Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

Estimation of viability of inner bark tissue of <Emphasis Type="Italic">Quercus serrata</Emphasis>, a substrate for log cultivation of <Emphasis Type="Italic">Lentinula edodes</Emphasis>, using the TTC assay method

In the log cultivation of Shiitake (Lentinula edodes), early colonization of this fungus is extremely retarded in living wood tissues, in particular in inner bark tissues. To estimate the viability of inner bark tissues of Quercus serrata, a substrate for log cultivation of Shiitake, we employed a colorimetric assay utilizing a tetrazolium salt (2,3,5-triphenyltetrazolium chloride, TTC) and investigated the relationships between degree of decrease in viability and increase in growth of L. edodes in the tissues. When the mixtures of different proportions of living and dead tissues were assayed, formazan production was proportional to the percentage of living tissues. When logs dried for various time periods were inoculated with L. edodes, the fungus grew more extensively in tissues with reduced formazan production. These results indicate that the TTC assay is a useful method for estimation of viability and thus can be used to decide the proper timing for inoculation of L. edodes.Contribution no. 372 from the Tottori Mycological Institute     

Tagged Mutagenesis and Gene-trap in the Moss, Physcomitrella patens by Shuttle Mutagenesis

The moss, Physcomitrella patens has been used as a useful materialin many fields, because of its simple body plan, ease of genetargeting, and other reasons. Although many mutants have beenreported, no method to isolate the corresponding genes was reported.We developed a gene tagging and gene-trap system in P. patensby using the shuttle mutagenesis technique, which has been usedin the budding yeast. In 5264 tagged lines, 203 mutants withaltered developmental or morphological phenotypes were obtained.In 129 of 4757 gene-trap lines, ß-glucuronidase (GUS)activity was detected in some tissue. Although multiple copiesof a tag were detected in many tagged lines by Southern analyses,most copies are likely integrated at the same locus accordingto PCR analyses.     

Development of a Novel Shock Wave Catheter Ablation System -The First Feasibility Study in Pigs-

Introduction

Radio-frequency catheter ablation (RFCA) using Joule heat has two fundamental weaknesses: the limited depth of treatment and the risk of thrombus formation. In contrast, focused shock wave (SW) therapy could damage tissues at arbitrary depths without heat generation. Thus, we aimed to develop a SW catheter ablation (SWCA) system that could compensate for the weaknesses of RFCA therapy.

Methods and Results

We developed a SWCA system where the SW generated by a Q-switched Holmium: yttrium aluminum garnet (YAG) laser beam was reflected by a reflector attached to 14-Fr catheter tip and then was converged onto the focus. We examined the feasibility of our system on pigs in vivo. When applied using the epicardial approach, the SWCA caused persistent spheroidal lesions with mild superficial injury than the RFCA. The lesions were created to a depth based on the focal length (2.0 mm) [2.36 ± 0.45 (SD) mm immediately after procedure, n = 16]. When applied to the atrioventricular (AV) node using the endocardial approach, the SWCA caused junctional escape rhythms in 2 pigs and AV block in 12 pigs (complete AV block in 9) in acute phase (n = 14). Nine of the 14 pigs survived with pacemakers for the long-term study, and the AV block persisted for 12.6 ± 3.9 (SD) days in all surviving pigs. Histological examination showed AV nodal cell body atrophy in the acute phase and fibrotic lesions in the chronic phase. Importantly, no acute or chronic fatal complications were noted.

Conclusions

Our novel SWCA system could be a promising modality as a non-thermal ablation method to compensate for the weaknesses of RFCA therapy. However, further research and development will be necessary as the current prototype still exhibited the presence of micro-thrombus formation in the animal studies.     

Soluble β-amyloid Precursor Protein Alpha Binds to p75 Neurotrophin Receptor to Promote Neurite Outgrowth

Background

The cleavage of β-amyloid precursor protein (APP) generates multiple proteins: Soluble β-amyloid Precursor Protein Alpha (sAPPα), sAPPβ, and amyloid β (Aβ). Previous studies have shown that sAPPα and sAPPβ possess neurotrophic properties, whereas Aβ is neurotoxic. However, the underlying mechanism of the opposing effects of APP fragments remains poorly understood. In this study, we have investigated the mechanism of sAPPα-mediated neurotrophic effects. sAPPα and sAPPβ interact with p75 neurotrophin receptor (p75^NTR), and sAPPα promotes neurite outgrowth.

Methods and Findings

First, we investigated whether APP fragments interact with p75^NTR, because full-length APP and Aβ have been shown to interact with p75^NTR in vitro. Both sAPPα and sAPPβ were co-immunoprecipitated with p75^NTR and co-localized with p75^NTR on COS-7 cells. The binding affinity of sAPPα and sAPPβ for p75^NTR was confirmed by enzyme-linked immunosorbent assay (ELISA). Next, we investigated the effect of sAPPα on neurite outgrowth in mouse cortical neurons. Neurite outgrowth was promoted by sAPPα, but sAPPα was uneffective in a knockdown of p75^NTR.

Conclusion

We conclude that p75^NTR is the receptor for sAPPα to mediate neurotrophic effects.     

Synthesis of 6,14-epoxymorphinan derivatives and their pharmacologies

A novel 6,14-epoxymorphinan benzamide derivative (NS22) that was previously reported showed opioid κ receptor agonistic activity and analgesic activity. The unsatisfactory κ selectivity of NS22 led us to synthesize its derivatives to improve the opioid κ receptor selectivity and the agonist activity. In the course of SAR of the various derivatives, 17-benzyl-6,14-epoxymorphinan derivatives (KNT-33, 53, 55, 80, 90, 133) were found to show high selectivities and affinities for the opioid κ receptor. In addition, KNT-33, 53, 55 showed dose-dependent analgesic effects in acetic acid writhing tests. Therefore, 17-benzyl substituents may play an important role for developing κ selectivity.     

SEX‐SPECIFIC CELL SURFACE STRUCTURE OF ANISOGAMETES: MORPHOLOGICAL CHANGES DURING FERTILIZATION OF BRYOPSIS MAXIMA (ULVOPHYCEAE,CHLOROPHYTA) REVEALED BY ULTRA–HIGH‐RESOLUTION FIELD EMISSION SEM1

Cell surfaces of biflagellate gametes and their morphological changes during fertilization of Bryopsis maxima Okamura were observed using a high‐resolution field emission scanning electron microscope. Male gametes have broad and narrow faces, which are divided into at least five morphologically distinct regions: 1) the apical plate is a plate‐like structure that is approximately 380–530 nm long and approximately 190 nm wide, in the center of the papilla and slightly protruded from the plasma membrane; 2) strips are smooth materials on ridges that originate from the basal part of the papilla and extend downward; 3) the lateral belt is a belt‐shaped structure on the center of the narrower faces; 4) the flagellar surface; and 5) the other region of the cell body has a fine‐grained appearance. In contrast, the entire female gamete surface is rough because of many granular or amorphous cell coats on the plasma membrane. When both gametes were mixed together, the initial fusion proceeded between the broader face of the male gamete and the anterior side of the female one near the basal bodies. Morphology of the male gamete's cell surface changed gradually as fusion proceeded and was covered by the granular materials; that surface closely resembled those of female gametes except for the apical plate. It was present until the planozygote attached itself to the substrate by the papilla. It finally disappeared after settlement. Therefore, these results indicate that gametes of B. maxima have sex‐specific surface structures that change their morphology during fertilization and settlement.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.