Synthesis of closo-Dodecaboryl Lipids and their Liposomal Formation for Boron Neutron Capture Therapy

High accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve efficient neutron capture therapy of cancers. We focused on liposomal boron delivery system to achieve a large amount of boron delivery to tumor. We succeeded in the synthesis of the double-tailed boron cluster lipids 4a–c and 5a–c, which has a B₁₂H₁₁S-moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols. Size distribution of liposomes prepared from the boron cluster lipid 4b, dimyristoylphosphatidylcholine, polyethyleneglycol-conjugated distearoylphosphatidylethanolamine, and cholesterol was determined as 100 nm in diameter by an electrophoretic light scattering spectrophotometer. Calcein-encapsulation experiments revealed that these boronated liposomes are stable at 37 °C in fetal bovine serum solution for 24 h.     

Biochemical Characterization and Phylogenetic Analysis Based on 16S rRNA Sequences for V-Factor Dependent Members of <Emphasis Type="Italic">Pasteurellaceae</Emphasis> Derived from Laboratory Rats

Phylogenetic analysis based on 16S rRNA sequences with sequence data of some bacterial species of Pasteurellaceae related to rodents deposited in GenBank was performed along with biochemical characterization for the 20 strains of V-factor dependent members of Pasteurellaceae derived from laboratory rats to obtain basic information and to investigate the taxonomic positions. The results of biochemical tests for all strains were identical except for three tests, the ornithine decarboxylase test, and fermentation tests of D(+) mannose and D(+) xylose. The biochemical properties of 8 of 20 strains that showed negative results for the fermentation test of D(+) xylose agreed with those of Haemophilus parainfluenzae complex. By phylogenetic analysis, the strains were divided into two clusters that agreed with the results of the fermentation test of xylose (group I: negative reaction for xylose, group II: positive reaction for xylose). The clusters were independent of other bacterial species of Pasteurellaceae tested. The sequences of the strains in group I showed 99.7–99.8% similarity and the strains in group II showed 99.3–99.7% similarity. None of the strains in group I had a close relation with Haemophilus parainfluenzae by phylogenetic analysis, although they showed the same biochemical properties. In conclusion, the strains had characteristic biochemical properties and formed two independent groups within the “rodent cluster” of Pasteurellaceae that differed in the results of the fermentation test of xylose. Therefore, they seemed to be hitherto undescribed taxa in Pasteurellaceae.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Uncovering the roles of PINK1 and parkin in mitophagy

Parkinson disease (PD) is the second most prevalent neurodegenerative disorder, and thus elucidation of the pathogenic mechanism and establishment of a fundamental cure is essential in terms of public welfare. Fortunately, our understanding of the pathogenesis of two types of recessive familial PDs—early-onset familial PD caused by dysfunction of the PTEN-induced putative kinase 1 (PINK1) gene and autosomal recessive juvenile Parkinsonism (ARJP) caused by a mutation in the Parkin gene—has evolved and continues to expand.Key words: PINK1, parkin, ubiquitin, mitochondria, autophagy, mitophagy, membrane potential, quality controlSince the cloning of PINK1 and Parkin, numerous papers have been published about the corresponding gene products, but the mechanism by which dysfunction of PINK1 and/or Parkin causes PD remain unclear. Parkin encodes a ubiquitin ligase E3, a substrate recognition member of the ubiquitination pathway, whereas PINK1 encodes a mitochondria-targeted serine-threonine kinase that contributes to the maintenance of mitochondrial integrity. Based on their molecular functions, it is clear that Parkin-mediated ubiquitination and PINK1 phosphorylation are key events in disease pathogenesis. The underlying mechanism, however, is not as well defined and claims of pathogenicity, until recently, remained controversial. Although Parkin''s E3 activity was clearly demonstrated in vitro, we were unable to show a clear E3 activity of Parkin in cell/in vivo. In addition, despite a predicted mitochondrial localization signal for PINK1, we were unable to detect PINK1 on mitochondria by either immunoblotting or immunocytochemistry. More confusingly, overexpression of nontagged PINK1 mainly localized to the cytoplasm under steady state conditions.Work by Dr. Youle''s group at the National Institutes of Health in 2008, however, offered new insights. They reported that Parkin associated with depolarized mitochondria and that Parkin-marked mitochondria were subsequently cleared by autophagy. Soon after their publication, we also examined the function of Parkin and PINK1 following a decrease in mitochondrial membrane potential. Our findings, described below (Fig. 1), have contributed to the development of a mechanism explaining pathogenicity.Open in a separate windowFigure 1Model of mitochondrial quality control mediated by PINK1 and Parkin. Under steady-state conditions, the mature 60 kDa PINK1 is constantly cleaved by an unknown protease to a 50 kDa intermediate form that is subsequently degraded, presumably by the proteasome (upper part). The protein, however, is stabilized on depolarized mitochondria because the initial processing event is inhibited by a decrease in mitochondrial membrane potential (lower part). Accumulated PINK1 recruits cytosolic Parkin onto depolarized mitochondria resulting in activation of its E3 activity. Parkin then ubiquitinates a mitochondrial substrate(s). As a consequence, damaged mitochondria are degraded via mitophagy. Ub, ubiquitin.(1) We sought to determine the subcellular localization of endogenous PINK1, and realized that endogenous PINK1 is barely detectable under steady-state conditions. However, a decrease in mitochondrial membrane-potential following treatment with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) results in the gradual accumulation of endogenous PINK1 on mitochondria. Importantly, when CCCP is washed out, the accumulated endogenous PINK1 rapidly disappears (within 30 min) both in the presence and absence of cycloheximide. These results support the hypothesis that PINK1 is constantly transported to the mitochondria, but is rapidly degraded in a membrane potential-dependent manner (see below for details). We speculate that PINK1 is stabilized by a decrease in mitochondrial membrane potential and as a result accumulates on depolarized mitochondria.(2) We examined the potential role of PINK1 in the mitochondrial recruitment of Parkin. In control MEFs (PINK1^+/+), Parkin is selectively recruited to the mitochondria following CCCP treatment, and subsequently results in the selective disappearance of the mitochondria via autophagy (called mitophagy). In sharp contrast, Parkin is not translocated to the mitochondria in PINK1 knockout (PINK1^−/−) MEFs following CCCP treatment, and subsequent mitochondrial degradation is also completely impeded. These results suggest that PINK1 is “a Parkin-recruitment factor” that recruits Parkin from the cytoplasm to damaged mitochondria in a membrane potential-dependent manner for mitophagy.(3) We monitored the E3 activity of Parkin using an artificial pseudo-substrate fused to Parkin in cells. Parkin''s E3 activity was repressed under steady-state conditions; however, we find that Parkin ubiquitinates the pseudo-substrate when it is retrieved to the depolarized mitochondria, suggesting that activation of the latent Parkin E3 activity is likewise dependent on a decrease in mitochondrial membrane potential.(4) PINK1 normally exists as either a long (approximately 60 kDa) or a short (approximately 50 kDa) protein. Because the canonical mitochondrial targeting signal (matrix targeting signal) is cleaved after import into the mitochondria, the long form has been designated as the precursor and the short form as the mature PINK1. However, our subcellular localization study of endogenous PINK1 following CCCP treatment shows that the long form is recovered in the mitochondrial fraction, suggesting that it is not the pre-import precursor form. Moreover, by monitoring the degradation process of PINK1 following recovery of membrane potential, we realized that the short form of PINK1 transiently appears soon after CCCP is washed out and then later disappears, suggesting that the processed form of PINK1 is an intermediate in membrane-potential-dependent degradation. In conclusion, these results imply that PINK1 cleavage does not reflect a canonical maturation process accompanying mitochondrial import as initially thought, but rather represents constitutive degradation in healthy mitochondria by a two-step mechanism; i.e., first limited processing and subsequent complete degradation probably via the proteasome.(5) PINK1 accumulation by decrease of membrane potential and subsequent recruitment of Parkin onto mitochondria are presumably etiologically important because they are impeded for the most part by disease-linked mutations of PINK1 or Parkin.These results, together with reports by other groups, strongly suggest that recessive familial PD is caused by dysfunction of quality control for depolarized mitochondria.At present, we do not know whether the aforementioned pathogenic mechanism of recessive familial PD can be generalized to prevalent sporadic PD. However, the clinical symptoms of recessive familial PD caused by dysfunction of PINK1 or Parkin resembles that of idiopathic PD except early-onset pathogenesis, and thus it is plausible that there is a common pathogenic mechanism. We accordingly believe that our results provide solid insight into the molecular mechanisms of PD pathogenesis, not only for familial forms caused by Parkin and PINK1 mutations, but also the major sporadic form of PD.To fully understand the molecular mechanism of PINK1-Parkin-mediated mitophagy, further details need to be addressed including: identifying the protease(s) that processes PINK1 in a mitochondrial membrane-potential dependent manner and that presumably monitors mitochondrial integrity; identifying a physiological substrate(s) of PINK1; determining the molecular mechanism underlying Parkin activation; and identifying the protein(s) linking Parkin-mediated ubiquitination to mitophagy. A detailed mechanism of the aforementioned events will be the focus of future research, however, we feel our conclusion that PINK1 and Parkin function in the removal of depolarized mitochondria is evident and hope that our studies will provide a solid foundation for further studies.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Two Types of FtsZ Proteins in Mitochondria and Red-Lineage Chloroplasts: The Duplication of FtsZ Is Implicated in Endosymbiosis

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Distribution of Zearalenone-Producing Fusarium Species in Japan

One hundred sixty-six isolates of Fusarium spp. from domestic cereal grains, feed, and other sources were examined for their ability to produce zearalenone on autoclaved moist rice grains. They belonged to the following species (number of producers/number tested): F. roseum (9/28), F. roseum (Culmorum) (3/4), F. roseum (Gibbosum) (2/5), F. roseum (Avenaceum) (1/2), F. roseum (Scirpi) (0/1), F. tricinctum (1/4), F. tricinctum (Sporotrichiella) (0/7), F. lateritium (1/1), F. episphaeria (0/2), F. moniliforme (0/3), F. oxysporum (0/12), F. rigidiusculum (0/4), F. solani (0/4), F. splendens (0/1), F. nivale (0/2), and Fusarium spp. (15/86). Zearalenone was isolated from molded rice by ethanol extraction and purified by column chromatography. Selected isolates of F. roseum M-3-2 and F. roseum (Gibbosum) A-O-2 produced 50 to 100 mg of zearalenone per kg of rice. Increased yields (250 to 407 mg/kg of rice) were obtained by F. roseum M-3-2 when the substrate was supplemented with 1% peptone.