Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart-muscle tissue sheets

Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.     

Early-age-dependent selective decrease of differentiation potential of hair-follicle-associated pluripotent (HAP) stem cells to beating cardiac-muscle cells

We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.     

Inhibitory effects of beta 2-microglobulin on in vitro calcification of osteoblastic cells

Elevated levels of aluminum and beta 2-microglobulin have been demonstrated in chronic dialysis patients. The role of aluminum in the pathogenesis of renal osteodystrophy has also been shown. We report on the effects of beta 2-microglobulin on calcification in vitro using osteoblastic cells, clone MC3T3-El. At concentrations comparable to those in plasma of chronic dialysis patients, both beta 2-microglobulin and aluminum suppressed calcification while collagen synthesis and alkaline phosphatase activity were maintained. These observations may be related to the impaired bone mineralization frequently observed in chronic dialysis patients.     

Proton nuclear magnetic resonance study of human plasma alpha-2-macroglobulin

A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS)     

Close evolutionary relationship between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase gene mediated by R plasmids

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of beta-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.     

Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

Preferential interactions of proteins with solvent components in aqueous amino acid solutions

The preferential interactions of proteins with solvent components in concentrated amino acid solutions were measured by high-precision densimetry. Bovine serum albumin and lysozyme were preferentially hydrated in all of the amino acids examined, glycine, α- and β-alanine, and betaine i.e., addition of these amino acids resulted in an unfavorable free energy change. It was shown that, for the former three amino acids, known to have a positive surface tension increment, their perturbation of the surface free energy of water is consistent with their preferential exclusion from the protein surface. In the case of betaine, which does not increase the surface tension of water, preferential exclusion from protein surface must reflect the chemical structure of this cosolvent, which is considerably more hydrophobic than that of the other three amino acids.     

Confirmation of direct angiotensin formation by kallikrein.

This study was undertaken to confirm our previous preliminary observation that hog pancreas kallikrein (EC 3.4.21.35) directly liberated an angiotensin-like substance from human plasma protein Cohn fraction IV-4 at an acidic pH of 4.0-5.0. First, the possibility of proangiotensin or des-Asp1-angiotensin being the pressor substance was ruled out by t.l.c. Secondly, the pressor substance was purified by Sephadex G-25 and Bio-Gel P-2 gel filtration, and finally by high-performance liquid chromatography. The amino acid composition of the isolated pressor substance (residues/mol) was: Asp, 1.03; Val, 1.03; Ile, 1.00; Tyr, 0.69; Phe, 1.04; His, 0.91; Arg, 0.86; Pro, 0.86. This composition was identical with that of angiotensin. Since the reaction mixture was not contaminated with common proteolytic enzymes, such as trypsin, chymotrypsin, renin, cathepsin D and proangiotensin-converting enzyme, and other enzymes activated by kallikrein, it is clear that hog kallikrein directly produces angiotensin in vitro.     

Isolation and amino acid composition of human angiotensin I

1. Angiotensin, the most powerful pressor substance known, suspected to be a causal substance in renal hypertension and previously isolated from animal sources, has now been isolated from human sources and the amino acid composition was analysed. 2. The procedures followed in the successful isolation of human angiotensin include: (a) preparation of stable materials to obtain maximum formation of human angiotensin; (b) a relatively selective adsorption of the formed angiotensin on Dowex 50W (X2); (c) gel filtration through Sephadex G-25; (d) cation-exchange chromatography on CM-cellulose; (e) anion-exchange gel filtration on DEAE-Sephadex A-25; (f) molecular-sieve chromatography through Bio-Gel P-2. 3. The homogeneity of the human angiotensin isolated was confirmed by paper and thin-layer chromatography and paper electrophoresis. 4. The biological activity observed indicates the substance isolated to be human angiotensin I. 5. The amino acid analysis suggested the following proportional composition: Asp, 1; Pro, 1; Val, 1; Ile, 1; Leu, 1; Tyr, 1; Phe, 1; His, 2; Arg, 1. This composition is similar to that of horse angiotensin I, i.e. isoleucine(5)-angiotensin I.