Detection and changes in levels of testosterone during spermatogenesis in the freshwater planarian Bdellocephala brunnea

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.     

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [³H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, α-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A₂ and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.     

Identification of SMARCAL1 as a Component of the DNA Damage Response

SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.     

Non-covalent modification of the heme-pocket of apomyoglobin by a 1,10-phenanthroline derivative

To expand the repertoire of artificial enzymes that are constructed by replacing the natural prosthetic group of hemoproteins with non-natural cofactors, we examined incorporation of a non-porphyrinic ligand (1) into the heme-pocket of apomyoglobin in a non-covalent fashion. Ligand 1 is a highly conjugated 1,10-phenanthroline derivative, which shares some structural features with protoporphyrin IX; for example, molecular size and arrangement of hydrophobic and anionic parts. Addition of apomyoglobin to a solution of 1 induces clear changes in the absorption spectrum of 1, suggesting one-to-one incorporation of 1 into the heme cavity of apomyoglobin with an affinity of 6.3 x 10(6)M(-1). We found that the hydrolytic activity of apomyoglobin toward p-nitrophenyl hexanoate was greatly suppressed because of the incorporation of 1 into the heme-pocket.     

Cepharanthine, an anti-inflammatory drug, suppresses mitochondrial membrane permeability transition

Cepharanthine (CEP), a biscocrourine alkaloid, has been widely used in Japan for the treatment of several disorders. Furthermore, accumulated evidence shows that CEP protects against some cell death systems but not others. Recently, it was found that mitochondria play an important role in a mechanism of apoptosis involving membrane permeability transition (MPT). Although CEP stabilizes the mitochondrial membrane structure and protects some functions of mitochondria from damage, the mechanism of action of CEP on MPT remains obscure. In this study, therefore, we examined the effect of CEP on Ca2+- and Fe2+/ADP-induced MPT of isolated mitochondria. CEP inhibited Ca2+-induced swelling, depolarization, Cyt.c release, and the release of Ca2+ in a concentration dependent manner. CEP also inhibited Ca2+-induced generation of reactive oxygen species and Fe/ADP-induced swelling and lipid peroxidation. Furthermore, CEP suppressed Ca2+-induced thiol modification of adenine nucleotide transloase (ANT). These results suggested that CEP suppressed MPT by a decrease in affinity of cyclophilin D for ANT. From these results it was concluded that the suppression of MPT by CEP might be due to its inhibitory action on Ca2+ release and antioxidant activity and that CEP might suppress the mechanism of apoptotic cell death when directly interacted with mitochondria in cells.     

Light-harvesting ionic dendrimer porphyrins as new photosensitizers for photodynamic therapy

Photodynamic therapy (PDT) is a promising therapeutic modality for treatment of solid tumors. In this study, third-generation aryl ether dendrimer porphyrins (DPs) with either 32 quaternary ammonium groups (32(+)DPZn) or 32 carboxylic groups (32(-)DPZn) were evaluated as a novel, supramolecular class of photosensitizers for PDT. DPs showed a different cell-association profile depending on the positive or negative charge on the periphery, and both DPs eventually localized in membrane-limited organelles. In contrast, protoporphyrin IX (PIX), which is a hydrophobic and relatively low molecular weight photosensitizer used as a control in this study, diffused through the cytoplasm except the nucleus. Confocal fluorescent imaging using organelle-specific dyes indicated that PIX induced severe photodamage to disrupt membranes and intracellular organelles, including the plasma membrane, mitochondrion, and lysosome. On the other hand, cells treated with DPs kept the characteristic fluorescent pattern of such organelles even after photoirradiation. However, notably 32(+)DPZn achieved remarkably higher (1)O(2)-induced cytotoxicity against LLC cells than PIX. Furthermore, both dendrimer porphyrins had far lower dark toxicity as compared with PIX, demonstrating their highly selective photosensitizing effect in combination with a reduced systemic toxicity.