Biomedical subjects

M E Lane

Publications and source records attributed to M E Lane.

At least 19 recordsLinked to original sources

PubMed2006

Use of a molecular form technique for the penetration of supersaturated solutions of salicylic acid across silicone membranes and human skin in vitro.

Permeation enhancement of salicylic acid (SA) from supersaturated solutions formed using a 'molecular form' technique was investigated. In a conventional cosolvent technique, two solvents are used, one in which the drug is considerably more soluble than the other. Propylene glycol and water have been predominantly used as cosolvents to create supersaturation in skin permeation enhancement. In this paper, we report the use of buffer solutions with different pHs as media for producing different molecular forms. Supersaturated solutions were prepared using pH 8:pH 2 (80:20 v/v), which gave a nominal pH when mixed of around 5. Model silicone membranes and human skin were used. Hydroxypropyl methyl cellulose (HPMC) was employed to stabilise the supersaturated states. Stability data showed that while the SA supersaturated solutions without HPMC crystallised between 15 min and 46 h depending on the degree of supersaturation, the solutions with HPMC were stable for more than 2 months. The flux of SA increased with the degree of saturation for solutions prepared in a 80:20 buffer pH 8/buffer pH 2 mixture. Although the fluxes of SA with and without HPMC were similar both through silicone membrane and human skin, HPMC was found to be effective in increasing the stability of supersaturated solutions of SA.

Algorithms↗

PubMed2006

Preparation and release of model drugs from thermally sensitive poly(N-isopropylacrylamide) based macrospheres.

Emulsion polymerization was employed to prepare poly(N-isopropylacrylamide) hydrogel spheres, which exhibited an LCST of 32 degrees C. The hydrogels were loaded with model drugs (benzoic acid (BA), sodium benzoate and diltiazem HCl (DHCl)) and release investigated at 25 degrees C and 37 degrees C. The temperature at which gel formation occurred was vital for successful hydrogel preparation, macrosphere formation not occurring when the temperature was close to the LCST. Sphere size increased on decreasing the stirring rate and on slowing the rate of addition of the aqueous phase. Pulsatile delivery was investigated using BA and DHCl. For both compounds a pulse was observed with a change in temperature. Pulsed release of the smaller model drug of lowest solubility, BA, was more successful. Drug release from hydrogel spheres was, therefore, found to be dependent on the physicochemical properties of the drugs, with pulsatile release of low molecular weight compounds, by temperature cycling, difficult to control.

Acrylic Resins↗

PubMed2001

A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells.

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents.

Adenocarcinoma↗

PubMed2001

Isolation and characterization of posteriorly restricted genes in the zebrafish gastrula.

In order to understand anteroposterior axis formation in vertebrates, we have used subtractive hybridization to clone genes expressed posteriorly in the zebrafish gastrula-stage embryo. Here we report the initial characterization of eight clones isolated from this screen. We find that all eight genes are expressed in posteriorly restricted domains, suggesting that they are involved in regulating posterior development during zebrafish embryogenesis.

Animals↗

PubMed2000

Downregulation of cyclin D1 alters cdk 4- and cdk 2-specific phosphorylation of retinoblastoma protein.

Progression of cells through the G1 phase of the cell cycle requires the assembly and activation of specific cyclin:cyclin-dependent kinase (cdk) complexes in a tightly regulated, sequential fashion. To more clearly define the temporal events leading to the G1/S transition, sequential changes in the expression of cyclin E and cdks 2, 4, and 6, as well as the phosphorylation of the retinoblastoma protein (pRb), were assayed in RA28 cells, a variant of human colon cancer RKO cells which were modified by transfection of an ecdysone-inducible antisense (AS) CD1 expression system. Induction of cyclin D1 antisense mRNA by the ecdysteroid, ponasterone A, resulted in a 55% decrease in cyclin D1 mRNA and a 58% decrease in CD1 protein levels. There was a 2.4-fold decrease in the ratio of hyperphosphorylated pRb (ppRb) to hypophosphorylated pRb, as well as a 60-75% decrease in cdk 2- and cdk 4-specific phosphorylated pRb proteins. Of interest, cyclin E-dependent phosphorylation (cdk2) decreased 2.5-fold at 3 h despite only a 30% decrease in cyclin E protein level. Levels of cdk 2, cdk 4, and cdk 6 decreased 40-70%, while levels of cyclin A and B were unaffected by induction of CD1 antisense. Induction of a CD1 antisense gene in a human colon cancer cell line resulted in rapid, concomitant changes in CD1 mRNA and protein, cyclin E, cdk2, cdk4, and cdk6, as well as the ratio of ppRb to pRb. In this system, growth regulatory events are tightly regulated and the perturbed expression of a single protein, CD1, rapidly alters expression of multiple regulatory proteins involved in the G1/S transition phase of cell cycle progression.

Blotting, Western↗

PubMed2000

A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2.

In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, we have screened in Drosophila melanogaster for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that we have identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2(Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed us to address the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19). The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, our results demonstrate that the equivalent sites in Cdk2 play no essential role.

Animals↗

PubMed1999

Analysis of zebrafish development using explant culture assays.

Two fundamental questions of developmental biology are when cells become committed to a certain lineage and what cell interactions are involved in establishing this commitment. These questions can be answered using explant or transplant assays. We have developed explant assays to study zebrafish development. These assays involve isolating by microdissection small regions of the embryo at specific times during development, and determining their fate after culture in isolation (lineage commitment assays) or after exposure to a putative inducing tissue (induction assays). In our laboratory, we have used these assays to address questions of neural development, including those of commitment to anterior and posterior neural lineages, and the signals involved in making these decisions. This chapter contains detailed guidelines for designing explant assays. These include suggestions for the isolation and successful culture of explants, and descriptions of the methods used to assay the final fate of explants after culture. Step-by-step protocols are given for the isolation of specific explants that can be used in specification and induction assays. The application of this technique is illustrated with descriptions of experiments. Explant assays will continue to generate key information concerning the establishment of lineage commitment of many embryonic tissues and will provide extremely valuable for analysis of new genes identified molecularly and in mutant screens.

Animals↗

PubMed1997

roughex down-regulates G2 cyclins in G1.

Cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. In roughex mutants, cells accumulate Cyclin A protein in early G1 and progress into S phase precociously. When Roughex is overexpressed in S/G2 cells, Cyclin A is mislocalized to the nucleus and degraded, preventing mitosis. Whereas Roughex inhibits Cyclin A accumulation, Cyclin E down-regulates Roughex protein in vivo. Roughex binds to Cyclin E and is a substrate for a Cyclin E-Cdk complex in vitro. These data argue that Roughex inhibits Cyclin A accumulation in early G1 by targeting Cyclin A for destruction. In late G1, Roughex is destabilized in a Cyclin E-dependent process, releasing Cyclin A for its role in S/G2.

Animals↗

PubMed1997

Cell cycle regulators in Drosophila: downstream and part of developmental decisions.

The molecular identification of an evolutionarily conserved set of cell cycle regulators in yeast, Xenopus egg extracts, and vertebrate cell culture has opened up a new perspective for understanding the mechanisms that regulate cell proliferation during metazoan development. Now we can study how the crucial regulators of eukaryotic cell cycle progression, the various cyclin/cdk complexes (for a recent review see Nigg (1995) BioEssays 17, 471-480), are turned on or off during development. In Drosophila, this analysis is most advanced, in particular in the case of the rather rigidly programmed embryonic cell cycles that generate the cells of the larvae. In addition, this analysis has revealed how the mitotic cycle is transformed into an endocycle which allows the extensive growth of larvae and oocytes. In contrast, we know little about cyclin/cdk regulation during the imaginal proliferation that generates the cells of the adult. Nevertheless, we will also consider this second developmental phase with its conspicuous regulative character, because it will be of great interest for the analysis of the molecular mechanisms that integrate growth and proliferation during development.

Animals↗

PubMed1996

Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development.

Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. We show that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest.

Amino Acid Sequence↗

PubMed1996

The relationship between rat intestinal permeability and hydrophilic probe size.

PURPOSE: The relationship between rat intestinal permeability (Papp) of a range of hydrophilic probe molecules and probe geometry was examined. METHODS: Molecules studies included mannitol, the polyethylene glycols (PEGs) 400, 900, and 4000, the dextran conjugated dye Texas Red (MW 3000) and the polysaccharide inulin (MW 5500). Molecular surface area, volume and cross-sectional diameter for each probe were determined from computer models. The effect of the bile salt sodium cholate, and bile salt: fatty acid mixed micelles on probe intestinal permeability was also studied. RESULTS: Of the size parameters tested, cross-sectional diameter correlated best with log intestinal permeability. The data was fitted to a relationship of the form Papp = Papp zero exp(-Krca) where rca is the molecular cross sectional radius. Papp zero and K are constants. Estimates of equivalent pore radii (R) were also made; the use of rca giving the most reasonable estimate of R. Absorption of all probes was enhanced by both simple and mixed micellar systems. CONCLUSIONS: For large hydrophilic probes and possibly protein drugs, cross sectional diameter is a more important size parameter than volume based values for evaluating size-related retarded absorption. The relationship established may be used as a tool to assess absorption enhancement potential of excipients.

Animals↗

PubMed1995

Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development.

Reduced protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic (dpp), wingless (wg), and patched (ptc) transcription that is independent of hedgehog (hh) gene activity. The resulting nonautonomous adult wing and leg pattern duplications are largely due to induced dpp and wg expression and resemble phenotypes elicited by ectopic hh expression. Inhibition of PKA in anterior cells close to the posterior compartment can substitute for hh activity to promote growth of imaginal discs, whereas overexpression of PKA can counteract transcriptional induction of ptc by hh in these cells. PKA therefore appears to be an integral component of the mechanism by which hh regulates the expression of key patterning molecules in imaginal discs.

Animals↗

PubMed1995

Localization and functions of protein kinase A during Drosophila oogenesis.

We have characterized the requirements for the Protein Kinase A (PKA) catalytic subunit, DC0, in Drosophila oogenesis. Intercellular bridges in egg chambers from PKA deficient females are unstable, leading to the formation of multinucleate nurse cells by fusions of adjacent cells. Germline clones of cells homozygous for null mutations of DC0 indicate that PKA acts autonomously in the germline. Highest levels of PKA catalytic subunit protein are associated with germ cell membranes, suggesting that targets of PKA are associated with the membrane or membrane skeleton and contribute to the stabilization of intercellular bridges. The migration of a subset of follicle cells, the border cells, is also disrupted by germline PKA mutations, implying that nurse cell junctions provide an essential path for border cell migrations.

Animals↗

PubMed1994

RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization.

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. We describe mutation affecting Protein Kinase A (PKA) that act in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of bicoid and oskar RNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition, oskar RNA fails to accumulate at the posterior, and bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns previously reported for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells.

Animals↗

PubMed1993

Genetic investigation of cAMP-dependent protein kinase function in Drosophila development.

The cAMP-dependent protein kinase (PKA) has been shown to mediate the vast majority of cellular responses to the intracellular second messenger, cAMP, in eukaryotes. To study the role of cAMP signal transduction in Drosophila development, we have isolated and molecularly characterized mutations of varying severity in the Drosophila PKA gene, DC0. Biochemical measurements indicate that DC0 is either the sole or the major PKA catalytic subunit gene in Drosophila. Adult females heterozygous for a strong and a weak DC0 allele fail to lay eggs and show a striking and novel defect in oogenesis that includes the formation of egg chambers containing multinucleate nurse cells. Females heterozygous for two weak DC0 alleles are fertile but produce offspring showing a variety of defects in embryogenesis, including preblastoderm arrest and alterations in cuticular patterning. Animals zygotically null for DC0 die as morphologically normal first-instar larvae, implying that maternally encoded protein, which perdures for at least 12 hr, suffices for embryogenesis. Animals hemizygous for weak DC0 alleles survive for several days as larvae but grow slowly. Mitotic recombination experiments in the adult eye indicate that the DC0 gene is not required autonomously either for cell viability or normal growth rates. These results argue that cAMP-mediated signal transduction is essential at a variety of stages during the development of a metazoan.

Animals↗

PubMed1989

Microporosity of the substratum regulates differentiation of MDCK cells in vitro.

We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

Animals↗

PubMed1987

Somatosensory evoked potentials following pudendal nerve stimulation as indicators of low sacral root involvement in a postlaminectomy patient.

Somatosensory cortical evoked potential studies (SEPs) following stimulation of the pudendal nerves proved useful in objectively documenting preoperative low sacral root involvement in a 33-year-old postlaminectomy patient, presenting with impending cauda equina syndrome and treated by piriformis muscle release with good clinical results. Preoperative P40(P1) latencies of SEPs were abnormally prolonged (47 msec) and returned to normal values (38 msec) postoperatively. This new noninvasive electrodiagnostic method has been described previously for normal subjects only. Further experience with this method is still needed, especially to elucidate the diagnostic reliability of various SEP parameters.

Adult↗

PubMed1984

Recent developments in the electrodiagnosis of radiculopathies.

The accuracy and reliability of electrodiagnosis in radiculopathy has improved in recent years. A better understanding of EMG findings coupled with the development of several tests that have recently become available in clinical practice (F wave studies, H reflex studies, and somatosensory evoked potential studies) have made it a valuable tool in the complete work-up for suspected radiculopathy. The use of this technique is reviewed and an illustrative case report is presented.

Adult↗