Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.     

Thermosensitive omsA mutation of Escherichia coli that causes thermoregulated release of periplasmic proteins.

A mutant of Escherichia coli with a thermosensitive defect, possibly in the outer membrane (omsA mutant), was isolated from E. coli K-12 by mutagenization and selection for thermosensitivity and beta-lactam supersensitivity of growth. The mutant also showed very high sensitivity to other antibiotics, such as macarbomycin, midecamycin, rifampin, and bacitracin. The mutation was recessive to the wild type and was mapped at about 4 min on the E. coli chromosome between fhuA and metD. The mutation caused rapid release into the medium of periplasmic enzymes such as RTEM penicillinase but practically no cytoplasmic enzyme when cells grown at 30 degrees C were transferred to 37 or 42 degrees C. Electron microscopic observations showed many large double-layered vesicles attached to the surface of cells incubated at 42 degrees C. We conclude that the mutant had a mutation that caused a temperature-dependent defect in the outer membrane structure or its assembly (named an oms mutation). The omsA mutant may be useful for production of periplasmic proteins, which it releases into the culture medium on shift up of temperature.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

The Sphingosine 1-Phosphate Transporter,SPNS2, Functions as a Transporter of the Phosphorylated Form of the Immunomodulating Agent FTY720

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P₁, and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.     

Polyphenols in alcoholic beverages activating constitutive androstane receptor CAR

The constitutive androstane receptor CAR is a xenosensing nuclear receptor that can be activated by natural polyphenols such as flavonoids and catechins. We examined alcoholic beverage phytochemicals for their ability to activate CAR. HepG2 cells were transfected with CAR expression vector and its reporter gene, and then treated with trans-resveratrol, ellagic acid, β-caryophyllene, myrcene, and xanthohumol. A luciferase assay revealed that ellagic acid and trans-resveratrol activated both human and mouse CAR. Since CAR regulates many genes involved in energy metabolism, the possibility exists that these polyphenols would reduce the risk of certain alcohol-induced metabolic disorders with the help of CAR.     

Acute Graft-Versus-Host Disease of the Kidney in Allogeneic Rat Bone Marrow Transplantation

Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD). However, the involvement of the kidney in acute GVHD is not well-understood. Acute GVHD was induced in Lewis rats (RT1^l) by transplantation of Dark Agouti (DA) rat (RT1^a) bone marrow cells (6.0×10⁷ cells) without immunosuppression after lethal irradiation (10 Gy). We examined the impact of acute GVHD on the kidney in allogeneic BMT rats and compared them with those in Lewis-to-Lewis syngeneic BMT control and non-BMT control rats. In syngeneic BMT and non-BMT control rats, acute GVHD did not develop by day 28. In allogeneic BMT rats, severe acute GVHD developed at 21–28 days after BMT in the skin, intestine, and liver with decreased body weight (>20%), skin rush, diarrhea, and liver dysfunction. In the kidney, infiltration of donor-type leukocytes was by day 28. Mild inflammation characterized by infiltration of CD3+ T-cells, including CD8+ T-cells and CD4+ T-cells, and CD68+ macrophages to the interstitium around the small arteries was noted. During moderate to severe inflammation, these infiltrating cells expanded into the peritubular interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. Renal dysfunction also developed, and the serum blood urea nitrogen (33.9±4.7 mg/dL) and urinary N-acetyl-β-D-glucosaminidase (NAG: 31.5±15.5 U/L) levels increased. No immunoglobulin and complement deposition was detected in the kidney. In conclusion, the kidney was a primary target organ of acute GVHD after BMT. Acute GVHD of the kidney was characterized by increased levels of urinary NAG and cell-mediated injury to the renal microvasculature and renal tubules.