Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro

OBJECTIVES: Pluripotent stem cells are proposed to be used in regenerative therapy and may exist in the human amniotic membrane. The present article is aimed at establishing a pluripotent stem cell line from human placenta. METHODS: HAM-1 (stem cell line derived from human amniotic membranes) was established by the colonial cloning technique using aMEM culture medium containing 10 ng/ml of EGF, 10 ng/ml of hLIF and 10% fetal bovine serum. RESULTS: HAM-1 cells appeared to maintain a normal karyotype indefinitely in vitro and expressed markers characteristic of stem cells from mice and human, namely alkaline phosphatase. Also, these cells contributed to the formation of chimeric mouse embryoid bodies and gave rise to cells of all germ layers in vitro. CONCLUSIONS: This study demonstrates that human amniotic membranes derived stem cells have a wide developmental capability and might be utilized to regenerate different types of cells or tissues for transplantation therapy.     

Catalytic mechanism of glycine N-methyltransferase

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.     

Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal tract

Studies have shown that bone marrow cells have the potential to differentiate into a variety of cell types. Here we show that bone marrow cells can repopulate the epithelia of the human gastrointestinal tract. Epithelial cells of male donor origin were distributed in every part of the gastrointestinal tract of female bone marrow transplant recipients. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract during epithelial regeneration after graft-versus-host disease or ulcer formation. Regeneration of gastrointestinal epithelia with donor-derived cells in humans shows a potential clinical application of bone marrow-derived cells for repairing severely damaged epithelia, not only in the gastrointestinal tract but also in other tissues.     

DIVING BEHAVIOR OF THE PACIFIC HARBOR SEAL (PHOCA VITULINA RICHARDII) IN MONTEREY BAY, CALIFORNIA

Physical environment and physiological characteristics of marine mammals potentially affect the duration and depth of diving. Härkönen (1987b) proposed a hypothesis that the harbor seal would gain maximum energy by foraging at intermediate depths. To investigate this hypothesis, we studied diving behavior of the Pacific harbor seal (Phoca vitulina ricbardii) during 1995 through 1997 in Monterey Bay, California. Dive depths (n = 13,063 dives) were recorded via time‐depth recorders. Approximately 80% of recorded dives were classified as square dives (type I), which typically were associated with foraging in pinnipeds. Approximately 11% of dives were V dives (type II; 1,402 dives), and the remainder (1,225 dives) were skewed dives (type III and IV). The deepest recorded dive was 481 m, while the greatest duration was 35.25 min. Body mass explained the variability of durations of long dives for females (95th percentile; D₉₅♂=‐5.47 + 0.18 × (mass♀), r²= 0.91, 95% CI for slope = [0.08, 0.28], n= 5) and for males (D₉₅♂=‐5.86 + 0.18 × (mass♀), r²= 0.83, 95% CI for slope = [0.12, 0.24], n= 11). The large proportion of variability in deep dives, however, was explained by body mass only for males (95th percentile; Z₉₅♂=‐363.9 + 6.05 × (mass♀), r²= 0.83, 95% CI for slope = [3.93, 8.17], n= 11) and not for females (Z₉₅,♂=?148.1 +3.11 × (mass♀), r²= 0.58, 95% CI for slope = [‐1.7, 7.9], n= 5, 95% CI for slope= [?1.7, 7.9]). Median depths of presumed foraging dives of harbor seals in the Monterey Bay area were between 5 and 100 m, which were within the range of the previously reported depths for other areas (< 100 m). Our findings generally supported Härkönen's hypothesis that harbor seals forage in the intermediate depth in their environment.     

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract     

Membrane-bound cytochrome c is an alternative electron donor for cytochrome aa3 in Nitrobacter winogradskyi.

We purified membrane-bound cytochrome c-550 [cytochrome c-550(m)] to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The cytochrome showed peaks at 409 and 525 nm in the oxidized form and peaks at 416, 521, and 550 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 18,400 on the basis of protein and heme c contents and 18,600 by gel filtration. The N-terminal amino acid sequence of cytochrome c-550(m) was determined to be A-P-T-S-A-A-D-A-E-S-F-N-K-A-L-A-S-A-?-A-E-?-G-A-?-L-V-K-P. We previously purified soluble cytochrome c-550 cytochrome c-550(s)] from N. winogradskyi and determined its complete amino acid sequence (Y. Tanaka, Y. Fukumori, and T. Y. Yamanaka, Biochim. Biophys. Acta 707:14-20, 1982). Although the sequence of cytochrome c-550(m) was completely different from that of cytochrome c-550(s), ferrocytochrome c-550(m) was rapidly oxidized by the cytochrome c oxidase of the bacterium. Furthermore, the liposomes into which nitrite cytochrome c oxidoreductase, cytochrome c oxidase, and nitrite were incorporated showed nitrite oxidase activity in the presence of cytochrome c-550(m). These results suggest that cytochrome c-550(m) may be an alternative electron mediator between nitrite cytochrome c oxidoreductase and cytochrome c oxidase.     

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.