Quantitative assessment of central and limbal epithelium after long-term wear of soft contact lenses and in patients with dry eyes: a pilot study.

PurposeAnalysis of microstructural alterations of corneal and limbal epithelial cells in healthy human corneas and in other ocular conditions.Patients and methodsUnilateral eyes of three groups of subjects include healthy volunteers (G1, n=5), contact lens wearers (G2, n=5), and patients with dry eyes (G3, n=5) were studied. Imaging of basal (BC) and intermediate (IC) epithelial cells from central cornea (CC), corneal limbus (CL) and scleral limbus (SL) was obtained by in vivo confocal microscopy (IVCM). An appropriate image analysis algorithm was used to quantify morphometric parameters including mean cell area, compactness, solidity, major and minor diameter, and maximum boundary distance.ResultsThe morphometric parameters of BC and IC demonstrated no significant differences (P>0.05) between groups. Comparison between three corneal locations (CC, CL, and SL) within the groups showed significant differences (P<0.05) with mean values of cell area, compactness, solidity, and major and minor diameter of BC that increase from CC to limbus. The BC were round and regular in the central cornea (P<0.05) compared with CL and SL.ConclusionsIVCM enables high-quality confocal images from central corneal and limbal epithelium. This quantitative study demonstrated morphological differences in the basal and intermediate epithelium between limbus and central cornea, and found no differences between contact lens wearers, dry eyes, and normal subjects.

Rational design and diversity-oriented synthesis of peptoid-based selective HDAC6 inhibitors.

A mini library of HDAC inhibitors with peptoid-based cap groups was synthesized using an efficient multicomponent approach. Four compounds were identified as potent HDAC6 inhibitors with a selectivity over other HDAC isoforms. The most potent HDAC6 inhibitor revealed remarkable chemosensitizing properties and completely reverted the cisplatin resistance in Cal27 CisR cells.

Influence of coronary artery disease on outcomes after liver transplantation.

Patients with coronary artery disease (CAD) who undergo liver transplantation (OLT) have been previously identified as a high-risk group. Since that identification, the management of CAD has undergone significant changes as has the cardiovascular screening and selection of patients for OLT. We retrospectively identified 42 patients with known CAD who underwent OLT to compare outcomes with a control group of 42 patients without CAD who were matched for gender, age, and primary liver disease. Mortality rates were higher in the CAD than the control group at 1 year (5 vs 1) and 3 years (11 vs 3; P < .05) although lower than previously reported (at 3 years, 26% vs 50%). New cardiovascular morbidity was also more frequent among the CAD than control group at 1 year (11 vs 3; P < .05) and 3 years (16 vs 4; P < .05). Although outcomes for patients with CAD undergoing OLT are improved from historical levels, they are still worse than those in patients without CAD despite current management and selection strategies.

Time course of learning to produce maximum cycling power.

The purpose of this investigation was to determine the time course and magnitude of learning effects associated with repeated maximum cycling power tests and to determine if cycle-trained men exhibit different learning effects than active men who are not cycle-trained. Cycle-trained (N = 13) and active men (N = 35) performed short maximal cycling bouts 4 times per day for 4 consecutive days. Inertial load cycle ergometry was used to measure maximum power and pedaling rate at maximum power. Maximum power of the cycle-trained men did not differ across days or bouts. Maximum power of the active men increased 7 % within the first day and 7 % from the mean of day one to day three. Pedaling rate at maximum power did not differ across days or bouts in either the cycle-trained or active men. These results demonstrate that valid and reliable results for maximum cycling power can be obtained from cycle-trained men in a single day, whereas active men require at least 2 days of practice in order to produce valid and reliable values.

Superficial femoral popliteal vein: An anatomic study.

OBJECTIVE: The superficial femoral popliteal vein (SFPV) has been used as an alternative conduit for both arterial and venous reconstructive surgery. Its popularity continues to grow, despite concern about the potential for venous morbidity after harvest. The purpose of this study was to determine an anatomic "safe" length of SFPV for harvest, assuming that the preservation of at least one valve and one significant collateral vein in the remaining popliteal vein (PV) segment can minimize venous morbidity. METHODS: Forty-four SFPVs were harvested from 39 cadaveric specimens. The length of both the superficial femoral vein (SFV) and PV was measured, and the number and location of valves and significant side branches (more than 2 mm in diameter) of the PV were measured. The Student two-tailed t test was used as a means of comparing vein lengths between the sexes. Correlation coefficients were determined for the effect of patient height on vein length, stratified by means of sex. RESULTS: Vein length (SFV mean, 24.4 +/- 4 cm; PV mean, 18.8 +/- 4 cm) varied with sex (male SFV mean, 28.1 +/- 5 cm; male PV mean, 21. 5 +/- 3 cm; female SFV mean, 22.6 +/- 4 cm; female PV mean, 18.4 +/- 3 cm; P =.01). Valve number (mean, 1.8 +/- 0.5) and location and collateral vein number (mean, 5 +/- 1.8) and location were variable and independent of height or sex. CONCLUSION: An anatomic "safe" length of SFPV for harvest to minimize venous morbidity would include all the SFV and 12 cm of PV in 95% of women and 15 cm of PV in 95% of men. We found that the male sex was a significant determinant for a longer safe length of vein that can be harvested.

The anion transporter and a 28 kDa protein are selectively photolabeled by p-azidobenzylphlorizin under conditions that alter RBC morphology, flexibility, and volume.

Tritiated p-azidobenzylphlorizin (p-AzBPhz) was photoactivated in the presence of red blood cells under conditions previously found to alter morphology, flexibility and volume. When less than 0.25 million molecules were added per cell, only a 28 kDa peptide was photolabeled: at 1-2 million molecules added, band 3 also incorporated significant radioactivity. When using leaky ghosts, other proteins became labeled, including those limited to the cytoplasm. Protein N-deglycosylation caused a shift of radiolabeled band 3 to higher Rf values on SDS-PAGE gels but not for the 28 kDa band; the latter was, however, susceptible to enzymatic digestion by NANase (N-acetylneuraminidase) III but not by NANase II. Inhibition of photoincorporation into both receptors by unlabeled p-AzBPhz was dose-dependent. Mercuric chloride and p-CMBS selectively blocked 28 kDa peptide labeling. DIDS partially blocked at band 3; after 15% inhibition, greater DIDS concentrations caused increased incorporation into the 28 kDa peptide. These results, and a temperature-dependent labeling pattern, suggest that: (i) cellular changes occur when p-AzBPhz binds to the exofacial sides of the anion transporter and 28 kDa peptide; (ii) these proteins may be physically associated in the native membrane; (iii) they mediate ligand-induced changes in morphology, flexibility, and volume.

Surface localization, regulation, and biologic properties of the 96-kDa alcohol/aldehyde dehydrogenase (EhADH2) of pathogenic Entamoeba histolytica.

The 96-kDa surface antigen of Entamoeba histolytica was demonstrated through extensive immunologic evaluation with monoclonal and monospecific antibodies to be identical to or an isoform of the amebic alcohol/aldehyde dehydrogenase (EhADH2). EhADH2 was secreted, excreted, or shed into the culture medium in quantities commensurate with amebic growth when studied in a novel culture system. Of importance, using RNase protection assays, specific mRNA coding for the EhADH2 gene product(s) was up-regulated by treatment of viable trophozoites with the enzyme substrate ethanol. These data provide insight into the biology of this enzyme and its regulation by appropriate stressors.

Band 3 antagonists, p-azidobenzylphlorizin and DIDS, mediate erythrocyte shape and flexibility changes as characterized by digital image morphometry and microfiltration.

Two nonpenetrating membrane probes, p-azidobenzylphlorizin (p-AzBPhz) and 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), have been shown in earlier studies to induce dose-dependent changes in red blood cell (RBC) shape and volume at the same low concentrations that inhibit anion transport. In the present work, these ligand-induced morphology and rheology changes were studied using video digital image morphometry (VDIM) and microfiltration techniques. The results of these experiments corroborate our earlier investigation. RBCs were filmed using a Nomarski optics microscope with video camera attachment and cell size and shape changes were computer analyzed using VDIM. Low microM p-AzBPhz or DIDS levels caused collapse of the cell's biconcave structure and cell flattening occurred within 1-2 sec after drug exposure. Higher doses of either agent converted cells to a new steady-state in which a concurrent limited increase in erythrocyte volume and blunt membrane protrusions were produced. These changes were reversed in less than 2 sec by washing the drug from the membrane. Both ligands increased the deformability of RBCs in a dose-dependent manner as determined by filtration through Nuclepore polycarbonate filters (3 microns pore diameter). The improvement in deformability of drug-treated sickle cells was much more dramatic than for normal cells at low p-AzBPhz concentrations. These results support our earlier conclusions that the ligands, through a common interaction with band 3, induce volume-associated cytoskeletal alterations which lead to changes in morphology and flexibility.

Morphology and volume alterations of human erythrocytes caused by the anion transporter inhibitors, DIDS and p-azidobenzylphlorizin.

p-Azidobenzylphlorizin (p-AzBPhz) is a potential photoaffinity labeling agent for the anion and glucose transporters in human RBCs. In the absence of light and at the same low concentrations which block these transport processes (only 1-2 million molecules bound/cell), this impermeable membrane probe produces rapid morphological and volume alterations. This high-affinity activity, called phase 1, can be rapidly and completely reversed by simply diluting the azide-treated cell suspension. Phase 2 effects, including formation of cells with multiple, long spicules (stage 3/4 echinocytes), followed by an influx of salt and water with eventual lysis, occur at two log units higher concentration by a different mechanism, probably by intercalating into and selectively expanding the outer lipid monolayer. Light scattering, electronic cell sizing, microhematocrit measurements and scanning electron microscopy have been employed to compare the effects of the azide and the anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), on red cells. DIDS produced only those changes analogous to the azide's low dose phase 1 action; cells swell, lose the ability to scatter 800 nm light and undergo a limited shape change (comparable to stage 1 echinocytosis). The mechanism by which the two ligands perturb the membrane is additive, suggesting that a Band 3-mediated transmembrane signaling is involved which leads to altered cytoskeleton dynamics.

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.

Heat shock-induced axenic growth of Bdellovibrio bacteriovorus.

The bdellovibrios are obligately predatory bacteria that attack other gram-negative bacteria. They grow only in the periplasmic space of prey unless they mutate to forms that can grow axenically. A culture medium that promoted enhanced growth of prey-independent bdellovibrios was developed. The ability of this medium to support the growth of prey-dependent bdellovibrios was tested under transcription-altering conditions. This approach tested the hypothesis that the inability to grow prey-dependent bdellovibrios in artificial media was rooted in both nutritional and transcriptional signal deficiencies. It was assumed that nutritional deficiencies had been resolved and that empirically applied artificial signals may evoke the expression of genes required for axenic growth of bdellovibrios. Prey-dependent bdellovibrios could be grown in PPYE medium (0.1% proteose peptone 3 and 0.03% Bacto yeast extract adjusted to pH 7.0 and supplemented with 3 mM MgCl2 and 2 mM CaCl2 after autoclaving) after heat shock, and subsequent rounds of growth occurred after additional heat shocks. Heat shock may have generated or simulated signals normally derived from prey.

Structural analysis and demonstration of the 29 kDa antigen of pathogenic Entamoeba histolytica as the major accessible free thiol-containing surface protein.

The 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine-rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non-pathogenic clinical isolates. To determine the structure and biochemical attributes of this protein, a repertoire of immunological techniques using monoclonal antibodies, and radiolabelling were employed. We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high-molecular-mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol-containing surface protein of E. histolytica. The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.

Research considerations. Geropsychiatry unit evaluation.

The specialized psychiatric units for geriatric patients are believed to be effective, yet no studies could be found documenting this. A method of evaluating the effectiveness of a geropsychiatric program is to measure changes in the cognitive and functional status of its patients. This study supports the hypothesis that there would be an improvement in the geropsychiatric patients' cognitive and functional assessment scores by discharge and after hospitalization.

An Accurate Quantum Monte Carlo Calculation of the Barrier Height for the Reaction H + H2 rarr H2 + H.

An improved quantum Monte Carlo method has been used to calculate the classical barrier height for the hydrogen exchange reaction H + H(2) --> H(2) + H with accuracies greater than previously attained. The method is exact in that, except for the easily estimated Monte Carlo statistical or sampling error, it requires no mathematical approximations or physical approximations beyond those of the Schrödinger equation. The minimum in the barrier, occurring for the collinear nuclear configuration with the protons separated by 1.757 bohrs, was found to be 9.61 +/- 0.01 kilocalories per mole above H + H(2).

Acquisition of apparently intact and unmodified lipopolysaccharides from Escherichia coli by Bdellovibrio bacteriovorus.

The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS). Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios. The relocalized trimers were found associated with the same LPS originally bound to them in the E. coli. The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios. This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey. One or two other bands were identical in migration to the LPS of prey-independent mutants of B. bacteriovorus and represented bdellovibrio-synthesized LPS. The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS. The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS. Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS.

Molecular and cellular characterization of the 29-kilodalton peripheral membrane protein of Entamoeba histolytica: differentiation between pathogenic and nonpathogenic isolates.

To further characterize the 29-kDa surface antigen of Entamoeba histolytica, we analyzed the complete nucleotide sequence and compared the immunoreactivity of this antigen in pathogenic and nonpathogenic strains. Five cDNA clones (one 1.0-kb full-length clone, designated p13, and four partial-length clones) encoding the antigen were analyzed for allelic variation. Comparison of the nucleotide sequences revealed several single-nucleotide substitutions in all five cDNAs, two of which resulted in amino acid differences. Localization of the antigen to the amebic surface in a previous report (B. E. Torian, B. M. Flores, V. L. Stroeher, F. S. Hagen, and W. E. Stamm, Proc. Natl. Acad. Sci. USA 87:6358-6362, 1990) was corroborated by transmission electron microscopy showing colloidal gold particles on the surface of the trophozoites. Computer analysis of the deduced amino acid sequence predicted that the protein encoded by p13 was a hydrophilic peripheral membrane protein, and these data were confirmed by a Triton X-114 membrane extraction showing the presence of the 29-kDa antigen primarily in the aqueous phase of the detergent partition. Monoclonal antibodies to a fusion peptide differentiated between pathogenic and nonpathogenic clinical strains of E. histolytica in immunoblots. Although no immunoreactive epitopes were detected on nonpathogenic strains, Northern (RNA) analysis and DNA-DNA hybridization with a 700-bp cDNA probe demonstrated that mRNA and the gene encoding the 29-kDa surface antigen were present in both pathogenic and nonpathogenic clinical isolates.

Detoxification of endotoxin-contaminated titanium and hydroxyapatite-coated surfaces utilizing various chemotherapeutic and mechanical modalities.

The surgical repair of the ailing implant may be complicated by the surface effects of pathogenic bacteria and their products. This study evaluated the ability of various chemotherapeutic modalities to detoxify endotoxin-contaminated titanium alloy and hydroxyapatite-coated test strips. Grit-blasted titanium alloy and hydroxyapatite-coated test strips were contaminated with purified outer membranes of Escherichia coli labeled with radioactive 14C. The titanium alloy strips were treated with citric acid, stannous fluoride, tetracycline HCl, chlorhexidine gluconate, hydrogen peroxide, chloramine T, sterile water, a plastic sonic scaler tip, and an air-powder abrasive unit. Hydroxyapatite-coated strips were treated with chloramine T, citric acid, or burnished with sterile water on cotton pellets. Residual lipopolysaccharide levels were measured by liquid scintillation spectrometry. The air-powder abrasive unit removed significantly greater amounts of lipopolysaccharide than all other treatment modalities on titanium samples (P < 0.05). A 60-second burnish with sterile water was able to remove significant amounts of lipopolysaccharide when compared with untreated controls (P < 0.05). Citric acid was superior in the removal of lipopolysaccharide from hydroxyapatite-coated surfaces when compared with the controls or chloramine T (P < 0.01). Detoxification of an implant infected surface may be beneficial when surgical repair of the ailing implant is indicated.

Fibroblastic growth and attachment on hydroxyapatite-coated titanium surfaces following the use of various detoxification modalities. Part II: Contaminated hydroxyapatite.

This study evaluated the ability of various chemotherapeutic and mechanical modalities to detoxify endotoxin-contaminated hydroxyapatite-coated dental implant surfaces as determined by the early attachment and growth of human gingival fibroblasts. Hydroxyapatite-coated test strips were contaminated with purified outer membranes of Escherichia coli and treated with citric acid, hydrogen peroxide, stannous fluoride, chlorhexidine gluconate, tetracycline HCl, polymyxin B, a plastic sonic scaler tip, or left untreated (contaminated and sterile controls). Human gingival fibroblasts were then seeded onto the test strips and incubated for 48 hours. The citric acid-treated strips showed greater cell growth than the other treatments. The plastic sonic scaler tip and the polymyxin B-treated samples exhibited greater cell coverage than the sterile control specimens. The use of citric acid and/or a modified plastic sonic scaler tip may be a valuable adjunct when surgical repair of an ailing hydroxyapatite-coated dental implant is contemplated.

Generation of substructure identification rules using feature-combinations from tandem mass spectra.

Software to interpret tandem mass spectra, entitled Method for Analyzing Patterns in Spectra (MAPS), has been developed to provide substructure information for an automated compound identification system, This software consists of several program modules which manipulate databases of tandem mass spectra and substructure information, generate substructure identification rules, and apply these rules to the tandem mass spectra of unknown compounds to identify components of their structure. The MAPS rule generation program has been modified to generate rules based on specific combinations of spectral features that occur concertedly. False positives are drastically reduced by searching for "feature-combinations" that have 100% uniqueness with respect to a reference database of compounds. Recall is increased by the determination of multiple feature-combinations indicative of the presence of a given substructure. Strategies were developed in the algorithm for the discovery of feature-combinations that avoid the computation "explosion" that occurs when working with a large number of spectral features. The rules developed have the form: "IF feature-eombination a (FC a) or FC b,..., or FC x, THEN substructure SSn is present."

Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides.

The associations of both rough and smooth lipopolysaccharides (LPS) with the OmpF porin of Escherichia coli K-12 were examined in galE strains deleted for ompC. Transformation with pSS37 and growth with galactose conferred the ability to assemble a Shigella dysenteriae O antigen onto the core oligosaccharide of E. coli K-12 LPS. The association of LPS with OmpF trimers was assessed by staining, autoradiography of LPS specifically labeled with [1-14C]galactose, and Western immunoblotting with a monoclonal antibody specific for OmpF trimers. These techniques revealed that the migration distances and multiple banding patterns of OmpF porin trimers in sodium dodecyl sulfate-polyacrylamide gels were dictated by the chemotype of associated LPS. Expression of smooth LPS caused almost all of the trimeric OmpF to run in gels with a slower mobility than trimers from rough strains. The LPS associated with trimers from a smooth strain differed from the bulk-phase LPS by consisting almost exclusively of molecules with O antigen.