Purification of megakaryocyte differentiation activity from a human fibrous histiocytoma cell line: N-terminal sequence homology with activin A.

We purified a protein possessing a potent ability to induce the differentiation of a murine megakaryoblastic cell line, L8057, from the supernatant of a human fibrous histiocytoma cell line, KHM-5M. The protein, a homodimer of a molecular weight of 25-kDa, has an N-terminal sequence identical with that of the beta A-chain of inhibin, indicating that it is identical to or highly homologous with activin A. Thus, it is speculated that activin A or its homologue is involved in megakaryocytic differentiation.     

Interactions of an antimicrobial peptide, tachyplesin I, with lipid membranes.

Tachyplesin I, isolated from the acid extracts of hemocytes of Tachypleus tridentatus, is a cyclic broad-spectrum antimicrobial peptide forming a rigid, antiparallel beta-sheet because of two intramolecular S-S linkages. The strong binding of the peptide to lipopolysaccharides cannot explain the susceptibilities of gram positive bacteria and fungi to the peptide. We found that tachyplesin I caused a rapid K+ efflux from Escherichia coli cells, concomitant with a reduced cell viability. This result suggests that the peptide-induced permeability enhancement of the bacterial membranes may be a plausible action mechanism. Thus, we studied the interactions of tachyplesin I with various large unilamellar vesicles (LUVs) to reveal the molecular machinery of the antimicrobial activity. Tachyplesin I induced the leakage of calcein, a trapped fluorescent marker, from LUVs of acidic phospholipids, especially phosphatidylglycerol (PG), but not from phosphatidylcholine LUVs. A detailed analysis found that the affinity of the peptide to the PG membranes is very strong and that the binding of one peptide molecule to approx. 200 lipid molecules leads to a significant leakage. The location of tachyplesin I in membranes was estimated by use of the Trp-2 fluorescence of the peptide. The presence of PG LUVs caused a blue shift of the maximum wavelength, an increase in the quantum yield, and a complete protection from fluorescence quenching by an aqueous quencher, acrylamide. Moreover, the degree of fluorescence quenching of the Trp residue by n-doxylstearates was in the order n = 5 greater than 7 greater than 12 approximately equal to 16. These results show that the Trp residue of tachyplesin I seems to locate in a hydrophobic environment near the surface of the PG bilayers.     

A comparative study on interactions of alpha-aminoisobutyric acid containing antibiotic peptides, trichopolyn I and hypelcin A with phosphatidylcholine bilayers.

Interactions of alpha-aminoisobutyric acid containing antibiotic peptides, trichopolyn I and hypelcin A with phosphatidylcholine bilayers were investigated to obtain some basic information on their bioactive mechanisms. Trichopolyn I as well as hypelcin A induced the leakage of a fluorescent dye, calcein, entrapped in sonicated egg yolk L-alpha-phosphatidylcholine vesicles. A quantitative analysis revealed that both the binding affinity and the 'membrane-perturbing activity' of trichopolyn I to the vesicles are about one-third of those of hypelcin A. The conformations and the orientations of the peptide and lipid molecules in the membranes were studied using polarized Fourier transform infrared-attenuated total reflection spectroscopy, circular dichroism, and differential scanning calorimetry. In phosphatidylcholine bilayers, both peptides mainly conformed to helical structures irrespective of the membrane physical state (gel or liquid-crystalline). The helix axes, penetrating the hydrophobic region of the bilayers, were oriented neither parallel nor perpendicular to the membrane normal. The disruption in the lipid packing induced by the peptide insertion seems to be responsible for the leakage by these peptides.     

Polyamine distributions in thermophilic eubacteria belonging to Thermus and Acidothermus

Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 A1, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.     

Interleukin-6 stimulates gonadotropin-releasing hormone secretion from rat hypothalamic cells

There are reports that both interleukin-1 beta and interleukin-6 (IL-6) stimulate the release of adrenocorticotropin through stimulation of hypothalamic corticotropin-releasing factor. We established a primary culture system for hypothalamic neurons producing gonadotropin-releasing hormone (GnRH) and examined whether IL-6 stimulated their GnRH secretion. We demonstrated immunohistochemically that some of these neurons contained GnRH-like immunoreactivity. In primary cultures of these GnRH neurons, we found that the calcium ionophore A23187 stimulated GnRH secretion in a dose- and time-dependent manner. These hypothalamic cells secreted IL-6 spontaneously, producing about 10 ng/l in 24 h, and their IL-6 secretion was significantly stimulated by E2 at 10(-9)-10(-8) mol/l. This stimulatory effect was observed within 3 h. IL-6 also stimulated the release of GnRH in a dose- and time-dependent manner, and these effects of IL-6 were significantly blocked by anti-IL-6 antiserum. These results suggest that the central action of IL-6 on the GnRH neurons may be an important physiological event in the hypothalamus.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor γII)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon γ, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.     

The origin and development of retinal pigment cells and melanophores analyzed by Xenopus black-white chimeras

At the 16 cell stage, three kinds of borealis–laevis and eight kinds of laevis–laevis chimeric embryos were produced by replacing a particular blastomere of albino embryos of Xenopus laevis with that of wild-type embryos of X. borealis or X. laevis , and then leaving the embryos to develop into frogs.
In the borealis–laevis chimera frogs, we found that all the melanized cells (retinal pigment cells and melanophores) were derived from a transplanted wild-type blastomere with a nuclear marker of X. borealis and that all the albino-mutant cells derived from the host did not become melanized. Thus, retinal pigment cells and melanophores differentiated according to their own genotype. We then examined the origin of these two types of cells, using melanin as a cell-marker in the borealis–laevis and laevis–laevis chimeras.
Retinal pigment cells derive from A1 (dorso-animal) and A2 (latero-animal) blastomeres. A1 of one side contributes to retinal pigment cells in both eyes. Though the blastomeres of one side contribute to the formation of bilateral melanophores, the major contribution is to melanophores of the same side. A1, A2 and V2 (latero-vegetal) form the anterior part of the neural fold, and A2 and V2 contribute to melanophores of the head region. The most anterior part of the neural fold derived from A1 does not make a significant contribution to melanophores. Though V2 is a vegetal blastomere, it forms the anterior part of the neural fold by upward movement against the downward movement for gastrulation. A3 forms the middle and posterior parts of the neural fold and contributes to melanophores of the trunk and hindlimbs. Melanophores of hindlimbs also come from A2, A4 and V2. It is to be noted that A4 contributes to melanophores of hindlimbs, despite no apparent contribution to the neural fold.
Development of the retinal pigment cells and melanophores is discussed from the point of pigmentation patterns of the chimeras.