No abstract

The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence. To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K. (1994) FEBS Lett. 351, 360-364).

This study reports the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of mouse embryonic stem (mES) cells, including undifferentiated single cells, embryoid bodies (EBs), and terminally differentiated cardiomyocytes. Gold nanoparticles (GNPs) were successfully delivered into all 3 mES cell differentiation stages without affecting cell viability or proliferation. Transmission electron microscopy (TEM) confirmed the localization of GNPs inside the following cell organelles: mitochondria, secondary lysosome, and endoplasmic reticulum. Using bright- and dark-field imaging, the bright scattering of GNPs and nanoaggregates in all 3 ES cell differentiation stages could be visualized. EB (an early differentiation stage) and terminally differentiated cardiomyocytes both showed SERS peaks specific to metabolic activity in the mitochondria and to protein translation (amide I, amide II, and amide III peaks). These peaks have been rarely identified in undifferentiated single ES cells. Spatiotemporal changes observed in the SERS spectra from terminally differentiated cardiomyocyte tissues revealed local and dynamic molecular interactions as well as transformations during ES cell differentiation.

The interest in cardiomyocytes derived from differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is increasing due to their potential for regenerative therapeutics and as a pharmaceutical model of drug screening. Characterization of ES or iPS derived cardiomyocytes is challenging and inevitable for the intended usage of such cells. In this paper we have outlined a novel, non-invasive method for evaluating in vitro beating properties of cardiomyocytes. The method is based on the analysis of time dependent variation in the total pixel intensities in derivative images obtained from the consecutive systolic and diastolic frames from the light microscopic video recordings of beating tissue. Fast Fourier transform (FFT) yielded the frequency domains for these images. The signal to noise ratio for the analysis met the Rose criterion. We have successfully applied our method for monitoring mouse ES cell (mESC) derived cardiac muscle cells to determine the initiation of beating, organization and maturation of beating tissue, calculating the beating rhythms in terms of beating interval or frequency and the strength of beating. We have shown the successful application of our image analysis method in direct monitoring of the responses of differentiated cardiomyocytes towards caffeine hydrate, p-hydroxyphenylacetamide and calcium chloride dehydrate - respectively as positive, neutral and negative inotropic agents. This non-invasive method of characterization will be useful in studying the response of these cells to various external stimulations, such as differentiation promoting agents or treatments, as well as in preliminary drug screening in a quick and inexpensive manner without needing much expertise.