 40 seconds and those exposed to the xenon light for 3, 6, or 9 seconds. However, xenon light exposures of 6 or 9 seconds were required to create bond strengths equal to those produced by the tungsten-quartz halogen light. The xenon light produced equivalent bond strengths at very short light exposures.

In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins.

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected approximately 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Delta mutation increases the petite-inducing phenotype of an abf2Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of rho(+) mtDNA.

Extracorporeal circulatory support in near-fatal flecainide overdose.

Flecainide, a class 1c antiarrhythmic, has a high mortality associated with significant overdose. We report the case of a 20-year-old female who took approximately 4 grams of flecainide and a small amount of paracetamol as an impulsive gesture. Circulatory failure unresponsive to pacing, inotropes and sodium bicarbonate was successfully treated with cardiopulmonary bypass (CPB). Resolution of her myocardial failure occurred over 24 hours and she was weaned from CPB 30 hours after its initiation. Coagulopathy and intravascular haemolysis were apparent during bypass and necessitated substantial use of blood products. Ischaemic renal dysfunction manifested early in her admission and required haemodiafiltration. Despite a prolonged period of unresponsiveness and pupillary dilatation during resuscitation and CPB she made a full recovery. We believe this is the first reported case of flecainide overdose, requiring extracorporeal circulatory support, not resulting in neurological deficit.

The effects of light intensity, temperature, and comonomer composition on the polymerization behavior of dimethacrylate dental resins.

One of the most common combinations for the organic phase of dental restorative materials is BisGMA (2,2bis[4-(2-hydroxy-3-methacryloyloxypropoxy) phenyl]propane) and TEGDMA (triethylene glycol dimethacrylate). However, this copolymer has some drawbacks, such as volume shrinkage during cure and lack of complete double-bond conversion. If the properties of this system are to be improved, an attempt must be made to understand the underlying kinetics of the reaction. This work examines the effects of light intensity, temperature, and composition on the polymerization behavior of BisGMA/TEGDMA copolymerizations. Using differential scanning calorimetry, we monitored the rates of photopolymerization for various experimental conditions. The BisGMA/TEGDMA copolymerization behaved similarly to other dimethacrylate systems and exhibited diffusion-controlled kinetics. It was found that the maximum rate of polymerization was significantly affected by the intensity of the light, and the temperature of the polymerization affected the conversion at which the maximum rate occurred. When the composition of the mixture was varied, it was discovered that the viscosity of the system played a significant role in the polymerization rate and the onset of reaction-diffusion-controlled termination. Mixtures which contained from 50 wt% to 75 wt% BisGMA displayed the highest maximum rate. This feature suggests that TEGDMA is an excellent diluent, since it increases the mobility of the reacting medium; however, the high reactivity is due to the presence of BisGMA. Therefore, based on compositional dependence, we conclude that the BisGMA portion of the mixture largely controls the polymerization mechanisms and kinetics.

Comparison of alterations of chromosome 17 in carcinoma of the ovary and of the breast.

Breast and ovarian carcinomas share a region of allelic loss on chromosome 17q25, suggesting that these tumours may arise by similar molecular pathways. We analysed paraffin-embedded tissues from 84 sporadic ovarian carcinomas and 42 sporadic infiltrating ductal carcinomas of the breast for abnormalities on chromosome 17. Loss of heterozygosity (LOH) of at least one informative marker on 17q was identified in 49 of 82 (60%) ovarian carcinomas, as against only 6 of 40 (15%) informative breast carcinomas (P<0.0001). In ovarian carcinoma, LOH was most commonly observed for GH on 17q23 (56%), and was also frequently observed at 17q21 (46%). In contrast, LOH of D17S 1330/CTT16 on 17q25 was observed in only 19% of ovarian tumours. LOH in breast carcinomas was most frequently observed at 17q21 (16%), less frequently at 17q23 (7%) and not identified at all at 17q25 in any breast cancers. Immunohistochemical analysis demonstrated overexpression of the p53 gene product in 38 of 84 (45%) ovarian carcinomas, as against 10 of 42 (24%) breast carcinomas (P = 0.0195). p53 immunoreactivity was significantly associated with LOH in ovarian and breast cancers. Immunohistochemical expression of HER2/neu was observed in 6 of 84 (7%) ovarian and 3 of 42 (7%) breast carcinomas. There was no relationship between HER2/neu immunoreactivity and LOH. Although sporadic carcinomas of breast and ovary share some regions of allelic loss on chromosome 17q, differences in other alterations on this chromosome suggest divergent pathways of tumour development.

Comparison of mutations of Ki-RAS and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors.

Ovarian tumors of low malignant potential ("borderline tumors") have been proposed variably to represent a distinctive type of malignancy, precursors of frank ovarian malignancy, or a nonmalignant process. We analyzed 81 malignant and 39 borderline ovarian tumors for p53 immunoreactivity and alterations in codon 12 of Ki-RAS in order to correlate these alterations with tumor and cell type. Diffuse p53 immunoreactivity was significantly more prevalent among malignant (36 of 81, 44%) than among borderline (3 of 39, 8%) tumors and was particularly prevalent among serous invasive carcinomas (16 of 26, 62%). Conversely, mutations in codon 12 of Ki-RAS were significantly more prevalent in borderline (16 of 39, 41%) than in malignant (9 of 81, 11%) ovarian tumors and were most prevalent among mucinous tumors. This preliminary molecular analysis suggests that serous borderline tumors have some molecular features usually associated with malignancy but are unlikely to represent a precursor of invasive serous carcinoma. In contrast, mucinous borderline tumors may represent a precursor or variant of mucinous carcinoma of the ovary.

Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.

Analysis of human papillomavirus infection and molecular alterations in adenocarcinoma of the cervix.

Although molecular alterations involved in the development of squamous cell carcinoma of the cervix have been extensively described, these genetic changes have not been as well characterized in the development of cervical adenocarcinoma. Twenty-seven paraffin-embedded adenocarcinomas of the cervix, including three cases of adenoma malignum, were analyzed for molecular alterations associated with other gynecologic malignancies. The presence of human papillomavirus (HPV) was assessed by polymerase chain reaction (PCR) using internally nested consensus primers. HPV types were identified by restriction endonuclease digestion of the PCR products, using DNA sequencing to confirm each digestion pattern. The presence of HPV was correlated with immunohistochemical expression of the p53 gene product, the presence of mutations in codon 12 of Ki-ras, and allelic deletion of markers associated with the development of other gynecologic carcinomas. HPV was identified in 16 (59%) of 27 cases, including type 18 in 7 tumors, type 16 in 7 tumors, and type 45 in 2 tumors. HPV types 16 and 45 were always identified in adjacent uninvolved cervical epithelium, but HPV type 18 was absent from the adjacent non-neoplastic epithelium in four of the seven positive cases. HPV was not identified in any of three cases of adenoma malignum. Diffuse immunohistochemical staining of the p53 gene product was present in only one (HPV-negative) tumor. A mutation in codon 12 of Ki-ras was observed in one endocervical adenocarcinoma (with an endometrioid pattern). Loss of heterozygosity was identified only for a marker on chromosome 6p in one mucinous endocervical carcinoma. Most endocervical adenocarcinomas lack molecular alterations characteristic of other histologically similar gynecologic malignancies, as well as those described in cervical squamous cell carcinomas.

Comparative analysis of histologic homologues of endometrial and ovarian carcinoma.

We compared molecular alterations in histologically homologous ovarian and uterine carcinomas, including the prevalence of allelic loss of markers on 17q (within and distal to the familial breast-ovarian cancer gene BRCA1), mutations of codon 12 of Ki-ras and immunohistochemical expression of the p53 and c-erbB2 gene products in endometrioid and papillary serous carcinomas occurring in the uterus and ovary. A total of 86 uterine and 28 ovarian endometrioid carcinomas, as well as 8 uterine and 26 ovarian papillary serous carcinomas, were evaluated. The prevalence of p53 gene product immunoreactivity was similar in papillary serous carcinomas occurring in the uterus (6 of 8, 75%) and ovary (16 of 26, 62%). Allelic loss on 17q also was seen in similarly high proportions of uterine (3 of 7, 43%) and ovarian (16 of 25, 64%) papillary serous carcinomas. In contrast, expression of the p53 gene product was seen in significantly more endometrioid tumors of the ovary (14 of 28, 50%) than in those occurring in the uterus (4 of 86, 5%) (p < 0.0001). Allelic loss on 17q also was present in significantly more ovarian (19 or 27, 70% than in uterine (2 of 72, 3%) endometrioid carcinomas (p < 0.0001). Immunohistochemical expression of c-erbB2 and mutations of codon 12 of Ki-ras were present in a minority of carcinomas. Endometrioid tumors of the ovary and endometrium, although histologically similar, may arise from different genetic events, whereas uterine papillary serous carcinoma shares with its ovarian counterpart several molecular alterations that may account for its aggressive clinical behavior.

A region of interstitial 17q25 allelic loss in ovarian tumors coincides with a defined region of loss in breast tumors.

Chromosomal regions of allelic imbalance in tumors are predicted to define the general location of tumor suppressor genes. We previously localized a putative breast tumor suppressor gene to a 3 cM region on 17q25 by deletion mapping of microsatellite markers in breast tumors. To determine if the same 17q25 region of loss is important in the genesis of other tumor types, 32 ovarian tumors and 24 prostate tumors, as well as 33 additional breast tumors, were analysed with 17q25 polymorphic microsatellite markers. While no significant loss was observed in prostate tumors, greater than half of ovarian tumors exhibited loss coincident with the candidate region previously defined in breast tumors. These results suggest that one or more novel tumor suppressor genes exist on 17q25 within a concordant region of interstitial loss defined in both breast and ovarian neoplasms.

Detection of hepatitis C by RT-PCR in formalin-fixed paraffin-embedded tissue from liver transplant patients.

The histopathologic alterations of hepatitis C virus (HCV) infection of the liver overlap with those of other diseases, making interpretation of liver biopsy specimens in some cases insufficient to render a diagnosis. Although HCV infection can be confirmed by detection of circulating anti-HCV antibodies, immunocompromised liver transplant recipients are often unable to mount an immunologic response to the virus, resulting in false-negative serologic testing. We describe the comparison of reverse transcription-polymerase chain reaction (RT-PCR) with histopathology, serology, and immunohistochemistry for the diagnosis of HCV. Sixty-three formalin-fixed, paraffin-embedded tissue samples (40 needle biopsy specimens and 23 native liver resection specimens) from 35 transplant patients were analyzed by use of a novel method of RNA extraction followed by nested PCR for HCV as well as albumin mRNA as an internal control. HCV was detected by RT-PCR in 50 of 51 (98%) paraffin sections of liver from transplant patients with circulating anti-HCV antibodies, 15 of which lacked characteristic histologic features of HCV infection. Overall, there were no false-negative results in 36 needle biopsy specimens from patients with hepatitis C infection, but three negative results were seen in end-stage cirrhotic native livers resected from HCV-infected patients. No false-positive test results were seen among 21 negative controls (10 liver samples from immunocompetent patients with abnormalities unrelated to hepatitis C and 11 liver biopsies from immunocompetent patients without histologic evidence of liver disease). In comparison, immunohistochemistry using antibody TORDJI-22 was positive for HCV in only 15 of 32 (47%) needle biopsies positive by RT-PCR. Our results indicate that RT-PCR is a more sensitive and specific method of detecting hepatitis C in routinely processed paraffin sections of formalin-fixed liver biopsy specimens than histopathologic examination or immunohistochemistry.

The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells.

The Caco-2 cell line is used by many investigators as a model of the intestinal epithelium to study nutrient uptake and transport. Our goal was to create an awareness of inherent variabilities in the Caco-2 cell line which may influence their suitability as a model or their application to specific problems. To study the influence of passage on the model, cultures were monitored from passage 20 to 109. Transepithelial electrical resistance (TEER) and sucrase activity (measured in 21-day-old cultures) increased through about passage 36. TEER values declined after about passage 60; sucrase remained elevated but variable. Cells at passage 22, 33, and 72 were grown simultaneously for 24 days. Older-passaged cells reached plateau phase sooner. Before Day 15, passage 72 cells had higher TEER and lower permeability to 14C-mannitol than passages 22 and 33; however, after Day 15 all passages showed similar permeability. On Day 21, passage 72 cells had significantly lower alkaline phosphatase activity than did the other passages. Electron microscopy did not reveal any major morphological differences between the passages; however, it did show that some areas of cells grown on membranes were not monolayers but were several cells thick with varied morphology. Investigation of the formation of these multilayered areas showed them to be an inherent part of cell growth under the conditions used. These results emphasize the inherent variability in Caco-2 cell models and emphasize the need to monitor closely the culture characteristics during growth and differentiation under specific experimental conditions.

Detection of human papillomavirus DNA in keratoacanthomas by polymerase chain reaction.

The etiology of keratoacanthomas is unknown, but human papillomavirus (HPV) has been suspected to be involved in the pathogenesis of this lesion because koilocytic changes may be observed and because HPV has been found in cutaneous squamous-cell carcinomas and premalignant keratoses in immunosuppressed patients. We analyzed DNA extracted from 39 keratoacanthomas from 22 "at-risk" patients (nine patients undergoing UV light and/or anthralin therapy for psoriasis, 10 solid organ transplant recipients, one patient with xeroderma pigmentosa, one patient with acquired immunodeficiency syndrome, and one patient undergoing therapy for non-Hodgkin's lymphoma) for the presence of HPV. The results were compared with analyses of DNA extracted from 30 keratoacanthomas from 28 patients at no known increased risk for these lesions. Using polymerase chain reaction (PCR) primers designed to detect multiple HPV types (including 6, 11, 16, 18, 31, and 33), HPV was detected in seven keratoacanthomas from six of the at-risk patients and in eight sporadic keratoacanthomas from eight patients without risk factors. HPV was also present in one of 26 nonlesional skin controls. Statistical analysis showed a significant difference in the prevalence of HPV DNA sequences found in keratoacanthomas compared to normal control skin (p = 0.038). The presence of virus by PCR could not be predicted by histologic evaluation. Sequence analysis showed the presence of HPV types 11, 13, 24, 33, and 57. Although these results confirm the frequent presence of HPV in keratoacanthomas, the role of this virus in the etiology and pathogenesis of these lesions remains to be elucidated.

Analysis of chronic endometritis for Chlamydia trachomatis by polymerase chain reaction.

Chronic endometritis is characterized histologically by plasma cells infiltrating endometrial stroma. Although it has been speculated that many instances of chronic endometritis are infectious, the origin of most cases is not apparent by routine histopathologic evaluation. Chlamydia trachomatis, an obligate intracellular bacterium, is known to cause chronic endometritis in the setting of pelvic inflammatory disease. The authors analyzed 43 specimens of histopathologically diagnosed chronic endometritis from 38 patients for C trachomatis by PCR using primers for the single-copy major outer membrane protein (MOMP) gene. C trachomatis was detected in only one such case in which dense plasma cell infiltrates were present and concurrent C trachomatis infection of the cervix was documented. Using serially diluted DNA from formalin-fixed, paraffin-embedded McCoy cells, the sensitivity of this method was shown to be equivalent to a single infected cell in a paraffin section. In conjunction with the results of other studies, these data indicate a limited role, if any, of C trachomatis in the origin of mild or moderate chronic endometritis.

Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR.

DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.

The influence of comonomer composition on dimethacrylate resin properties for dental composites.

During the polymerization of multifunctional monomers for dental restorations, typical final double-bond conversions range from 55 to 75%. The low conversion results in a large amount of extractable monomer, reduced adhesion to the filler, and the potential for increased swelling. In this work, the ability to increase the maximum conversion by optimizing the copolymer composition is explored. A series of multi-ethylene glycol dimethacrylate monomers of various lengths was used as a model system to determine how the copolymer composition affects the final conversion, the mechanical properties, and the predicted shrinkage. It was found that the ultimate conversion can be significantly increased, shrinkage decreased, and mechanical properties maintained. It was found that up to 30 wt% of poly(ethylene glycol) 600 dimethacrylate could be added to diethylene glycol dimethacrylate without reducing the strength and increasing the conversion. Results for other comonomer combinations were similar.

Histologic progression of recurrent hepatitis C in liver transplant allografts.

The incidence and severity of recurrent hepatitis C virus (HCV) infection in liver transplant recipients vary widely, and the long-term sequelae of recurrent infection are not known. To better define the biology of recurrent HCV in liver transplant patients, we reviewed the histology of recurrent HCV in serial biopsies of 19 patients with pretransplant polymerase chain reaction (PCR) evidence of HCV infection. All posttransplant (post-TX) biopsies (n = 81) were reviewed, and RNA was extracted from at least one paraffin-embedded biopsy from each patient. RNA was analyzed for HCV by nested, reverse transcription-PCR (RT-PCR) using primers for the 5' non-coding region of HCV as well as for albumin (as an internal control). All post-TX biopsies tested (12-1,677 days post-TX) were positive for HCV RNA by RT-PCR, while normal control biopsies were negative. Fifteen of 19 patients developed recurrent chronic hepatitis typical of HCV. Many of these patients showed a progression from early biopsies with acute lobular hepatitis to later biopsies with chronic hepatitis with portal lymphoid aggregates. An acute lobular hepatitis typified by sinusoidal lymphocytosis, acidophil bodies, and lobular disarray was seen an average of 135 days post-TX, with a range of 39-279 days. The time post-TX between this and earlier non-hepatitis biopsies was significantly different (p < 0.0004, Student's t test). Chronic hepatitis with portal lymphoid aggregates was seen an average of 356 days post-TX, with a range of 89-1,365 days. The time post-TX was significantly longer than for acute lobular hepatitis (p < 0.03, Student's t test). Fifty-three percent of HCV TX patients progressed from acute lobular hepatitis to chronic hepatitis with lymphoid aggregates within 1 year of TX, and 79% showed these changes within 4 years. Six patients had progressive fibrosis; one die of liver failure and two became cirrhotic. Recurrent HCV appears to progress from an acute lobular hepatitis to chronic hepatitis with lymphoid aggregates in the majority of patients. Significant scarring occurred in 32% of patients and 16% developed end-stage liver disease from recurrent HCV. These later findings suggest that the long-term course of recurrent HCV in liver allografts may not be as indolent as first thought.

High zinc concentrations in culture media affect copper uptake and transport in differentiated human colon adenocarcinoma cells.

Previous studies suggest that high dietary zinc reduces the copper status of animals by reducing copper transport across the intestinal mucosa. The present study used an enterocyte mimic, the Caco-2 cell, grown on porus membranes as a model to assess the effects of various concentrations of zinc (6 to 200 micromol/L) in the culture and assay media on 67Cu uptake and transport. Differentiated cells incubated for 7 d in a culture medium containing 50 micromol zinc + 0.7 micromol copper/L transported significantly less 67Cu across the monolayer than similar cells exposed to 6 micromol zinc/L. However, cells exposed to 200 micromol zinc/L for the same period transported significantly more 67Cu than those exposed to 6 micromol zinc/L. Cells exposed for only 1 h to 200 micromol zinc/L in the uptake-transport medium alone did not show lower 67Cu uptake or transport, suggesting that time of exposure of the cells to high zinc was a contributing factor. Caco-2 cells exposed to 50 through 200 micromol zinc/L had higher cellular metallothionein (MT) than those exposed to 6 micromol zinc/L. As the amount of MT in cells increased upon exposure to 50 and 100 micromol zinc/L, the rate of 67Cu transport decreased. At higher zinc concentrations in the medium, there was even more MT in the cells, but a greater rate Of 67Cu transport. These studies demonstrate the use of the Caco-2 cell as a model for copper uptake-transport studies, but the conditions must be rigorously defined and controlled.