Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo

Summary We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O₂ ^– production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

Carbon stock and cycling in a bambooPhyllostachys bambusoides stand

Gross production and carbon cycling in aPhyllostachys bambusoides stand in Kyoto Prefecture, central Japan, were determined, and then a compartment model showing the carbon stock and cycling within the ecosystem was developed. Aboveground carbon stock was 52.3 tC ha⁻¹, increasing at a rate of 3.6 tC ha⁻¹ year⁻¹. Belowground carbon stock was 20.8 tC ha⁻¹ in the root system and 92.0 tC ha⁻¹ in the soil. Aboveground net production was 11.2 tC ha⁻¹ year⁻¹. Belowground net production was crudely estimated at 4.5 tC ha⁻¹ year⁻¹. The gross production was estimated at 41.8 tC ha⁻¹ year⁻¹ by summing the amount of outflow to the environment and the increment in biomass. Leaves consumed 13.7 tC ha⁻¹ year⁻¹ by respiration; the rest (41.8−13.7=28.1 tC ha⁻¹ year⁻¹) was surplus production of the leaves and flowed into the other compartments. The amounts of construction and maintenance respiration of the aboveground compartments were 3.4 and 18.5 tC ha⁻¹ year⁻¹, respectively. The annual amount of soil respiration was 11.2 tC ha⁻¹ year⁻¹. Soil respiration levels of 4.3 and 3.1 tC ha⁻¹ year⁻¹ were estimated for the flow of root respiration and root detritus. The proportion of net to gross production was 37%, which fell within the range of young and mature forests. A shorter life span of culms, compared to tree trunks, resulted in smaller biomass accumulation ratio (biomass/net production) in the ecosystem, of 4.66.     

Contribution of K(ir)2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca2(+) concentration ([Ca2(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca2(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca2(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ～15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba2(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba2(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba2(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca2(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.     

Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis

Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.     

Isolation of tissue progenitor cells from duct-ligated salivary glands of swine

Tissue stem cells participate in the repopulation of tissue after injury. Tissue injury stimulates the normally quiescent tissue stem cells to differentiate and proliferate, in the process of replacing and/or repairing the damaged cells, and hence effecting tissue regeneration. The salivary glands retain the ability for frequent regeneration. Previously, we isolated progenitor cells from the injured salivary glands of mice and rats that differentiated into hepatic and pancreatic lineages. The isolated progenitors were CD49f-positive and intracellular laminin-positive, and proliferated on type I collagen while maintaining their multipotency. In this study, we analyzed the tissue stem cells induced by ligating the main excretory duct of the salivary gland in swine. After duct ligation of the gland, acinar cells receded due to apoptosis, and epithelial cells subsequently proliferated. We cultured cells obtained from the duct-ligated salivary gland and purified the cells by limited dilution. The isolated cells were positive for CD29, CD49f, intracellular laminin, AFP, CK19, CK18, and Thy-1(CD90), and weakly positive for c-Kit (CD117). After three-dimensional formation, the cells expressed insulin and albumin. We designated the cells as swine salivary gland-derived progenitor cells. Gene expression of insulin and albumin was significantly increased (five-fold) and that of insulin was also increased (3.8-fold) with differentiation medium with nicotinamide and/or GLP-1 treatment in spherical culture. The expressions of albumin and insulin were 1/10-fold and 1/4-fold compared to porcine hepatocytes and pancreatic endocrine cells. The differentiated SGP cells could release insulin, which were stimulated by glucose and potassium. These results indicate that swine SGP cells could differentiate into hepatocytes and beta-cells, functionally. Swine SGP cells were useful tools for therapy and analyzing endodermal regenerative models in large animals.     

Bacteriorhodopsin analog regenerated with 13-desmethyl-13-iodoretinal

The retinal analog 13-desmethyl-13-iodoretinal (13-iodoretinal) was newly synthesized and incorporated into apomembranes to reconstitute bacteriorhodopsin analog 13-I-bR. The absorption maximum was 598 nm and 97% of the chromophore was an all-trans isomer in the dark- and light-adapted state. Upon flash illumination, 13-I-bR underwent a transient spectral change in which a shorter wavelength intermediate (lambda(max) = 426 nm) similar to the M species of the native bR developed. Also, 13-I-bR showed light-induced proton pumping with rates and extents comparable to those seen in the native bR. The ultraviolet circular dichroism (CD) spectrum originating from the aromatic groups was different from that of the native bR, indicating that the substituted bulky iodine atom strongly interacts with neighboring amino acids. A projection difference Fourier map showed the labeled iodine was in the vicinity of helix C. 13-I-bR is an advantageous specimen for kinetic investigations of light-induced structural changes associated with the proton pumping cycle by x-ray diffraction.