Robert Hard

Profound Apoptosis-Mediated Regional Myocyte Loss and Compensatory Hypertrophy in Pigs With Hibernating Myocardium

BACKGROUND Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. METHODS AND RESULTS Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). CONCLUSIONS These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Newt Lung Epithelial Cells: Cultivation, Use, and Advantages for Biomedical Research

Publisher Summary This chapter reviews the cultivation, structure, advantages, and potential uses of ciliated and nonciliated newt lung epithelial cells (NLC). When compared to cells from other organisms, newt cells—especially pneumocytes and ciliated epithelia—derived from the lung-possess a number of advantages for examining cellular processes at various levels of microscopic organization. However, with the exception of the fibroblastic TV 1 cell line, derived from newt iris, there are no established caudate cell lines. In this respect, determining the optimal culture conditions for caudate cell survival and proliferation remains an important area for future research. The relatively long cilia, their planar waveform, and the development of models to study movement at various levels of organization makes newt lung ciliated epithelium an ideal material for functional studies of ciliary movement and mucociliary transport (MCT). The favorable optical properties and exceedingly thin nature of the NP prove valuable in the development of same cell-correlative light microscopic (LM)-EM methods, utilizing advanced techniques of cryofixation.

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this posttranslational modification, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.

Tools to Rapidly Produce and Screen Biodegradable Polymer and Sol-Gel-Derived Xerogel Formulations:

A new method to rapidly produce and screen biodegradable polymer- and xerogel-based formulations is described. The approach is based on a high-speed pin printer and imaging with an epi-fluorescence microscope/charge-coupled device detector. By using this approach we can produce and screen over 600 formulations/h and rapidly identify lead formulations and/or compositions that are the most useful for the development of biodegradable devices or (bio)sensors.

Targeted gene disruption of dynein heavy chain 7 of Tetrahymena thermophila results in altered ciliary waveform and reduced swim speed.

Tetrahymena thermophila swims by the coordinated beating of hundreds of cilia that cover its body. It has been proposed that the outer arm dyneins of the ciliary axoneme control beat frequency, whereas the inner arm dyneins control waveform. To test the role of one of these inner arms, dynein heavy chain 7 protein (Dyh7p), a knockout mutant was generated by targeted biolistic transformation of the vegetative macronucleus. Disruption of DYH7, the gene which encodes Dyh7p, was confirmed by PCR examination of both genomic and cDNA templates. Both intact and detergent extracted, reactivated cell model preparations of these mutants, which we call DYH7neo3, displayed swim speeds that were almost half that of wild-type cells. Although the DYH7neo3 mutants were slower than wild type, they were able to modulate their swim speed and show ciliary reversal in response to depolarizing stimuli. High-speed video microscopy of intact, free-swimming DYH7neo3 mutants revealed an irregular pattern of ciliary beat and waveform. The mutant cilia appeared to be engaging in less coordinated, swiveling movements in which the typical shape, periodicity and coordination seen in wild-type cilia were absent or disturbed. We propose that the axonemal inner arm dynein heavy chain 7 proteins contribute to the formation of normal ciliary waveform, which in turn governs the forward swimming velocity of these cells.

Analysis of the Initial Burst of Drug Release Coupled with Polymer Surface Degradation

AbstractPurpose. Local pH effect on the release of a model pH-inert hydrophobic drug coupled with polymer degradation is described at the induction phase of biodegradable polymer erosion for better understanding the nature of initial burst of a drug. Methods. Using a novel approach with time-of-flight secondary ion mass spectrometry, both surface concentration of Ph3N and degradation kinetics of PLLA are simultaneously and independently determined from a model Ph3N/PLLA (20:80 wt%) blend matrix (t ≈ 0.4 μm on 1.0 cm2). In vitro hydrolysis of the model blend matrix is investigated for short-term periods (<24 h) at physiologic pH and temperature and compared to basic pH. Results. The rate of PLLA degradation is accelerated by a factor of ∼3 when using basic pH in vitro, but the rate of Ph3N accumulation at the surface is accelerated by a factor of ∼6. Conclusions. A new quantitative method has been developed to examine the earliest stages of polymer degradation and drug release. It was applied to a model system that could not be examined by traditional in vitro methods. For the model system studied the release of a low molecular weight hydrophobic drug at the induction phase of polymer erosion is related to but not singularly dependent on degradation kinetics.

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.

Determining the protein drug release characteristics and cell adhesion to a PLLA or PLGA biodegradable polymer membrane

Biodegradable polymers are of interest for developing controlled protein drug delivery platforms. In this study, two poly (alpha-hydroxy) esters were formulated with Aerosol-OT, a surfactant stabilizer, to encapsulate the protein keratinocyte growth factor (KGF) for controlled release KGF is involved in a number of crucial biologic processes, most notably epithelial growth and repair. The concentration of KGF that caused a biological response in vitro was determined (optimally 10 ng/mL) and compared with the release of KGF from the two biodegradable polymer membrane formulations. Each polymer formulation released biologically relevant levels, 10 ng/mL of active KGF, although with different times release kinetics. The membrane composed of PLGA/AOT/KGF exhibited a faster release rate of KGF into solution after 120 h of degradation time than the release rate of the PLLA/AOT/KGF matrices. Cell seeding assays showed that both polymer matrices, when formulated with AOT, sustained cell growth. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used to characterize the distribution of AOT and KGF through the polymer membrane. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Phase separation at the surface of poly(ethylene oxide)-containing biodegradable poly(L-lactic acid) blends.

The surface chemistry and in-depth distribution of the composition of a poly(ethylene oxide) (PEO)-containing biodegradable poly(L-lactic acid) (PLLA) blend matrix system have been investigated using X-ray photoelectron spectroscopy (XPS). This study reports detailed quantitative compositional information using a novel numerical method for determining depth profiles. The PEO system studied is an amphiphilic Pluronic P104 surfactant, PEO-b-poly(propylene oxide) (PPO)-b-PEO. The extent of phase separation is analyzed by determining the surface enrichment of the PEO component via measurement of chemical composition at the polymer-air interface. For this blend system, the combination of the PPO component in the Pluronic surfactants drives the formation of a surface excess of Pluronic in the blends with PLLA. The surface excess profile shows a rapid increase in Pluronic surface composition versus bulk Pluronic mass fractions of 1-5%, but the profile levels off above bulk Pluronic mass fractions of 5%.

Interleukin-1 Facilitates Airway Epithelial Migration in Response to Injury†

Objective To test the hypothesis that interleukin (IL)‐1 plays a permissive role in respiratory epithelial cell migration and proliferation.