1.Preservation of cardiomyocytes from the adult heart.
Abi-Gerges N1, Pointon A, Pullen GF, Morton MJ, Oldman KL, Armstrong D, Valentin JP, Pollard CE. J Mol Cell Cardiol. 2013 Nov;64:108-19. doi: 10.1016/j.yjmcc.2013.09.004. Epub 2013 Sep 16.
Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and N-benzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes.
2.Gramicidin channels that have no tryptophan residues.
Fonseca V1, Daumas P, Ranjalahy-Rasoloarijao L, Heitz F, Lazaro R, Trudelle Y, Andersen OS. Biochemistry. 1992 Jun 16;31(23):5340-50.
In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels.
3.Carrier mediated uptake of L-tyrosine and its competitive inhibition by model tyrosine linked compounds in a rabbit corneal cell line (SIRC)--strategy for the design of transporter/receptor targeted prodrugs.
Balakrishnan A1, Jain-Vakkalagadda B, Yang C, Pal D, Mitra AK. Int J Pharm. 2002 Oct 24;247(1-2):115-25.
The objective of this study was to investigate the presence of amino acid transporters on the corneal epithelium and to enhance corneal drug absorption through prodrug modification targeted to the amino acid transporters. SIRC was used as a model cell line representing the corneal epithelium. Uptake studies were carried out using [3H] L-tyrosine at 37 degrees C. Temperature, energy and pH dependence studies were carried out. The uptake seems to be composed of a major saturable and minor non-saturable component (V(max) =2.9+/-0.62 nmoles/min/mg protein, K(m) =71+/-21 microM, K(d) =2.6+/-0.6 nl/min/mg protein). No significant inhibition of uptake was observed in the presence of metabolic inhibitors or in the absence of sodium. Competitive inhibition studies were performed in the presence of various amino acids and model tyrosine conjugates (p-nitro and p-chloro benzyl ether conjugate of L-tyrosine). Uptake was inhibited by neutral aromatic and large neutral aliphatic amino acids.