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Biomedical subjects

Michael E Johnson

Publications and source records attributed to Michael E Johnson.

At least 19 recordsLinked to original sources
PubMed2006

Expression profiling identifies altered expression of genes that contribute to the inhibition of transforming growth factor-beta signaling in ovarian cancer.

Ovarian cancer is resistant to the antiproliferative effects of transforming growth factor-beta (TGF-beta); however, the mechanism of this resistance remains unclear. We used oligonucleotide arrays to profile 37 undissected, 68 microdissected advanced-stage, and 14 microdissected early-stage papillary serous cancers to identify signaling pathways involved in ovarian cancer. A total of seven genes involved in TGF-beta signaling were identified that had altered expression >1.5-fold (P < 0.001) in the ovarian cancer specimens compared with normal ovarian surface epithelium. The expression of these genes was coordinately altered: genes that inhibit TGF-beta signaling (DACH1, BMP7, and EVI1) were up-regulated in advanced-stage ovarian cancers and, conversely, genes that enhance TGF-beta signaling (PCAF, TFE3, TGFBRII, and SMAD4) were down-regulated compared with the normal samples. The microarray data for DACH1 and EVI1 were validated using quantitative real-time PCR on 22 microdissected ovarian cancer specimens. The EVI1 gene locus was amplified in 43% of the tumors, and there was a significant correlation (P = 0.029) between gene copy number and EVI1 gene expression. No amplification at the DACH1 locus was found in any of the samples. DACH1 and EVI1 inhibited TGF-beta signaling in immortalized normal ovarian epithelial cells, and a dominant-negative DACH1, DACH1-Delta DS, partially restored signaling in an ovarian cancer cell line resistant to TGF-beta. These results suggest that altered expression of these genes is responsible for disrupted TGF-beta signaling in ovarian cancer and they may be useful as new and novel therapeutic targets for ovarian cancer.

Female↗
PubMed2006

Temporal changes in the tear menisci following a blink.

This study investigated tear meniscus formation after a blink. Multiple images of the inferior and superior menisci, viewed en-face, were sequentially captured using a video-slitlamp that permitted the measurement of tear meniscus height (TMH). In a similar manner, tangentially viewed images were obtained of the inferior meniscus so changes in cross-sectional radius of curvature (TMR) with time could be determined. Additionally, the relationship between inferior TMH and tear volume was quantified by the successive instillation of small aliquots of fluid. In this latter experiment the inflow and outflow of tears were controlled by anaesthetizing the ocular surface and inner nose, and use of punctal plugs. The TMH of both menisci rose after a blink (p<0.01) by a similar amount (p=0.59). This suggests that capillarity is dominant over gravity in tear drainage, however, it may simply reflect that these influences are small compared to the large flow resistances that occur in thin films, hydraulic connectivity between the two menisci, or differences in their anterior-posterior width leading to unequal volume-height relationships. TMR increased with time after a blink (p<0.0001); immediately on eye opening the curvature of the inferior meniscus was approximately circular but this soon became eccentric, with the radius of its upper half exceeding that of its lower half. The growth rate of the two menisci slowed with time and this, based on theoretical considerations, is predicted to have occurred primarily because localized thinning of the tear film adjacent to the menisci markedly raised flow resistance. Inferior TMH increased linearly with the cumulative volume of instilled fluid (r2=0.98), supporting its use in the diagnosis and classification of tear anomalies, although the gradient of this relationship varied between subjects.

Adult↗
PubMed2005

Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods.

We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of alpha and C-terminal end of beta) of erythroid spectrin. The values of the inverse line width parameter (deltaH0(-1)) from a family of Sp alphaI-1-368delta peptides scanning residues 21-30 exhibited a periodicity of approximately 3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C', a helix with a heptad pattern sequence. The residues with low deltaH0(-1) values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the alpha spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the deltaH0(-1) values from a family of spin-labeled Sp betaI-1898-2083delta peptides also exhibited a periodicity of approximately 3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high deltaH0(-1) values and a hydrophobic side with low deltaH0(-1) values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in alphabeta spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies.

Cyclic N-Oxides↗
PubMed2005

Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors.

Design, synthesis, and biological evaluation of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease (SARS-3CLpro) inhibitors for severe acute respiratory syndrome coronavirus (SARS-CoV) are described. These inhibitors exhibited antiviral activity against SARS-CoV in infected cells in the micromolar range. An X-ray crystal structure of our lead inhibitor (4) bound to SARS-3CLpro provided important drug-design templates for the design of small-molecule inhibitors.

Animals↗
PubMed2005

Effectiveness of sodium hyaluronate eyedrops in the treatment of dry eye.

BACKGROUND: Dry eye is a common condition, affecting approximately 10-20% of the adult population. Artificial tears are often effective in relieving symptoms in mild and moderate dry eye by replenishing deficient tear volume. Sodium hyaluronate has been proposed as a component in artificial tears, due to its viscoelastic rheology. This paper reports on a study carried out to assess the efficacy of two recently developed eyedrops containing 0.1% and 0.3% sodium hyaluronate (SH) in the treatment of moderate dry eye. METHODS: Thirteen subjects were recruited with moderate dry eye. Forty microlitres of 0.1% SH, 0.3% SH, or 0.9% saline were instilled in both eyes, and the subjects' symptom intensity and non-invasive break-up time (NIBUT) were measured at 5, 15, 30, 45, and 60 min, and then hourly, until 6 h after drop instillation. This was repeated twice following an interval of 7(+/-1) days, but with a different treatment so that at the end of the final visit each subject had trialled all products. Drop allocation was randomized and double-masked. RESULTS: Both symptoms and NIBUT improved with all treatments. These changes were of a larger magnitude and longer duration with the SH containing eyedrops than with saline. SH of 0.3% tended to perform better than 0.1% SH and achieved statistical significance (P=0.04) for NIBUT when considered over the whole 6-h study period. CONCLUSIONS: Sodium hyaluronate of 0.1% and 0.3% reduces symptoms of ocular irritation and lengthens NIBUT in subjects with moderate dry eye more effectively than saline, in terms of peak effect and duration of action.

Administration, Topical↗
PubMed2005

The Effect of instilled fluorescein solution volume on the values and repeatability of TBUT measurements.

PURPOSE: To investigate how the volume of instilled fluorescein solution alters invasive tear breakup time (TBUT) and the standard deviation (SD) of multiple recordings, and to determine the volume of fluid supplied by traditional fluorescein-impregnated strips (FS) and a micropipette calibrated to dispense 1 microL. METHODS: TBUT was measured 3 times in the right eyes of 46 subjects following the instillation of 1, 2.7, and 7.4 microL of fluorescein solution by micropipette and the use of a wetted FS. The volume of fluid delivered to an eye by FS and a micropipette set for 1 microL was estimated by weighing each before and after use. RESULTS: The volume of fluorescein solution instilled before measurement influenced TBUT (P < 0.001). Increasing the delivered volume from 1 to 2.7 microL lengthened TBUT (P < 0.001), but an additional increase in volume to 7.4 microL was not associated with a further change in TBUT (P = 0.50). Differences between TBUT values measured after using FS and a micropipette calibrated for 1 microL were not significant (P = 0.95). No significant differences were found in the repeatability of TBUT recordings between the instillation techniques used in this study (P = 0.18), although measurement variability increased with mean TBUT for all techniques. With the FS instillation technique described herein, the average volume of fluorescein solution they supplied was less than that delivered by a micropipette set for 1 microL (P = 0.017), but the range of volumes transferred by FS was greater. CONCLUSIONS: Values of TBUT are dependent on the volume of fluorescein solution instilled before measurement; thus, attempts should be made to standardize this variable. Accurate estimation of TBUT requires more recordings to be averaged than are normally taken, unless breakup time is very short.

Adolescent↗
PubMed2004

Design, synthesis, and evaluation of oxyanion-hole selective inhibitor substituents for the S1 subsite of factor Xa.

We have designed, synthesized, and evaluated the factor Xa inhibitory activities of p-amidinophenyl-sulfones, amines, and alcohols intended to take advantage of the polarity and hydrogen-bonding potential of the oxyanion hole region of the S1 specificity pocket. We demonstrate that placement of an anionic group within the oxyanion hole region of the catalytic site substantially enhances activity, with small flexible groups favored over bulkier ones. Ab initio pKa calculations suggest that the hydroxyl substituent frequently used for benzamidine moieties may be ionized to form an anionic group, consistent with the general trend. One nonamidine based substituent also shows promising activity.

Alcohols↗
PubMed2004

Changes in the tear film and ocular surface from dry eye syndrome.

Dry eye syndrome (DES) refers to a spectrum of ocular surface diseases with diverse and frequently multiple aetiologies. The common feature of the various manifestations of DES is an abnormal tear film. Tear film abnormalities associated with DES are tear deficiency, owing to insufficient supply or excessive loss, and anomalous tear composition. These categorizations are artificial, as in reality both often coexist. DES disrupts the homeostasis of the tear film with its adjacent structures, and adversely affects its ability to perform essential functions such as supporting the ocular surface epithelium and preventing microbial invasion. In addition, whatever the initial trigger, moderate and severe DES is characterized by ocular surface inflammation, which in turn becomes the cause and consequence of cell damage, creating a self-perpetuating cycle of deterioration. Progress has been made in our understanding of the aetiology and pathogenesis of DES, and these advances have encouraged a proliferation of therapeutic options. This article aims to amalgamate prevailing ideas of DES development, and to assist in that, relevant aspects of the structure, function, and production of the tear film are reviewed. Additionally, a synopsis of therapeutic strategies for DES is presented, detailing treatments currently available, and those in development.

Animals↗
PubMed2004

Mitochondrial injury and caspase activation by the local anesthetic lidocaine.

BACKGROUND: Lidocaine, a local anesthetic, can be neurotoxic. However, the cellular mechanisms of its neurotoxicity at concentrations encountered during spinal anesthesia remain unclear. METHODS: The authors examined the mechanisms of lidocaine neurotoxicity in the ND7 cell line derived from rat dorsal root ganglion. Individual neurons were assayed by flow cytometry or microscopy using fluorescent probes of plasma membrane integrity, mitochondrial membrane potential, caspase activity, phospholipid membrane asymmetry, and mitochondrial cytochrome c release. RESULTS: In the ND7 cell line, lidocaine at 185 mm x 10 min to 2.3 mm x 24 h caused necrosis or late apoptosis. Equimolar Tris buffer and equipotent tetrodotoxin controls were not toxic, indicating that neither osmotic nor Na-blocking effects explain lidocaine neurotoxicity. The earliest manifestation of lidocaine neurotoxicity was complete loss of mitochondrial membrane potential within 5 min after exposure to lidocaine at a concentration of 19 mm or greater. Consistent with these data, 37 mm lidocaine (1%) induced release of mitochondrial cytochrome c into the cytoplasm, as well as plasma membrane blebbing, loss of phosphatidylserine membrane asymmetry, and caspase activation, with release of mitochondrial cytochrome c to the cytoplasm within 2 h. Treatment with z-VAD-fmk, a specific inhibitor of caspases, prevented caspase activation and delayed but did not prevent neuronal death, but did not inhibit the other indicators of apoptosis. CONCLUSIONS: Collectively, these data indicate that lidocaine neurotoxicity involves mitochondrial dysfunction with activation of apoptotic pathways.

Anesthetics, Local↗
PubMed2003

Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering.

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.

Amino Acid Sequence↗
PubMed2003

Design and synthesis of highly constrained factor Xa inhibitors: amidine-substituted bis(benzoyl)--diazepan-2-ones and bis(benzylidene)-bis(gem-dimethyl)cycloketones.

Two conformationally constrained templates have been designed to provide selective inhibitors of the coagulation cascade serine protease, Factor Xa (FXa). The most active inhibitor, 2,7-bis[(Z)-p-amidinobenzylidene)]-3,3,6,6-tetramethylcycloheptanone, exhibits a K(i) of 42 nM against FXa, with strong selectivity against thrombin (1000-fold), trypsin (300-fold) and plasmin (900-fold). With only two freely rotatable bonds, molecular modeling suggests that one amidine group is positioned into the S1 pocket, forming hydrogen bonds with the side chain of Asp189, similar to other amidine-based inhibitors, with the second benzamidine positioned into the S4 pocket in a position to form strong cation-pi bonding with the S4 aryl cage. We suggest that this interaction plays an important role in the specificity of these inhibitors against other serine proteases.

Antithrombin III↗
PubMed2003

Solution structural studies on human erythrocyte alpha-spectrin tetramerization site.

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid alpha-spectrin. The first 20 residues preceding the first helix (helix C') are in a disordered conformation. The subsequent three helices (helices A1, B1, and C1) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C', the partial domain involved in alpha- and beta-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C' is connected to helix A1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of alpha- and beta-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying alpha- and beta-subunit self-association affinities in their formation of the various spectrin tetramers.

Animals↗
PubMed2003

Regioselective covalent modification of hemoglobin in search of antisickling agents.

Although the molecular defect in sickle hemoglobin that produces sickle cell disease has been known for decades, there is still no effective drug treatment that acts on hemoglobin itself. In this work, a series of diversely substituted isothiocyanates (R-NCS) were examined for their regioselective reaction with hemoglobin in an attempt to alter the solubility properties of sickle hemoglobin. Electrospray mass spectrometry, molecular modeling, X-ray crystallography, and conventional protein chemistry were used to study this regioselectivity and the resulting increase in solubility of the modified hemoglobin. Depending on the attached R-group, the isothiocyanates were found to react either with the Cysbeta93 or the N-terminal amine of the alpha-chain. One of the most effective compounds in the series, 2-(N,N-dimethylamino)ethyl isothiocyanate, selectively reacts with the thiol of Cysbeta93 which, in conjunction with the cationic group, was seen to perturb the local hemoglobin structure. This modified HbS shows an approximately 30% increase in solubility for the fully deoxygenated state, along with a significant increase in oxygen affinity. This compound and a related analogue appear to readily traverse the erythrocyte membrane. A discussion of the relation of these structural changes to inhibition of gelation is presented. The dual activities of increasing HbS oxygen affinity and directly inhibiting deoxy HbS polymerization, in conjunction with facile membrane traversal, suggest that these cationic isothiocyanates show substantial promise as lead compounds for development of therapeutic agents for sickle cell disease.

Anemia, Sickle Cell↗
PubMed2002

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin.

Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the N-terminal region of alpha-spectrin, with the Arg28 position considered to be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Sp alpha 1-156, consisting of the first 156 residues in the N-terminal region of alpha-spectrin (alpha N). The association of these alpha-spectrin peptides that have single amino acid replacements with a beta-spectrin model peptide, consisting of the C-terminal region of beta-spectrin (beta C), was determined, and structural changes due to amino acid replacements were monitored by nuclear magnetic resonance (NMR). We found evidence for similar and very localized structural changes in Sp alpha 1-156Arg45Thr and Sp alpha 1-156Arg45Ser, although these 2 mutant peptides associated with beta-spectrin peptide with significantly differing affinities. The Sp alpha 1-156Arg28Ser peptide showed an affinity for the beta-spectrin peptide comparable to that of Sp alpha 1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that both Arg45 replacements induce only minor structural perturbations in the first helix of Sp alpha 1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results also indicate that the mechanism for reduced spectrin tetramerization is through mutation-induced changes in molecular recognition at the alpha beta-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.

Amino Acid Substitution↗
PubMed2002

An oxyanion-hole selective serine protease inhibitor in complex with trypsin.

p-amidinophenylmethylphosphinic acid (AMPA) was designed, synthesized and crystallized in complex with trypsin to study interactions with the oxyanion hole at the S1 site. In comparison to benzamidine, AMPA shows improved activity, which the crystal structure demonstrates to result from hydrogen bonds between the negatively charged phosphinic acid group and the catalytic residues at the oxyanion hole.

Anions↗