Fenofibrate subcellular distribution as a rationale for the intracranial delivery through biodegradable carrier.

Fenofibrate, a well-known normolipidemic drug, has been shown to exert strong anticancer effects against tumors of neuroectodermal origin including glioblastoma. Although some pharmacokinetic studies were performed in the past, data are still needed about the detailed subcellular and tissue distribution of fenofibrate (FF) and its active metabolite, fenofibric acid (FA), especially in respect to the treatment of intracranial tumors. We used high performance liquid chromatography (HPLC) to elucidate the intracellular, tissue and body fluid distribution of FF and FA after oral administration of the drug to mice bearing intracranial glioblastoma. Following the treatment, FF was quickly cleaved to FA by blood esterases and FA was detected in the blood, urine, liver, kidney, spleen and lungs. We have also detected small amounts of FA in the brains of two out of six mice, but not in the brain tumor tissue. The lack of FF and FA in the intracranial tumors prompted us to develop a new method for intracranial delivery of FF. We have prepared and tested in vitro biodegradable poly-lactic-co-glycolic acid (PLGA) polymer wafers containing FF, which could ultimately be inserted into the brain cavity following resection of the brain tumor. HPLC-based analysis demonstrated a slow and constant diffusion of FF from the wafer, and the released FF abolished clonogenic growth of glioblastoma cells. On the intracellular level, FF and FA were both present in the cytosolic fraction. Surprisingly, we also detected FF, but not FA in the cell membrane fraction. Electron paramagnetic resonance spectroscopy applied to spin-labeled phospholipid model-membranes revealed broadening of lipid phase transitions and decrease of membrane polarity induced by fenofibrate. Our results indicate that the membrane-bound FF could contribute to its exceptional anticancer potential in comparison to other lipid-lowering drugs, and advocate for intracranial delivery of FF in the combined pharmacotherapy against glioblastoma.

Molecular dynamics of ions in two forms of an electroactive polymer.

Molecular dynamics (MD) are performed in all-atom simulations of two polymer species based on polythiophene. In one case the amphiphilic polymer forms a monolayer interface between a vacuum and an aqueous layer containing ions. The electroactive nature of the polymer is invoked by conferring a negative charge on it to compensate for charge imbalance in the Na(+) and Cl(-) concentrations of the aqueous layer. The effects of hydrostatic pressure and charge imbalance on the stability of the monolayer are investigated. In another simulation a polythiophene oligomer is wound into a helix where it serves as an ion channel between two aqueous regions on both sides of a phospholipid bilayer membrane.

Cleavage stage versus blastocyst stage embryo transfer in assisted conception.

BACKGROUND: Recent advances in cell culture media have led to a shift in IVF practice from early cleavage embryo transfer to blastocyst stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and self selection of viable embryos thus resulting in higher implantation rates. OBJECTIVES: To determine if blastocyst stage embryo transfers (ETs) affect live birth rate and associated outcomes compared with cleavage stage ETs and to investigate what factors may influence this. SEARCH STRATEGY: Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Controlled Trials Register (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was January 2007. SELECTION CRITERIA: Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers. DATA COLLECTION AND ANALYSIS: Of the 50 trials that were identified, 18 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed. The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation. Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines. MAIN RESULTS: Evidence of a significant difference in live-birth rate per couple between the two treatment groups was detected in favour of blastocyst culture (9 RCTs; OR 1.35, 95% CI 1.05 to 1.74 (Day 2/3: 29.4% versus Day 5/6: 36.0%)). This was particularly for trials with good prognosis patients, equal number of embryos transferred (including single embryo transfer) and those in which the randomisation took place on Day 3. Rates of embryo freezing per couple was significantly higher in Day 2 to 3 transfers (9 RCTs; OR 0.45, 95% CI 0.36 to 0.56). Failure to transfer any embryos per couple was significantly higher in the Day 5 to 6 group (16 RCTs; OR 2.85, 95% CI 1.97 to 4.11 (Day 2/3: 2.8% versus Day 5/6: 8.9%)) but was not significantly different for good prognosis patients (9 RCTs; OR 1.50, 95% CI 0.79 to 2.84). AUTHORS' CONCLUSIONS: This review provides evidence that there is a significant difference in pregnancy and live birth rates in favour of blastocyst transfer with good prognosis patients with high numbers of eight-cell embryos on Day three being the most favoured in subgroup for whom there is no difference in cycle cancellation. There is emerging evidence to suggest that in selected patients, blastocyst culture maybe applicable for single embryo transfer.

The merits of blastocyst versus cleavage stage embryo transfer: a Cochrane review.

UNLABELLED: This paper is based on a Cochrane review published in The Cochrane Library, issue 2, 2002 (see www.CochraneLibrary.net for information) with permission from The Cochrane Collaboration and John Wiley and Sons. Cochrane reviews are regularly updated as new evidence emerges and in response to comments and criticisms, and The Cochrane Library should be consulted for the most recent version of the review. BACKGROUND: The aim of this study was to determine the relative merits of blastocyst versus cleavage stage embryo transfer, concerning the chance of pregnancy, live birth, multiple pregnancy and the factors contributing to these primary outcomes, from the best available evidence. METHODS: A systematic review employing the principles of the Cochrane Menstrual Disorders and Subfertility Group was undertaken. Fourteen randomized controlled trials, all comparing day 2/3 with day 5/6 embryo transfer, were included in a meta-analysis. RESULTS: For day 2/3 versus day 5/6 transfer, there was no significant difference in the odds of pregnancy [odds ratio (OR) = 0.91, 95% confidence interval (CI) 0.71-1.17] nor of live birth (OR = 0.83, 95% CI 0.48-1.42) per treated couple. These results were similar whether all trials, only trials with transfer of equal numbers of day 2/3 versus day 5/6, or only trials with transfer of fewer day 5/6 than day 2/3 embryos, were pooled. There was no significant difference in the odds of multiple pregnancy for day 2/3 versus day 5/6 transfer overall (OR 0.77, 95% CI 0.52-1.13) nor when fewer day 5/6 than day 2/3 embryos were transferred (day 2/3 versus day 5/6 OR 0.69, 95% CI 0.42-1.12). CONCLUSION: The current evidence fails to support a widespread change of practice from cleavage stage to blastocyst stage embryo transfer in couples undergoing IVF.

Antibody-based sensors for heavy metal ions.

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25+/-11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.

Laser zona pellucida thinning--an alternative approach to assisted hatching.

BACKGROUND: The purpose of this study was to assess the efficacy and hatching characteristics of in-vitro cultured human embryos subjected to laser zona pellucida thinning. METHOD: Zona thinning was performed on 110 embryos using a non-contact 1.48 microm diode laser and the hatch rate in vitro was compared with 42 control embryos. Variation of zona thickness and degree of zona expansion was assessed. Scanning electron microscopy was performed on embryos entrapped during hatching to identify the site of hatching. RESULTS: The rate of hatching was significantly higher in laser thinned blastocysts compared with control embryos (68 versus 33% respectively, P < 0.01). Laser thinning increased the variation of zona thickness in embryos from 11.6-27.3%. Natural zona thinning occurred in 92% of laser thinned hatching blastocysts and 100% of control embryos. CONCLUSION: These results suggest that laser zona thinning is effective and may provide significant advantages over conventional assisted hatching techniques, which create holes.

One-step competitive immunoassay for cadmium ions: development and validation for environmental water samples.

A rapid, simple, and reliable competitive immunoassay was developed and validated for measurement of Cd(II) in environmental water samples. This assay employed a monoclonal antibody that recognizes Cd(II)-EDTA complexes as capture reagent and a Cd(II)-EDTA conjugate of horseradish peroxidase as an enzyme label. The assay depended on a competitive binding reaction between the enzyme conjugate and Cd(II)-EDTA complexes, derived from the environmental water sample, for the binding sites of the immobilized antibody. The concentration of Cd(II) in the sample was quantified by the ability of its EDTA complexes to inhibit the binding of the enzyme conjugate to the antibody and, subsequently, color formation in the assay. The assay was specific to Cd(II), with a limit of detection of 0.3 ppb. Ca(II), Mg(II), and Fe(III), the metal ions commonly found in ambient water at relatively high concentrations, did not interfere with the assay. Mean analytical recovery of added Cd(II) was 100.29 +/- 3.60. The precision of the assay was satisfactory; coefficients of variation were 3.6-10.9 and 4.81-10.21% for intra- and interassay precision, respectively. The assay compared favorably with graphite furnace atomic absorption spectroscopy in its ability to accurately measure Cd(II) spiked into water samples from a Louisiana bayou.

Inhibition of retinal angiogenesis by peptides derived from thrombospondin-1.

PURPOSE: Thrombospondin (TSP)1 is a tumor suppressor with activity that is associated with its ability to inhibit neovascularization. Previous studies have mapped this antiangiogenic activity to the type 1 repeats and the amino-terminal portion of the molecule within the procollagen-like domain. The present study was performed to investigate the ability of TSP-1 and peptides derived from the type 1 repeats to inhibit retinal angiogenesis. METHODS: TSP-1 and peptides with tryptophan-rich, heparin-binding sequences and transforming growth factor (TGF)-beta1 activation sequences were evaluated in two models of retinal angiogenesis: a retinal explant assay and a rat model of retinopathy of prematurity (ROP). RESULTS: Platelet-derived TSP-1 inhibited angiogenesis in both experimental models. Peptides from the native TSP-1 sequence, which contained both the tryptophan-rich repeat and the TGF-beta1 activation sequence, were the most potent inhibitors of endothelial cell outgrowth in the retinal explant assay. In contrast, a peptide containing only the tryptophan-rich, heparin-binding sequence was most active in inhibiting neovascular disease in the rat ROP model. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain two subdomains that may independently influence the process of neovascularization, and that peptides derived from these type 1 repeats may be promising pharmacologic agents for treatment of retinal angiogenesis.

Intra-cytoplasmic sperm injection versus partial zona dissection, subzonal insemination and conventional techniques for oocyte insemination during in vitro fertilisation.

BACKGROUND: In vitro fertilisation (IVF) and embryo transfer as treatment for male factor infertility is associated with lower fertilisation and pregnancy rates than for other indications. Since the late 1980s several assisted fertilisation techniques have emerged and have been rapidly developed to try to enhance results for couples with male factor infertility, or to help couples with severe male factor for whom conventional IVF was not possible. The technique of partial zona dissection (PZD) was developed to increase the probability that a sperm capable of fertilisation comes in contact with the oocyte. Although this method improved conventional IVF results, the improvement was only marginal and relatively large numbers of sperm are still required. This drawback applied less to the subsequent technique of subzonal microinjection of spermatozoa into the perivitelline space (SUZI). However, for all of these techniques fertilisation rates remained low, rates of polyspermic fertilisation were increased, and cases with a very limited number of spermatozoa in the ejaculate could still not be treated. The advent of intra-cytoplasmatic sperm injection (ICSI) of a single sperm (or sperm head or nucleus) into the oocyte appears to be an important breakthrough. OBJECTIVES: To investigate whether ICSI improves fertilisation and/or pregnancy rates in comparison to other fertilisation techniques. SEARCH STRATEGY: The Menstrual Disorders and Subfertility Group search strategy (see Review Group details) was used to identify trials that had compared ICSI with other infertility techniques, such as PZD, SUZI, conventional IVF and additional IVF. SELECTION CRITERIA: Trials were included if they compared the effects of these techniques on fertilisation and pregnancy outcomes. Only randomised studies were included in this review. DATA COLLECTION AND ANALYSIS: Ten studies met the inclusion criteria for this review. Eight studies compared ICSI with conventional IVF. One study compared ICSI with SUZI and one study compared ICSI with additional IVF. Data was extracted independently by two reviewers. Where relevant data was missing or unclear, the authors had been consulted. Male participants were classified according to their semen parameters, i.e. normal semen (concentration >20 million per ml, motility >50%, morphology >14%), borderline semen (concentration 10-20 million per ml, motility 30-50%, morphology 4-14% normal forms) and very poor semen (concentration <10 million per ml, motility <30%, morphology <4% normal forms). MAIN RESULTS: For couples with normal semen there is no evidence of a difference in fertilisation rates per retrieved oocyte or pregnancy rates between ICSI and conventional IVF. On the other hand, for fertilisation rate per inseminated oocyte, ICSI appears to result in better outcomes than IVF for normal semen. For couples with borderline semen ICSI results in higher fertilisation rates (all) than IVF. Couples with very poor semen will have better fertilisation outcomes with ICSI than with SUZI or additional IVF. REVIEWER'S CONCLUSIONS: There is evidence from this systematic review that fertilisation rates are significantly better with ICSI than IVF in couples with borderline semen. When the semen parameters are normal there is insufficient evidence of a difference in effectiveness between ICSI and IVF when retrieved oocytes were the unit of randomisation. However, there was a small but statistically significant increase in fertilisation rate when inseminated oocytes were the unit of randomisation. Total fertilisation rates were significantly reduced in ICSI cycles than IVF but there were no damaged oocytes in IVF cycles regardless of the semen parameters.

Binding properties of a monoclonal antibody directed toward lead-chelate complexes.

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel.

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution.

A method was developed for the quantification of protein-ligand interactions in which the free protein present in homogeneous reaction mixtures was separated and quantified using a KinExA immunoassay instrument. Separation was achieved by rapid percolation of the reaction mixture over a column of microbeads whose surfaces were coated with an immobilized form of the ligand. The protein thus captured was quantified using a fluorescently labeled anti-protein antibody. The features of this new method were illustrated using a model system in which each of the principal reagents was covalently labeled with a different fluorescent molecule: mouse monoclonal anti-biotin primary antibody (fluorescein), biotin (B-phycoerythrin), and goat anti-mouse polyclonal secondary antibody (indodicarbocyanin). Values for the equilibrium and kinetic rate constants for the binding between the anti-biotin antibody and biotin conjugated with B-phycoerythrin were determined and shown to be independent of whether the fluorescent label was located on the primary or secondary antibody. Equilibrium binding experiments conducted with (F(AB))(2) and corresponding F(AB) fragments showed that the valency of the binding protein had no influence on the value of the dissociation constant. The values of the equilibrium and rate constants obtained by this new method are those for the binding reaction in homogeneous solution; the immobilized ligand is only a tool exploited for the separation and quantification of the free protein.

The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes.

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

Do men undergoing sterilizing cancer treatments have a fertile future?

This study was designed to assess the effect of cancer treatments on the natural and assisted reproductive potential of men. A cohort of men with cancer, in whom radiotherapy and/or chemotherapy was planned, were invited to participate. Twenty-two pre- and post-treatment semen samples were analysed. The reproductive potential of participants was assessed with respect to the current range of fertility treatment options available. Abnormal sperm concentrations were found in 27% of patients pre-treatment compared to 68% post-treatment following a mean latency of 20 months from treatment. Fifty-nine percent of patients experienced a clinically significant decrease in sperm, concentration following radiotherapy and/or chemotherapy; 23% developed azoospermia following treatment. Eighty-two percent of patients with testicular malignancy had oligo- or azoospermia post-treatment. Only one patient had a clinically significant reduction in the percentage of motile spermatozoa post-treatment. Cryopreservation of semen prior to treatment improved the fertility prospects of 55% of patients. Intracytoplasmic sperm injection (ICSI) enhanced the fertility prospects of a further 14%. In the absence of, or after depletion of, cryopreserved semen, ICSI could enhance the fertility prospects of 45% of patients. Fertilization has been achieved by ICSI using spermatozoa retrieved by testicular biopsy from an azoospermic testicular cancer survivor 8 years after chemotherapy. It was concluded that chemotherapy and/or radiotherapy may depress semen concentration to the extent of rendering a man infertile. The severity of the reduction in sperm concentration following treatment is unpredictable but likely to be most severe in those with testicular malignancy and those treated with radiotherapy or alkylating chemotherapy agents. Not all men are keen to undergo an appraisal of their post-treatment fertility potential, for reasons which are unclear. Improving awareness and education of patients concerning the effects of both cancer and cancer treatments on reproductive potential is essential. With the advent of ICSI, it is possible to offer a very reasonable chance of conception in all men with cancer who present for cryopreservation of semen prior to treatment in whom spermatozoa (even in very low concentrations) are present in the ejaculate.

Relationship between National Institutes of Health research awards to US medical schools and managed care market penetration.

CONTEXT: Medical research conducted in academic medical centers is often dependent on support from clinical revenues generated in these institutions. Anecdotal evidence suggests that managed care has the potential to affect research conducted in academic medical centers by challenging these clinical revenues. OBJECTIVE: To examine whether empirical evidence supports a relationship between managed care and the ability of US medical schools to sustain biomedical research. DESIGN: Data on annual extramural research grants awarded to US medical schools by the National Institutes of Health (NIH) from fiscal years 1986 to 1995 were obtained, and each medical school was matched to a market for which information about health maintenance organization (HMO) penetration in 1995 was available. MAIN OUTCOME MEASURES: Growth in total NIH awards, traditional research project (R01) awards, R01 awards to clinical and basic science departments, and changes in institutional ranking by NIH awards were compared among schools located in markets with low, medium, and high managed care penetration. RESULTS: Medical schools in all markets had comparable rates of growth in NIH awards from 1986 to 1990. Thereafter, medical schools in markets with high managed care penetration had slower growth in the dollar amounts and numbers of NIH awards compared with schools in markets with low or medium managed care penetration. This slower growth for schools in high managed care markets was associated with loss of share of NIH awards, equal to $98 million in 1995, and lower institutional ranking by NIH awards. Much of this revenue loss can be explained by the slower growth of R01 awards to clinical departments in medical schools in high managed care markets. CONCLUSIONS: These findings provide evidence of an inverse relationship between growth in NIH awards during the past decade and managed care penetration among US medical schools. Whether this association is causal remains to be determined.

Matrix stimulates the proliferation of human corneal endothelial cells in culture.

PURPOSE: Extracellular matrices were tested for their ability to support the adhesion and proliferation of human corneal endothelial cells. METHODS: Human corneal endothelial cells were plated onto tissue culture dishes coated with purified fibronectin or a matrix elaborated by cultured bovine corneal endothelial cells. The presence of human cells in the cultures was confirmed by karyotyping. Cell size at increasing passage number was analyzed, and cellular response to growth factors was assessed using a 96-well microtiter plate assay. RESULTS: When tissue culture dishes were coated with fibronectin, the cells attached to the dish but grew slowly. Human corneal endothelial cells plated onto the matrices elaborated by bovine corneal endothelial cells attached to the culture dish and grew to fill the flask. At confluence, the cells had a hexagonal morphology similar to that seen in vivo. Karyotype analysis showed that the cells were of human and not bovine origin. The time required for senescence in culture was dependent on the age of the donor cornea. The bovine matrices enhanced the proliferative response of human corneal endothelial cell cultures to endothelial cell growth supplement and keratinocyte growth factor. Epidermal growth factor and hepatocyte growth factor stimulated human cell proliferation in a dose-dependent fashion, regardless of the substratum on which the cells were plated. CONCLUSIONS: The use of substratum elaborated by bovine corneal endothelial cells has proved useful in the preparation of human endothelial cell cultures from juvenile and adult donors. The method has been used to establish cultures from more than 50 donors from age 1 day to 76 years.

Rapid size-exclusion chromatography of proteoglycans.

The separation of intact proteoglycans using high-performance liquid chromatography is not trivial because the high molarity denaturing buffers required to maintain proteoglycans in the disaggregated state create back-pressures higher than the limits of many HPLC systems. Until recently, low back-pressure requirements of HPLC size-exclusion columns precluded their use for the separation of intact proteoglycans. In this study we show that rapid size-exclusion chromatography is possible in 8 M urea buffers using a Dionex BioLC system equipped with a Bio-Rad BioSil Sec-400 column. This technique reduced the time required for size-exclusion chromatography of intact proteoglycans from approximately 18 h (Sepharose CL4B) to 25 min and in some cases improved resolution of the sample.

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.